Formulations for improving gut health

Abstract

To provide active substances and methods for improving gut health in animals and humans.SOLUTION: Active substances include riboflavin, vitamin A, vitamin C, vitamin D, vitamin E, vitamin K, folic acid, β-carotene, vitamin B1, niacin, vitamin B5, vitamin B6, biotin, vitamin B12, ω-3 fatty acids and combinations thereof. Benefits include increased concentration of at least one short-chain fatty acid or salt thereof in the intestinal tract, increased microbiota diversity in the intestinal tract, increased beneficial bacteria in the intestinal tract, increased butyrate synthesis pathway in the intestinal tract, improved intestinal barrier function, reduced intestinal redox potential, reduced amount of gas produced in the intestinal tract; and stimulation of intestinal immune response.SELECTED DRAWING: None

Description

Detailed Description of the Invention

[0001] [Technical field] The present invention relates to a formulation for improving the gut health of animals and humans. In particular, the formulation of the present invention can be used to increase the concentration of short chain fatty acids (SCFAs) in the lower gastrointestinal tract and / or to increase the butyric acid synthesis pathway in the lower gastrointestinal tract. The formulation of the present invention can also be used to increase or regulate the microbiota diversity and / or abundance of beneficial bacteria, such as Bifidobacterium, Akkermansia and Faecalibacterium, in the lower gastrointestinal tract. The present invention also relates to methods and compositions for improving intestinal barrier function and stimulating gut immune response. [background]

[0002] Human and animal microbiota are the collection of microorganisms that live on and in the human / animal body, with the largest and most diverse cluster of microorganisms residing in the gut. The gut microbiota has co-evolved with the host, providing a stable environment for the microorganisms, which also provide a wide variety of functions to the host, such as digestion of complex dietary macronutrients, production of nutrients and vitamins, defense against pathogens, and maintenance of the immune system. Recent data reveal that abnormal gut microbiota composition is associated with various diseases, including immune deficiency, allergies, metabolic disorders (such as type 2 diabetes), and immune-mediated bowel disease (IBD). One of the mechanisms by which the microbiota influences human health and disease is its ability to produce either harmful metabolites that are associated with disease development or beneficial metabolites that protect against disease. Proteins and peptides, as well as dietary fibers that escape digestion by host enzymes in the upper intestinal tract, are metabolized by the microbiota in the cecum and colon. The major products from the fermentation activity of gut bacteria are SCFAs, especially acetate, propionate, and butyrate. A growing body of evidence suggests that SCFAs in the human or animal intestinal tract have beneficial effects on overall gut health, including host metabolic performance such as glucose metabolism, the immune system, gastrointestinal integrity and barrier, and gastrointestinal motility.

[0003] WO 2007 / 058523 A1 describes the use of n-3 docosapentanoic acid for the preparation of a nutritional pharmaceutical composition for improving intestinal barrier integrity, enhancing barrier function, stimulating intestinal maturation and / or reducing intestinal barrier permeability.

[0004] WO 2016 / 053085 A1 describes a composition comprising a uridine source and a butyrate-generating fiber for use in the prevention or treatment of constipation, transit time dysfunction or abnormal colon length.

[0005] US 2010 / 247489 A1 discloses the use of delayed release compositions containing certain minerals, such as tungsten, to reduce gas formation in the colon. The compositions may further contain vitamins or strains of acetogenic or butyrogenic bacteria.

[0006] Tan et al. (2016) Cell Reports, Vol. 15, No. 12, 2809-2824 uses mice to examine the beneficial role of dietary fiber in peanut allergy. Tan et al. report that this effect includes remodeling the gut microbiota and increasing the levels of short-chain fatty acids and the activity of the receptors GPR43 and GPR109a.

[0007] WO 2010 / 117274 A1 describes carbohydrates that increase the production of C5 and / or C6 short chain fatty acids.

[0008] WO 2017 / 182347 A1 describes microcapsules comprising a core containing vitamin B3, which are characterized by a coating layer system comprising two shellac layers and a pH adjusting substance disposed between the two shellac layers.

[0009] US Pat. No. 9,433,583 B2 describes a colon-targeted single dosage form containing vitamin D and optionally further vitamins for the prevention of colon adenomatous polyps and colon cancer.

[0010] WO 2014 / 070014 describes the use of riboflavin (vitamin B2) to stimulate populations of Faecalibacterium prausnitzii.

[0011] [Summary of the Invention] The inventors of the present application have found that intestinal microbiota produces more SCFAs in the presence of certain micronutrients. In addition, an increase in the butyrate synthesis pathway was also observed. In further tests, it was found that micronutrients can also increase intestinal microbiota diversity and microbial abundance, improve intestinal barrier function, and reduce gas production. Thus, the present invention relates to the following items [1] to

[67] : [1] An active substance as claimed in claim 1 for use in improving the intestinal health of an animal; or an active agent selected from the group consisting of riboflavin, vitamin A, vitamin C, vitamin D, vitamin E, vitamin K, folic acid, beta-carotene, vitamin B1, niacin, vitamin B5, vitamin B6, biotin, vitamin B12, omega-3 fatty acids, and combinations thereof, wherein the improvement is characterized by: (i) increasing the concentration of at least one short chain fatty acid or a salt thereof in the intestinal tract; (ii) increasing the diversity of the gut microbiota; (iii) increasing the abundance of microbiota in the intestinal tract; (iv) promotion or increase of the butyric acid synthesis pathway in the intestinal tract; (v) Improved intestinal barrier function, (vi) a decrease in the redox potential of the intestinal tract; (vii) reducing the amount of gas produced in the intestinal tract; and / or (viii) Stimulation of intestinal immune responses comprising or consisting of wherein said use comprises administering to an animal a delayed release formulation comprising an effective dose of an active agent, wherein release of said active agent is delayed until the intestine, preferably the large intestine.

[0012] [2] The method according to claim [1], wherein the improvement comprises or consists of an increase in the concentration of at least one short chain fatty acid or a salt thereof in the intestinal tract, wherein the at least one short chain fatty acid is selected from the group consisting of butyric acid, acetic acid, propionic acid, and combinations thereof.

[0013] [3] The active substance for use according to item [2], wherein the at least one short chain fatty acid is butyric acid.

[0014] [4] The active substance for use according to item [2], wherein the at least one short chain fatty acid is acetic acid.

[0015] [5] The active substance for use according to item [2], wherein the at least one short chain fatty acid is propionic acid.

[0016] [6] The active substance for use according to any one of the preceding items, wherein the use comprises or consists of the treatment or prevention of a disorder associated with low concentrations of one or more SCFAs in the lower gastrointestinal tract.

[0017] [7] The method according to item [6], wherein the disorder associated with a low concentration of one or more SCFAs in the lower gastrointestinal tract is an inflammatory disease.

[0018] [8] The active substance for use according to item [7], wherein the inflammatory disease is characterized by chronic inflammation.

[0019] [9] The active substance for use according to item [6], wherein the disorder associated with a low concentration of one or more SCFAs in the lower gastrointestinal tract is atherogenesis.

[0020]

[10] The active substance for use according to item [6], wherein the disorder associated with a low concentration of one or more SCFAs in the lower gastrointestinal tract is a metabolic disorder.

[0021]

[11] The method according to item

[10] , wherein the metabolic disorder is type 2 diabetes and / or obesity.

[0022]

[12] The method according to item [1], wherein the improvement comprises or consists of an increase in the microbiota diversity in the intestinal tract.

[0023]

[13] An active substance for use according to item

[12] , wherein the use comprises or consists of treating or preventing a disorder associated with insufficient microbiota diversity in the intestinal tract.

[0024]

[14] The method according to item [1], wherein the improvement comprises or consists of increasing the abundance of at least one microorganism selected from the group consisting of Bifidobacterium, Akkermansia and Faecalibacterium in the human intestinal tract.

[0025]

[15] An active substance for the use according to item

[14] , wherein the use comprises or consists of treating or preventing disorders associated with insufficient Bifidobacterium, Akkermansia and Faecalibacterium in the human intestinal tract, such as IBD, metabolic diseases including obesity and type 2 diabetes, and other inflammatory diseases.

[0026]

[16] The method according to item [1], wherein the improvement comprises or consists of increasing the abundance of at least one microorganism selected from the group consisting of Bacteroides, Alistipes, and Faecalibacterium.

[0027]

[17] The method according to claim 1, wherein the improvement comprises or consists of promoting or increasing the butyric acid synthesis pathway in the intestinal tract.

[0028]

[18] An active substance for use according to item

[17] , wherein the use comprises or consists of treating or preventing disorders associated with an insufficient butyric acid synthesis pathway in the intestinal tract.

[0029]

[19] An active agent selected from the group consisting of riboflavin, vitamin A, vitamin C, vitamin D, vitamin E, vitamin K, folic acid, beta-carotene, vitamin B1, niacin, vitamin B5, vitamin B6, biotin, vitamin B12, omega-3 fatty acids and combinations thereof, for use in the treatment or prevention of a condition associated with impaired intestinal barrier function in an animal, preferably a mammal, said use comprising administering to the animal a delayed release formulation comprising an effective dose of the active agent, wherein release of the active agent is delayed until the large intestine.

[0030]

[20] The method according to item

[19] , wherein the condition associated with impaired barrier function of the intestinal tract of an animal is characterized by leaky gut.

[0031]

[21] The active substance for use according to items

[19] or

[20] , wherein the condition associated with impaired intestinal barrier function is caused by malnutrition.

[0032]

[22] The method according to item

[19] or

[20] , wherein the condition associated with impaired barrier function of the intestinal tract of an animal is inflammatory bowel disease.

[0033]

[23] The method according to item

[22] , wherein the inflammatory bowel disease is Crohn's disease.

[0034]

[24] The method according to item

[22] , wherein the inflammatory bowel disease is ulcerative colitis.

[0035]

[25] The active substance for use according to item

[19] , wherein the animal is a monogastric animal.

[0036]

[26] The active substance for use according to item

[19] , wherein the animal is a poultry or a pig.

[0037]

[27] The active substance for use according to any one of items [1] to

[26] , wherein the delayed release formulation is a sustained release formulation.

[0038]

[28] The active substance for use according to any one of items [1] to

[26] , wherein the release of the active substance is delayed until the large intestine.

[0039]

[29] The active substance according to any one of items [1] to

[26] , wherein the release of the active substance is delayed until the colon.

[0040]

[30] The active substance for use according to any one of items [1] to

[29] , wherein the active substance comprises riboflavin.

[0041]

[31] The active substance for use according to any one of items [1] to

[29] , wherein the active substance is vitamin A.

[0042]

[32] The active substance for use according to any one of items [1] to

[29] , wherein the active substance is vitamin C.

[0043]

[33] The active substance for use according to any one of items [1] to

[29] , wherein the active substance is vitamin D.

[0044]

[34] The active substance according to any one of items [1] to

[29] , wherein the active substance is vitamin E.

[0045]

[35] The active substance for use according to any one of items [1] to

[29] , wherein the active substance is vitamin K.

[0046]

[36] The active substance for use according to any one of items [1] to

[29] , wherein the active substance comprises folic acid.

[0047]

[37] The active substance for use according to any one of items [1] to

[29] , wherein the active substance is β-carotene.

[0048]

[38] The active substance for use according to any one of items [1] to

[29] , wherein the active substance is vitamin B1.

[0049]

[39] The active substance for use according to any one of items [1] to

[29] , wherein the active substance consists of niacin.

[0050]

[40] The active substance for use according to any one of items [1] to

[29] , wherein the active substance is vitamin B5.

[0051]

[41] The active substance for use according to any one of items [1] to

[29] , wherein the active substance is vitamin B6.

[0052]

[42] The active substance for use according to any one of items [1] to

[29] , wherein the active substance comprises biotin.

[0053]

[43] The active substance for use according to any one of items [1] to

[29] , wherein the active substance is vitamin D.

[0054]

[44] The active substance for use according to any one of items [1] to

[29] , wherein the active substance is composed of at least one omega-3 fatty acid.

[0055]

[45] The method according to item

[44] , wherein the active substance is docosahexaenoic acid (DHA), eicosapentanoic acid (EPA), or a combination thereof.

[0056]

[46] The active substance for use according to item

[44] , wherein the active substance consists of DHA.

[0057]

[47] The active substance for use according to item

[44] , wherein the active substance consists of EPA.

[0058]

[48] ​​The active substance for use according to item

[44] , wherein the active substance consists of a combination of DHA and EPA.

[0059]

[49] The active substance for use according to any one of items [1] to

[29] , wherein the active substance consists of a combination of riboflavin, vitamin A, vitamin C, vitamin D, vitamin E, vitamin K, folic acid, β-carotene, vitamin B1, niacin, vitamin B5, vitamin B6, biotin, vitamin B12, DHA and EPA.

[0060]

[50] The active substance contains up to 2.9 mg of riboflavin per kg of body weight, up to 42.9 μg of vitamin A per kg of body weight, up to 3.6 μg of vitamin D per kg of body weight, up to 28.6 mg of vitamin C per kg of body weight, up to 14.3 mg of vitamin E per kg of body weight, up to 143 μg of vitamin K per kg of body weight, up to 14.3 μg of folic acid per kg of body weight, up to 0.4 mg of beta-carotene per kg of body weight, up to 1.4 mg of vitamin B1, up to 2 mg of vitamin B2, up to 2 mg of vitamin C per kg of body weight, up to 2 mg of vitamin D per kg of body weight, up to 2 mg of vitamin E per kg of body weight, up to 2 mg of vitamin B1, up to 2 mg of vitamin B1, up to 2 mg of vitamin B1, up to 2 mg of vitamin C per kg of body weight ... B1, up to 2 mg of vitamin B1, up to 2 mg of vitamin B1, up to 2 mg of vitamin B1, up to 2 mg of vitamin B1, up to 2 mg of vitamin B1, up to 2 mg of vitamin B1, up to 2 mg of vitamin B1, up to 2 mg of vitamin B1, up to 2 mg of The active substance for use according to any one of the preceding items is administered to the animal, preferably a mammal, in a daily dose of 1.4 mg niacin, up to 14.3 mg vitamin B5, up to 1.4 mg vitamin B6, up to 35.7 μg biotin, up to 43 μg vitamin B12, 71.4 mg omega-3 fatty acids or combinations thereof, wherein preferably EPA is at a maximum of 25.7 mg per kg body weight and DHA is at a maximum of 14.3 mg per kg body weight.

[0061]

[51] A delayed release formulation for delivering an active agent to the intestinal tract, comprising an active agent and an enteric layer or shell, wherein the active agent is comprised of up to 200 mg riboflavin, up to 3000 μg vitamin A, up to 2000 mg vitamin C, up to 250 μg vitamin D, up to 1000 mg vitamin E, up to 10 mg vitamin K, up to 1 mg folic acid, up to 25 mg beta-carotene, up to 100 mg vitamin B1, up to 1500 mg niacin, up to 1000 mg vitamin B5, up to 100 mg vitamin B6, up to 2500 μg biotin, up to 3000 μg vitamin B12, up to 5000 mg omega-3 fatty acids or combinations thereof, preferably wherein EPA is up to 1800 mg and DHA is up to 1000 mg, and wherein release of the active agent is delayed to the intestinal tract.

[0062]

[52] A delayed release formulation for delivering an active agent to the intestinal tract, comprising an active agent and an enteric layer or shell, wherein the active agent is comprised of 1-85 mg riboflavin, 0.2-0.4 mg vitamin A, 400-600 mg vitamin C, 5-80 μg vitamin D, 80-120 mg vitamin E, 80-140 μg vitamin K, 0.3-0.5 mg folic acid, 80-120 mg omega-3 fatty acids or combinations thereof, wherein release of the active agent is delayed to the intestinal tract.

[0063]

[53] The delayed release formulation according to item

[51] or

[52] , wherein the active substances consist of 70-80 mg of riboflavin, 0.25-0.35 mg of vitamin A, 450-550 mg of vitamin C, 15-25 μg of vitamin D, 90-110 mg of vitamin E, 100-120 μg of vitamin K, 0.35-0.45 mg of folic acid, 90-110 mg of omega-3 fatty acids or a combination thereof.

[0064]

[54] The composition according to any one of items

[51] to

[53] , wherein the release of the active substance is delayed until the colon.

[0065]

[55] Non-therapeutic use of an active agent selected from the group consisting of riboflavin, vitamin A, vitamin C, vitamin D, vitamin E, vitamin K, folic acid, beta-carotene, vitamin B1, niacin, vitamin B5, vitamin B6, biotin, vitamin B12, omega-3 fatty acids, and combinations thereof, to improve the condition or health of the intestinal tract in an animal, preferably a mammal, monogastric animal, or poultry, not suffering from an intestinal disorder or condition.

[0066]

[56] The non-therapeutic use according to item

[55] , wherein the mammal is a healthy human.

[0067]

[57] The non-therapeutic use according to item

[55] , wherein the mammal is a healthy adult.

[0068]

[58] The non-therapeutic use according to any one of items

[55] to

[57] , wherein the improvement comprises or consists of an increase in the concentration of at least one short-chain fatty acid or a salt thereof in the intestinal tract.

[0069]

[59] The non-therapeutic use according to any one of items

[55] to

[57] , wherein the improvement comprises or consists of an increase in the microbiota diversity in the intestinal tract.

[0070]

[60] The non-therapeutic use according to any one of items

[55] to

[57] , wherein the improvement comprises or consists of an increase in Bifidobacterium, Akkermansia and / or Faecalibacterium in the intestinal tract.

[0071]

[61] The non-therapeutic use according to any one of items

[55] to

[57] , wherein the improvement comprises or consists of promotion or increase of the butyric acid synthesis pathway in the intestinal tract.

[0072]

[62] The non-therapeutic use according to any one of items

[55] to

[57] , wherein the improvement comprises or consists of an improvement in intestinal barrier function, and wherein the active substance does not comprise an omega-3 fatty acid.

[0073]

[63] The non-therapeutic use according to any one of items

[55] to

[62] , wherein the active substance does not include vitamin D3.

[0074]

[64] The non-therapeutic use according to any one of items

[55] to

[63] , wherein the active substance does not contain omega-3 fatty acids.

[0075]

[65] Non-therapeutic use of an active agent selected from the group consisting of riboflavin, vitamin A, vitamin C, vitamin D, vitamin E, vitamin K, folic acid, beta-carotene, vitamin B1, niacin, vitamin B5, vitamin B6, biotin, vitamin B12, omega-3 fatty acids, and combinations thereof, for maintaining or improving gastrointestinal function in a healthy mammal.

[0076]

[66] A method for improving intestinal health in an animal, said improvement comprising: (i) increasing the concentration of at least one short chain fatty acid or a salt thereof in the intestinal tract; (ii) increasing the diversity of the gut microbiota; (iii) increasing the abundance of microbiota in the intestinal tract; (iv) promotion or increase of the butyric acid synthesis pathway in the intestinal tract; (v) Improved intestinal barrier function, (vi) a decrease in the redox potential of the intestinal tract; and / or (vii) A reduction in the amount of gas produced in the intestinal tract; comprising or consisting of administering to the animal a delayed release formulation comprising an effective dose of the active agent, wherein release of the active agent is delayed until the intestinal tract or a feed comprising a supraphysiological dose of the active agent, present in the intestinal tract in an effective amount; The method of claim 1, wherein the active agent is selected from the group consisting of: riboflavin, vitamin A, vitamin C, vitamin D, vitamin E, vitamin K, folic acid, beta-carotene, vitamin B1, niacin, vitamin B5, vitamin B6, biotin, vitamin B12, omega-3 fatty acids, and combinations thereof.

[0077]

[67] A poultry or non-human feed comprising a supraphysiological dose of an active agent selected from the group consisting of riboflavin, vitamin A, vitamin C, vitamin D, vitamin E, vitamin K, folic acid, beta-carotene, vitamin B1, niacin, vitamin B5, vitamin B6, biotin, vitamin B12, omega-3 fatty acids, and combinations thereof, wherein a sufficient amount of the active agent is present in the intestinal tract.

[0078] The present invention further relates to the following embodiments: (1) An active agent selected from the group consisting of riboflavin, vitamin A, vitamin C, vitamin D, vitamin E, vitamin K, folic acid, beta-carotene, vitamin B1, niacin, vitamin B5, vitamin B6, biotin, vitamin B12, omega-3 fatty acids, and combinations thereof, for use in increasing the concentration of at least one short chain fatty acid (SCFA) or salt thereof in the intestinal tract of a mammal, said use comprising administering to said mammal an effective amount of a delayed release formulation comprising said active agent, wherein release of said active agent is delayed to the intestinal tract.

[0079] (2) The active substance for use according to embodiment (1), wherein the at least one short chain fatty acid is selected from the group consisting of butyric acid, acetic acid, propionic acid and combinations thereof.

[0080] (3) The active substance for use according to embodiment (2), wherein the at least one short chain fatty acid is butyric acid.

[0081] (4) The active substance for use according to embodiment (2), wherein the at least one short chain fatty acid is acetic acid.

[0082] (5) The active substance for use according to embodiment (2), wherein the at least one short chain fatty acid is propionic acid.

[0083] (6) The active substance for use according to any one of the preceding embodiments, wherein said use comprises or consists of the treatment or prevention of a disorder associated with low concentrations of one or more SCFAs in the lower gastrointestinal tract.

[0084] (7) The active substance for use according to embodiment (6), wherein the disorder associated with a low concentration of one or more SCFAs in the lower gastrointestinal tract is an inflammatory disease.

[0085] (8) The active substance for use according to embodiment (7), wherein the inflammatory disease is characterized by chronic inflammation.

[0086] (9) The active substance for use according to embodiment (6), wherein the disorder associated with a low concentration of one or more SCFAs in the lower gastrointestinal tract is atherogenesis.

[0087] (10) The active substance for use according to embodiment (6), wherein the disorder associated with a low concentration of one or more SCFAs in the lower gastrointestinal tract is a metabolic disorder.

[0088] (11) An active agent selected from the group consisting of riboflavin, vitamin A, vitamin C, vitamin D, vitamin E, vitamin K, folic acid, omega-3 fatty acids and combinations thereof for use in the treatment or prevention of a condition associated with impaired intestinal barrier function in a mammal, said use comprising administering to said mammal an effective amount of a delayed release formulation comprising said active agent, wherein release of said active agent is delayed to the intestinal tract.

[0089] (12) The active substance for use according to embodiment (12), wherein the condition associated with impaired barrier function of the intestinal tract of a mammal is characterized by leaky gut.

[0090] (13) The active substance for use according to embodiment (12) or (13), wherein the condition associated with impaired intestinal barrier function is caused by malnutrition.

[0091] (14) The active substance for use according to embodiment (12) or (13), wherein the condition associated with impaired barrier function of the intestinal tract of an animal is inflammatory bowel disease.

[0092] (15) The active substance for use according to embodiment (15), wherein the inflammatory bowel disease is Crohn's disease.

[0093] (16) The active substance for use according to embodiment (15), wherein the inflammatory bowel disease is ulcerative colitis.

[0094] (17) The active substance for use according to any one of embodiments (1) to (17), wherein the delayed release formulation is a sustained release formulation.

[0095] (18) The active substance for use according to any one of embodiments (1) to (17), wherein the release of the active substance is delayed until the large intestine.

[0096] (19) The active substance according to any one of embodiments (1) to (17), wherein the release of the active substance is delayed until the colon.

[0097] (20) The active substance for use according to any one of the preceding embodiments, wherein the active substance consists of riboflavin.

[0098] (21) The active substance for use according to any one of the preceding embodiments, wherein the active substance consists of vitamin A.

[0099] (22) The active substance for use according to any one of the preceding embodiments, wherein the active substance consists of vitamin C.

[0100] (23) The active substance for use according to any one of the preceding embodiments, wherein the active substance consists of vitamin D.

[0101] (24) The active agent of any one of the preceding embodiments, wherein the active agent consists of Vitamin E.

[0102] (25) The active substance for use according to any one of the preceding embodiments, wherein the active substance consists of vitamin K.

[0103] (26) The active substance for use according to any one of the preceding embodiments, wherein the active substance consists of folic acid.

[0104] (27) The active substance for use according to any one of the preceding embodiments, wherein the active substance is composed of at least one omega-3 fatty acid.

[0105] (28) The active substance for use according to any one of the preceding embodiments, wherein the active substance consists of docosahexaenoic acid (DHA), eicosapentanoic acid (EPA), or a combination thereof.

[0106] (29) The active substance for use according to any one of the preceding embodiments, wherein the active substance consists of DHA.

[0107] (30) The active substance for use according to any one of the preceding embodiments, wherein the active substance consists of EPA.

[0108] (31) The active substance for use according to any one of the preceding embodiments, wherein the active substance consists of a combination of DHA and EPA.

[0109] (32) The active substance for use according to any one of the preceding embodiments, wherein the active substance consists of a combination of riboflavin, vitamin A, vitamin C, vitamin D, vitamin E, vitamin K, folic acid, beta-carotene, vitamin B1, niacin, vitamin B5, vitamin B6, biotin, vitamin B12, DHA and EPA.

[0110] (33) The active agent for use according to any one of the preceding embodiments, wherein the active agent is administered to the mammal in a daily amount of 0.01-1.2 mg riboflavin per kg body weight, 2.9-5.7 μg vitamin A per kg body weight, 0.1-1.1 μg vitamin D per kg body weight, 0.2-1.7 mg vitamin E per kg body weight, 1.1-2.0 μg vitamin K per kg body weight, 4.3-7.1 μg folic acid per kg body weight, 1.1-1.7 mg omega-3 fatty acids per kg body weight, or a combination thereof.

[0111] (34) The active agent for use according to any one of the preceding embodiments, wherein the active agent is administered to the mammal in a daily amount of 1.0-1.1 mg riboflavin per kg body weight, 3.6-5.0 μg vitamin A per kg body weight, 0.2-0.4 μg vitamin D per kg body weight, 1.3-1.6 mg vitamin E per kg body weight, 1.4-1.7 μg vitamin K per kg body weight, 5.0-6.4 μg folic acid per kg body weight, 1.3-1.6 mg omega-3 fatty acids per kg body weight, or a combination thereof.

[0112] (35) A delayed release formulation for delivering an active agent to the intestinal tract, comprising the active agent and an enteric coating, wherein the active agent is comprised of 1-85 mg riboflavin, 0.2-0.4 mg vitamin A, 400-600 mg vitamin C, 5-80 μg vitamin D, 80-120 mg vitamin E, 80-140 μg vitamin K, 0.3-0.5 mg folic acid, 80-120 mg omega-3 fatty acids or combinations thereof, wherein release of the active agent is delayed to the intestinal tract.

[0113] (36) The delayed release formulation according to embodiment (36), wherein the active substances consist of 70-80 mg of riboflavin, 0.25-0.35 mg of vitamin A, 450-550 mg of vitamin C, 15-25 μg of vitamin D, 90-110 mg of vitamin E, 100-120 μg of vitamin K, 0.35-0.45 mg of folic acid, 90-110 mg of omega-3 fatty acids or a combination thereof.

[0114] (37) The delayed release formulation according to embodiment (36) or (37), wherein the delayed release formulation is a sustained release formulation.

[0115] (38) The delayed release formulation according to embodiment (36) or (37), wherein the release of the active substance is delayed until the large intestine, preferably the colon.

[0116] (39) The delayed release formulation according to embodiment (36) or (37), wherein the release of the active substance is delayed until the colon.

[0117] (40) The delayed release formulation according to any one of embodiments (36) to (40), comprising riboflavin as the only active substance.

[0118] (41) The delayed release formulation according to any one of embodiments (36) to (40), comprising vitamin A as the only active substance.

[0119] (42) The delayed release formulation according to any one of embodiments (36) to (40), comprising vitamin C as the only active substance.

[0120] (43) The delayed release formulation according to any one of embodiments (36) to (40), comprising vitamin D as the only active substance.

[0121] (44) The delayed release formulation according to any one of embodiments (36) to (40), comprising vitamin E as the only active substance.

[0122] (45) The delayed release formulation according to any one of embodiments (36) to (40), comprising vitamin K as the only active substance.

[0123] (46) The delayed release formulation according to any one of embodiments (36) to (40), comprising folic acid as the only active substance.

[0124] (47) The delayed release formulation according to any one of embodiments (36) to (40), comprising β-carotene as the only active substance.

[0125] (48) The delayed release formulation according to any one of embodiments (36) to (40), comprising vitamin B1 as the only active substance.

[0126] (49) The delayed release formulation according to any one of embodiments (36) to (40), comprising niacin as the only active substance.

[0127] (50) The delayed release formulation according to any one of embodiments (36) to (40), comprising vitamin B5 as the only active substance.

[0128] (51) The delayed release formulation according to any one of embodiments (36) to (40), comprising vitamin B6 as the only active substance.

[0129] (52) The delayed release formulation according to any one of embodiments (36) to (40), comprising biotin as the only active substance.

[0130] (53) The delayed release formulation according to any one of embodiments (36) to (40), comprising vitamin B12 as the only active substance.

[0131] (54) The delayed release formulation according to any one of embodiments (36) to (40), comprising one or more omega-3 fatty acids as the only active substance.

[0132] (55) Non-therapeutic use of an active agent selected from the group consisting of riboflavin, vitamin A, vitamin D, vitamin C, vitamin E, vitamin K, folic acid, beta-carotene, vitamin B1, niacin, vitamin B5, vitamin B6, biotin, vitamin B12, omega-3 fatty acids, and combinations thereof, to increase the concentration of at least one short-chain fatty acid or a salt thereof in the intestinal tract of a mammal not suffering from an intestinal disorder or pathology.

[0133] (56) The non-therapeutic use according to embodiment (49), wherein the mammal is a healthy human.

[0134] (57) Non-therapeutic use of an active agent selected from the group consisting of riboflavin, vitamin A, vitamin C, vitamin D, vitamin E, vitamin K, folic acid, beta-carotene, vitamin B1, niacin, vitamin B5, vitamin B6, biotin, vitamin B12, omega-3 fatty acids, and combinations thereof, for maintaining or improving intestinal function in a healthy mammal.

[0135] (58) The non-therapeutic use according to embodiment (51), wherein the mammal is a human. [Brief description of the drawings]

[0136] [Figure 1] Acetate production after 48 h fermentation from administration of the different test formulations, either as aqueous solutions (top) or dissolved in ethanol (bottom), is shown. Negative controls of various stock solutions were also included. RTG = riboflavin table grade, R5P = riboflavin-5-phosphate, AA = ascorbic acid, RTG+AA = riboflavin table grade + ascorbic acid, FA = folic acid, VK1 = vitamin K1, 0080EE = DHA, 4535EE = DHA+EPA, 8000EE = EPA. Each compound was tested at three doses (0.2x, 1x and 5x) (Table 1). [Diagram 2]Propionate production after 48 h fermentation from administration of the different test formulations, either as aqueous solutions (top) or dissolved in ethanol (bottom), is shown. Negative controls of various stock solutions were also included. RTG = riboflavin table grade, R5P = riboflavin-5-phosphate, AA = ascorbic acid, RTG+AA = riboflavin table grade + ascorbic acid, FA = folic acid, VK1 = vitamin K1, 0080EE = DHA, 4535EE = DHA+EPA, 8000EE = EPA. Each compound was tested at three doses (0.2x, 1x and 5x) (Table 1). [Diagram 3] Butyrate production after 48 h fermentation from administration of the different test formulations, either as aqueous solutions (top) or dissolved in ethanol (bottom), is shown. Negative controls of various stock solutions were also included. RTG = riboflavin table grade, R5P = riboflavin-5-phosphate, AA = ascorbic acid, RTG+AA = riboflavin table grade + ascorbic acid, FA = folic acid, VK1 = vitamin K1, 0080EE = DHA, 4535EE = DHA+EPA, 8000EE = EPA. Each compound was tested at three doses (0.2x, 1x and 5x) (Table 1). [Figure 4] Total SCFA production after 48 h fermentation from administration of the different test formulations, either as aqueous solutions (top) or dissolved in ethanol (bottom), is shown. Negative controls of various stock solutions were also included. RTG = riboflavin table grade, R5P = riboflavin-5-phosphate, AA = ascorbic acid, RTG+AA = riboflavin table grade + ascorbic acid, FA = folic acid, VK1 = vitamin K1, 0080EE = DHA, 4535EE = DHA+EPA, 8000EE = EPA. Each compound was tested at three doses (0.2x, 1x and 5x) (Table 1). [Diagram 5]Schematic of Caco-2 and goblet cells co-cultured in a transwell plate. Measurements of transepithelial electrical resistance (TEER) provide information on the integrity of tight junctions formed between polarized cells. "Leaky gut" is characterized by an increase in intestinal permeability and can be achieved in vitro by stressing cells. Addition of apical or basolateral stressors results in a significant time-dependent decrease in TEER and an increase in Lucifer Yellow paracellular flux rates compared to control cells. [Figure 6] Figure 1 shows the effect of vitamins and omega-3 fatty acids on gut health in a healthy intestinal cell model. Values ​​are expressed as the difference between the transepithelial electrical resistance (TEER) of cells treated with 48 h fermentation samples and the TEER of cells treated with 0 h fermentation samples. Each compound was tested at three doses (0.2x, 1x and 5x) (Table 1). [Figure 7] Figure 1 shows the effect of vitamins and omega-3 fatty acids on gut integrity in the basolateral induction of leaky gut cell model. Values ​​are expressed as the difference between the transepithelial electrical resistance (TEER) / Lucifer Yellow of cells treated with 48 h fermentation samples and the TEER / Lucifer Yellow of cells treated with 0 h fermentation samples. Each compound was tested at three doses (0.2x, 1x and 5x) (Table 1). [Figure 8] Figure 1 shows the effect of vitamins and omega-3 fatty acids on gut integrity in the apical induction of leaky gut cell model. Values ​​are expressed as the difference between the transepithelial electrical resistance (TEER) / Lucifer Yellow of cells treated with 48h fermentation samples and the TEER / Lucifer Yellow of cells treated with 0h fermentation samples. Each compound was tested at three doses (0.2x, 1x and 5x) (Table 1). [Figure 9] The Shannon diversity index after 24 h fermentation from administration of the different test formulations, either as aqueous solutions (top) or dissolved in ethanol (bottom), is shown. Negative controls of the various stock solutions were also included. Each compound was tested at three doses (0.2x, 1x and 5x) (Table 1). [Figure 10]The relative abundance (%) of Bifidobacterium after 24 h fermentation from administration of the different test formulations, either as aqueous solutions (top) or dissolved in ethanol (bottom), is shown. Negative controls of various stock solutions were also included. Each compound was tested at three doses (0.2x, 1x and 5x) (Table 1). [Figure 11] The relative abundance (%) of genes involved in acetyl-CoA fermentation to the butyrate pathway 24 h after administration of the different test formulations, either as aqueous solutions (top) or dissolved in ethanol (bottom), is shown. Negative controls of the various stock solutions were also included. Each compound was tested at three doses (0.2x, 1x and 5x) (Table 1). [Figure 12] FIG. 1 shows the effect of vitamins and omega-3 fatty acids on GROa-CXCL1 production by HT29 cells. Data are expressed as pg / ml. [Figure 13] FIG. 1 shows the effect of vitamins and omega-3 fatty acids on IL-8 production by HT29 cells. Data are expressed as pg / ml. [Figure 14] FIG. 1 shows the effect of vitamins and omega-3 fatty acids on MIP3A-CCL20 production by HT29 cells. Data are expressed as pg / ml. [Figure 15] Gas pressure over different time periods (0-6 h, 6-24 h, and 24-48 h) from administration of different test formulations, either as aqueous solutions (top) or dissolved in ethanol (bottom), is shown. Negative controls of various stock solutions were also included. Each compound was tested at three doses (0.2x, 1x, and 5x) (Table 1). [Figure 16] Total SCFA production over different time periods (0-6 h, 6-24 h, and 24-48 h) from administration of different test formulations for either Donor C (top) or Donor D (bottom) is shown. A negative control (blank) was also included. Each compound was tested at three doses (0.2x, 1x, and 5x) (Table 2). [Figure 17]FIG. 1 shows the abundance of Bifidobacteriaceae (log(cells / ml)) in control incubations and incubations with test formulations after 24 h incubation with fecal inocula from donor C and donor D. [Figure 18] The Bray-Curtis distance (β diversity) between water as a control and vitamin supplementation is shown. The dotted line indicates the Bray-Curtis distance between the two controls at 24 h, which serves as a reference point. [Figure 19] Figure 1 shows the Shannon diversity index (α diversity) between water as a control and vitamin supplementation. The dotted line indicates the α diversity of the water control at 24 h. Diversity index values ​​higher than the baseline control are expressed as increased diversity. [Figure 20] Figure 1 shows a Principal Coordinate Analysis (PCoA) based on Weighted UniFrac distances of the cecal microbiota profiles of broilers from groups A and BA (green shading), B (gold shading) and C (pink shading). Samples are depicted as points filled with different colour shades, from light (early samples) to dark (late samples), according to the colour legend provided. The first and second coordinate axes are recorded for each plot; the % variability of the data set explained by each axis is recorded. [Figure 21] A-F Principal coordinate analysis (PCoA) based on weighted UniFrac distances of cecal microbiota profiles of broilers taken at time point T1. Samples are depicted as points filled with different colors, similar to Fig. 1, i.e., green for group A, gold for group B, and pink for group C. The first and second coordinate axes are recorded for each plot; the % variation of the data set explained by each axis is recorded. [Figure 22]Alpha diversity, measured as Faith's PD index (A-C) and number of observed OTUs (D-F), in the cecal microbiota of broilers sampled at time points T1 (A, D), T2 (B, E), and T3 (C, F). Box-and-whisker distributions of the diversity index in all samples are depicted. Green, gold, and pink shading are used to distinguish chickens from groups A, B, and C, respectively. *, statistically significant difference (P<0.0001). [Figure 23] Figure 1 shows the phylum level microbiota profile of the broiler cecal ecosystem. For each available time point (T1, T2, T3), the average phylogenetic profile of the cecal microbiota of broilers in groups A, B, and C is provided as a phylum level pie chart; bacterial taxa were filtered for graphical representation as >0.1% out of at least 1% of all available samples. [Figure 24] Family-level microbiota profile of broiler cecal ecosystem. For each available time point (T1, T2, T3), the average phylogenetic profile of the cecal microbiota of broilers from groups A, B, and C is provided as a family-level pie chart; bacterial taxa were filtered for graphical representation as >0.5% out of at least 1% of all available samples. [Diagram 25] Figure 1 shows the relative abundance distribution of bacterial families in the cecal microbiota of broilers. Box-and-whisker distributions of relative abundance (%) in all samples at the three time points (left to right) are drawn for families showing significant differences between the three groups (A, B, C) at at least one time point. Green, gold and pink shading are used to distinguish chickens in groups A, B and C, respectively. Bejamini-Hocherg correlation P-values ​​are recorded when statistical significance (P<0.05) is reached. [Figure 26]Figure 1 shows the relative abundance distribution of bacterial genera in the cecal microbiota of broilers. Box-and-whisker distributions of relative abundance (%) in all samples at the three time points (from left to right) are drawn for genera showing significant differences between the three groups (A, B, C) at at least one time point. Green, gold and pink shading are used to distinguish chickens in groups A, B and C, respectively. Bejamini-Hocherg correlation P-values ​​are recorded when statistical significance (P<0.05) is reached. [Figure 27] Figure 1 shows Principal Coordinate Analysis (PCoA) based on Weighted (A) and Unweighted (B) UniFrac distances of ileal microbiota profiles of broilers from groups A (green shading), B (gold shading) and C (pink shading). Samples are depicted as triangles filled with different color shades, from light (early samples) to dark (late samples), according to the color legend provided. The first and second coordinate axes are recorded for each plot, and the % variability of the data set explained by each axis is recorded. [Figure 28] Principal coordinate analysis (PCoA) based on weighted (A-C) and unweighted (D-E) UniFrac distances of broiler ileal microbiota profiles taken at time points T1 (A,D), T2 (B,E), and T3 (C,F). Samples are depicted as triangles filled with different colors, similar to FIG. 27, i.e., group A green, group B gold, and group C pink shades. The first and second coordinate axes are recorded for each plot; the % variation of the data set explained by each axis is recorded. [Figure 29] Alpha diversity, measured as Faith's PD index (A-C) and number of observed OTUs (D-F), in the ileal microbiota of broilers sampled at time points T1 (A,D), T2 (B,E), and T3 (C,F). Box-and-whisker distributions of the diversity index in all samples are depicted. Green, gold, and pink shading are used to identify chickens of groups A, B, and C, respectively. Statistically significant differences (Kruskal Wallis test) are displayed with the corresponding P-values. [Diagram 30]Figure 1 shows the phylum level microbiota profile of the broiler ileal ecosystem. For each available time point (T1, T2, T3), the average phylogenetic profile of the ileal microbiota of broilers from groups A, B, and C is provided as a pie chart of phylum level bacteria; taxa were filtered for graphical representation as >0.1% in at least one of all available samples. [Diagram 31] Family-level microbiota profile of the broiler ileal ecosystem. For each available time point (T1, T2, T3), the average phylogenetic profile of the ileal microbiota of broilers from groups A, B, and C is provided as a pie chart of family-level bacteria; taxa were filtered for graphical representation as >0.5% in at least one of all available samples. [Diagram 32] Figure 1 shows the relative abundance distribution of the family Peptostreptococcaceae in the ileal microbiota of broilers. Box-and-whisker distributions of the relative abundance in all samples are depicted at the three time points (from left to right). Green, gold and pink shading are used to distinguish chickens of groups A, B and C, respectively. Bejamini-Hocherg correlation P-values ​​are recorded when statistical significance (P<0.05) was reached. [Diagram 33] Figure 1 shows Principal Coordinate Analysis (PCoA) based on Weighted (A) and Unweighted (B) UniFrac distances of waste microbiota profiles of broilers from groups A (green shading), B (gold shading) and C (pink shading). Samples are depicted as boxes filled with different color shades, from light (early samples) to dark (late samples). The first and second coordinate axes are recorded for each plot; the % variation of the data set explained by each axis is recorded. [Diagram 34] Figure 1 shows the phylum level microbiota profile of the waste ecosystem. For each available time point (T1, T2, T3), the average phylogenetic profile of the waste microbiota of broilers from groups A, B and C is provided as a pie chart of phylum level bacteria; taxa were filtered for graphical representation as >0.1% in at least one of all available samples. [Diagram 35] Figure 2 shows the family-level microbiota profile of the waste ecosystem. For each available time point (T1, T2, T3), the average phylogenetic profile of the waste microbiota of broilers from groups A, B, and C is provided as a family-level bacterial pie chart; bacterial taxa were filtered for graphical representation as >0.5% in at least one of all available samples. [Diagram 36] FIG. 1 shows the H-NMR spectrum of the water extract of DSM cecal sample C281 pH 7.13. The vertical axis in A has been expanded to better detect small signals in the downfield region (aromatic region). [Figure 37] A) PCA score plot of binned and normalized 1H-NMR spectra of all samples and B) PCA scores without outlier samples (graphs performed using R software). [Figure 38] A) PLS-DA score plot of binned and normalized 1H-NMR spectra of 61 samples, where it can be seen that the three groups are linearly separable along the time points. B) PLS-DA score plot considering the control [A] and treated groups [B+C] (graphs are generated using Python software). [Figure 39] Box plots of the integral of the uracil signal at 5.793 ppm (d); samples are compared according to the following time points: A) T0 corresponding to 14 days of age; B) T1 corresponding to 28 days of age and C) T2 corresponding to 42 days of age. [Diagram 40] Box plot of the integral of the glutamate signal at 2.0447 ppm (m); samples are compared according to time points: A) TO corresponding to 14 days of age; B) T1 corresponding to 28 days of age and C) T2 corresponding to 42 days of age. Uracil appears to increase its concentration up to 42 days for all treatments, especially sample C. [Diagram 41]Box plots of the integral of the alanine signal at 1.470 ppm (d); samples are compared according to the following time points: A) T0 corresponding to 14 days; B) T1 corresponding to 28 days of age and C) T2 corresponding to 42 days of age. [Diagram 42] Box plots of the integral of the creatine signal at 3.920 ppm(s); samples are compared according to the following time points: A) T0 corresponding to 14 days; B) T1 corresponding to 28 days of age and C) T2 corresponding to 42 days of age. [Diagram 43] Box plot of the integral of the butyrate signal at 0.883 ppm (t); samples are compared according to the following time points: A) T0 corresponding to 14 days; B) T1 corresponding to 28 days of age and C) T2 corresponding to 42 days of age. [Diagram 44] Box plots of the integral of the propionate signal at 1.0446 ppm (m); samples are compared according to the following time points: A) T0 corresponding to 14 days; B) T1 corresponding to 28 days of age and C) T2 corresponding to 42 days of age. [Diagram 45] Box plots of the integral of the 1.0909 ppm(s) acetate signal; samples are compared according to the following time points: A) T0 corresponding to 14 days; B) T1 corresponding to 28 days of age and C) T2 corresponding to 42 days of age. [Diagram 46] LogNormal fits of mean metabolite signal integral versus time are shown. Comparisons are made between treatment groups using standard deviations as error bars. Translucent areas define the 95% confidence intervals of the fits. [Figure 47] PCA score plot of all normalized functional characteristics (6361 data) for all samples (63 samples). [Figure 48] PCA score plots of normalized functional characteristics of treatment groups (groups B and C) compared to the control group (group A) for each time point. [Figure 49] The primary KEGG metabolic pathways in the cecum of broilers of three different groups A, B and C are shown for each available time point (T1, T2, T3). [Figure 50]The relative abundance (KEGG database) of environmental information processing and metabolic pathways in the broiler cecum of three different groups A, B and C is shown for each available time point (T1, T2, T3) along with the statistically significant relative abundance. Green and red boxes refer to groups B and C, respectively, and blue boxes to group A. P values ​​are indicated at the top right of each graph. [Figure 51] For each available time point (T1, T2, T3), secondary KEGG metabolic pathways in the broiler cecum of three different groups A, B and C are shown, where the relative abundance is ≧0.5%. [Figure 52] Four different pathways for butyrate synthesis and the corresponding genes (protein names) are displayed. The major substrates are indicated. The marginal genes are highlighted in red. L2Hgdh, 2-hydroxyglutarate dehydrogenase; Gct, glutaconate CoA transferase (α, β subunits); HgCoAd, 2-hydroxy-glutaryl-CoA dehydrogenase (α, β, γ subunits); Gcd, glutaconyl-CoA decarboxylase (α, β subunits); Thl, thiolase; hbd, β-hydroxybutyryl-CoA dehydrogenase; Cro, crotonase; Bcd, butyryl-CoA dehydrogenase (electron transfer protein α, β subunits); KamA, lysine-2,3-aminomutase; KamD, E, _β-lysine-5,6-aminomutase (α, β subunits); Kdd, 3,5- Diaminohexanoate dehydrogenase; Kce, 3-keto-5-aminohexanoate cleavage enzyme; Kal, 3-aminobutyryl-CoA ammonia lyase; AbfH, 4-hydroxybutyrate dehydrogenase; AbfD, 4-hydroxybutyryl-CoA dehydratase; Isom, vinylacetyl-CoA 3,2-isomerase (same protein as AbfD); 4Hbt, butyryl-CoA:4-hydroxybutyrate CoA transferase; But, butyryl-CoA:acetate CoA transferase; Ato, butyryl-CoA:acetoacetate CoA transferase (α,β subunits); Ptb, phosphate butyryltransferase; Buk, butyrate kinase. Cosubstrates for individual butyryl-CoA transferases are shown. (Vital et al. 2014).

[0137] [Definition] As used throughout this specification and claims, the term "beneficial bacteria" refers to bacteria from the following genera: Acidaminococcus, Akkermansia, Bacteroides, Bifidobacterium, Blautia, Clostridium, Collinsella, Dorea, Escherichia, and the like. The beneficial bacteria include at least one bacterial taxon selected from the genera: Eubacterium, Faecalibacterium, Lachnospira, Lactobacillus, Parabacteroides, Raoultella, Roseburia, Ruminococcus, Prevotella, and Christensenella. The following families may also include beneficial bacterial taxa: Erysipelotrichaceae, Coriobacteriaceae, and Barnesiellaceae.

[0138] As used throughout this specification and claims, the term "niacin" is not the only active agent. If present, it is in combination with at least one other active agent, and preferably niacin is not present.

[0139] "Supraphysiological" dose means the same as "supernutritional", i.e., a dosage higher than the amount of active agent recommended for an animal, such that at least an effective amount of the active agent is present in the intestinal tract of the animal, preferably in the lower gastrointestinal tract and / or colon, and can directly nourish the bacterial population.

[0140] By "only active agent" it is meant that the only active agent present in the intestinal tract, preferably the colon, is the one indicated.

[0141] As used herein, the term "intestinal (gut) tract" refers to that portion of the gastrointestinal tract consisting of the small intestine and the large intestine.

[0142] The term "vitamin A" as used herein refers to a compound selected from retinol, retinal, retinoic acid, and provitamin A carotenoids (especially β-carotene). In a preferred embodiment, vitamin A is retinol. Any concentration or amount of vitamin A used herein refers to retinol equivalents.

[0143] The term "vitamin E" refers to compounds with alpha-tocopherol activity. In a preferred embodiment, vitamin E is alpha-tocopherol and its esters, particularly alpha-tocopherol acetate. Any concentration or amount of vitamin E recited herein refers to alpha-tocopherol equivalent.

[0144] The term "vitamin D" as used herein means vitamin D3. 25-hydroxyvitamin D3 can be used instead of or in addition to vitamin D3, preferably in species other than humans. The relative potency of 25-hydroxyvitamin D3 to vitamin D3 is about 40:1, so dosing of 25-hydroxyvitamin D3 must be adjusted accordingly.

[0145] The term "riboflavin" includes riboflavin and its esters, particularly riboflavin-5'-phosphate.

[0146] The term "folate" refers to folic acid and its pharma- ceutically acceptable salts.

[0147] The term "vitamin C" refers to ascorbic acid, pharma- ceutically acceptable salts thereof (eg, sodium ascorbate and calcium ascorbate) and pharma- ceutically acceptable salts thereof (particularly ascorbyl palmitate).

[0148] The term "vitamin K" includes vitamin K1 and vitamin K2.

[0149] The term "β-carotene" refers to β-carotene or provitamin A.

[0150] The term "vitamin B1" refers to thiamine, aneurysm, and its pharmaceutical salts (e.g., thiamine mononitrate).

[0151] The term "niacin" means nicotinic acid, vitamin B3, pyridine-3-carboxylic acid, and includes nicotinamide or niacinamide, which may be present in combination with at least one other active agent, but is specifically excluded from the present invention when it is the only active agent.

[0152] The term "vitamin B5" refers to pantothenic acid and its pharma- ceutically acceptable salts (eg, calcium pantothenate), including its derivatives pantothenol or panthenol.

[0153] The term "vitamin B6" means pyridoxal phosphate and includes pyridoxal-5'-phosphate.

[0154] The term "biotin" refers to vitamin B7, vitamin H, coenzyme R, and biopeiderm.

[0155] The term "vitamin B12" means cobalamin and includes all forms of vitamin B12 (eg, cyanocobalamin and methylcobalamin).

[0156] An "omega (ω) 3 fatty acid" is a polyunsaturated fatty acid with a double bond at the n-3 position. In a preferred embodiment, the omega-3 fatty acid is eicosapentaenoic acid (EPA) or docosahexaenoic acid (DHA) or a combination of both.

[0157] [Detailed Description] In one aspect, the present invention provides an active agent selected from the group consisting of: riboflavin, vitamin A, beta-carotene, vitamin C, vitamin D, vitamin E, vitamin K, folic acid, beta-carotene, vitamin B1 niacin (if present), vitamin B5, vitamin B6, biotin, vitamin B12, omega-3 fatty acids, and combinations thereof, for use in improving the intestinal health of an animal, wherein the improvement is due to: (i) increasing the concentration of at least one short chain fatty acid or a salt thereof in the intestinal tract; (ii) increasing the diversity of the gut microbiota; (iii) increasing the abundance of beneficial bacterial microbiota in the intestinal tract; (iv) promotion or increase of the butyric acid synthesis pathway in the intestinal tract; (v) Improved intestinal barrier function, (vi) a decrease in the redox potential of the intestinal tract; (vii) reducing the amount of gas produced in the intestinal tract; and (vii) Stimulation of intestinal immune responses Containing or consisting of; wherein said use comprises: a) a formulation containing an effective amount of an active agent, the active agent being delivered to the large intestine; or b) a supraphysiological dose of the active agent, such that at least an effective amount of the active agent is present in the intestinal tract of the animal; to the animal, However, when niacin is the active substance, it is not the only active substance; when riboflavin is present, it is an active substance that has an improving effect other than increasing the amount of Faecalibacterium prausnitzii.

[0158] Typically, the use involves administering to an animal, which may be a mammal, a delayed release formulation comprising an effective dose of an active agent, where release of the active agent is delayed until the large intestine or until the colon, or by a parenteral route where the agent is delivered directly to the colon. Delayed release formulations and their preparation are described in more detail below.

[0159] If the animal is other than human, such as monogastric livestock or poultry, administration can be by either a delayed release formulation, in which release of the active agent is delayed until the large intestine, or by the use of supraphysiological doses in the feed, such that an effective amount of the active agent is present in the intestinal tract. If the animal is human, administration can be by a number of methods, as detailed herein, including administration of a delayed release formulation, administration via a parenteral route, and / or the use of supraphysiological doses.

[0160] In some embodiments, niacin is not considered one of the actives of the present invention.

[0161] [Increase in short-chain fatty acids / promotion of butyric acid synthesis] As used herein, the term "short chain fatty acid (SCFA)" refers to a fatty acid having 2 to 6 carbon atoms. SCFAs include formic acid, acetic acid, propionic acid, butyric acid, 2-methylpropionic acid, 3-methylbutanoic acid, and hexanoic acid. The most important SCFAs are acetic acid, propionic acid, and butyric acid.

[0162] The "increase" in the concentration of one or more SCFAs in the colon of an animal is compared to the SCFA concentration of an animal to which the active agent is not administered. The animal to which the active agent is not administered is referred to herein as a "control animal". Preferably, the "increase" in the concentration of one or more SCFAs in the colon of an animal is compared to the SCFA concentration of the same animal to which the active agent is not administered. That is, the control animal in this context is typically the same animal before the start of administration of the active agent. Preferably, the control animal has not received the active agent described herein in the form of a nutritional supplement for at least 28 days. In some embodiments, the animal is a mammal; in some embodiments, the animal is a monogastric animal, such as a pig. In some embodiments, the animal is a poultry.

[0163] In one embodiment, the concentration of acetic acid increases upon administration of the active agent. In another embodiment, the concentration of propionic acid increases upon administration of the active agent. In another embodiment, the concentration of butyric acid increases upon administration of the active agent. In yet another embodiment, the concentrations of acetic acid, propionic acid, and butyric acid increase upon administration of the active agent.

[0164] In one embodiment, the concentration of one or more SCFAs is increased upon a single administration of the active substance. In another embodiment, the concentration of one or more SCFAs is increased upon two administrations of the active substance, which are applied on two consecutive days. In another embodiment, the concentration of one or more SCFAs is increased upon seven administrations of the active substance, which are applied on seven consecutive days. In another embodiment, the concentration of one or more SCFAs is increased upon 14 administrations of the active substance, which are applied on 14 consecutive days. Preferably, the concentration of one or more SCFAs (e.g., acetate, propionate, and / or butyrate) is increased upon 21 administrations of the active substance, which are applied on 21 consecutive days. Most preferably, the concentration of one or more SCFAs (e.g., acetate, propionate, and / or butyrate) is increased upon 28 administrations of the active substance, which are applied once a day for 28 consecutive days.

[0165] The concentration of SCFAs in the colon may be increased by at least 5%, preferably at least 10%, or at least 15%, or at least 20% compared to the concentration of SCFAs in the colon of a control to which no active agent is administered.

[0166] The concentration of SCFAs in the colon can be determined by obtaining a fecal sample from a mammal administered an active agent and measuring the concentration of one or more SCFAs.

[0167] Methods for measuring the concentration of commonly known SCFAs, for example by gas chromatography, are well known to those skilled in the art. For example, De Weird et al. (2010) (DOI:10.1111 / j.1574-6941.2010.2010.00974.x) describes a suitable method.

[0168] Alternatively, the increase in one or more SCFAs upon administration of an active agent can be determined using reactant and fecal suspensions as in vitro models, as described in the Examples of this application.

[0169] It was further found that administration of the active substance leads to an increase in the relative abundance of genes involved in acetyl-CoA fermentation to the butyrate pathway.The present invention therefore relates to the use of the active substance for the purpose of activating or increasing the butyrate synthesis pathway.The relative abundance of genes involved in acetyl-CoA fermentation to the butyrate pathway can be determined as described in the Examples.

[0170] The active agents described herein are micronutrients effective in increasing the concentration of at least one SCFA in the colon of a mammal and / or improving the intestinal barrier function. It has been found that vitamin A, vitamin C, riboflavin, vitamin D, vitamin E, vitamin K, folic acid and omega-3 fatty acids can increase the concentration of at least one SCFA in the colon of a mammal when administered at certain concentrations.

[0171] Preferred active agents for increasing the butyrate pathway are vitamin A, vitamin C, riboflavin, vitamin E, folic acid, beta-carotene, omega-3 fatty acids, and combinations thereof. Conditions that may be treated, ameliorated, or prevented by this action include metabolic disorders, type 2 diabetes, obesity, chronic inflammation, and atherogenesis.

[0172] Accordingly, another embodiment of the present invention is a method of increasing at least one short chain fatty acid and / or increasing butyrate synthesis pathway activity in an animal, including a human, experiencing a condition selected from the group consisting of metabolic disorders, type 2 diabetes, obesity, chronic inflammation and atherogenesis, comprising administering at least one active agent selected from the group consisting of vitamin A, vitamin C, riboflavin, vitamin E, folic acid, beta-carotene, omega-3 fatty acids and combinations thereof.

[0173] Another embodiment of the present invention is an active substance for use in improving intestinal health in an animal, including a human, wherein the improvement is an increase in the concentration of at least one short chain fatty acid or salt thereof in the intestinal tract; or a promotion or increase in the butyric acid synthesis pathway in the intestinal tract, wherein the short chain fatty acid is selected from the following: acetic acid, propionic acid and butyric acid or salts thereof.

[0174] Another embodiment of the present invention is an active agent selected from the group consisting of: vitamin A, beta-carotene, vitamin C, riboflavin, vitamin D, vitamin E, vitamin K, folic acid, omega-3 fatty acids, and combinations thereof, for use in increasing the concentration of at least one short chain fatty acid or salt thereof in the intestinal tract.

[0175] Another embodiment of the present invention is an active substance for use in improving the intestinal health of an animal, including a human, wherein the improvement is an increase in the concentration of at least one short chain fatty acid or salt thereof in the intestinal tract; or a promotion or increase in the butyric acid synthesis pathway in the intestinal tract, wherein the animal, including a human, is experiencing a condition selected from the group consisting of: metabolic disorders, type 2 diabetes, obesity, chronic inflammation and atherogenesis.

[0176] [Enhancing microbial diversity and beneficial bacteria] Furthermore, it has been found that administration of the active substance increases gut microbiota diversity.The present invention therefore relates to the use of the active substance for increasing gut microbiota diversity, preferably colonic microbiota diversity.Microbiota diversity can be determined as described in the examples for humans, pigs and poultry.

[0177] In particular, the inventors have found that supplementation with the active substance increases the relative abundance of beneficial bacteria, preferably Lactobacillus, Bifidobacterium, Akkermansia and Faecalibacterium, in humans.The present invention therefore relates to the use of the active substance in the intestine, preferably in the colon, for increasing the relative abundance of beneficial bacteria, preferably beneficial bacteria comprising at least one of Lactobacillus, Bifidobacterium, Akkermansia and / or Faecalibacterium.

[0178] For example, in poultry, Bacteroides and Faecalibacterium are increased. Thus, another embodiment is a method of increasing beneficial bacteria in an animal, which comprises administering a supraphysiological dose of an active agent to the animal.

[0179] Increasing the abundance of beneficial bacteria can treat, prevent, or alleviate the symptoms of irritable bowel syndrome, metabolic disorders, obesity, and inflammatory disorders.

[0180] Preferred substances are vitamin B1, riboflavin, vitamin B5, vitamin B12, β-carotene, biotin, vitamin C, vitamin E, vitamin D, folic acid, vitamin K, omega-3 fatty acids, and combinations thereof. However, when riboflavin is the active substance, it is present for a purpose other than increasing the abundance of F. prausnitzii.

[0181] Thus, another embodiment of the present invention is a method of treating, preventing, or alleviating symptoms of irritable bowel syndrome, metabolic disorders, obesity, or inflammatory disorders in an animal in need thereof, including a human, comprising administering to the colon of the animal an amount of an active agent that increases beneficial bacteria; the active agent is selected from the following: vitamin B1, riboflavin, vitamin B5, vitamin B12, β-carotene, biotin, vitamin C, vitamin E, vitamin D, folic acid, vitamin K, omega-3 fatty acids, and combinations thereof; with the proviso that when riboflavin is the active agent, it is present for a purpose other than increasing the abundance of F. prausnitzii.

[0182] Another embodiment of the invention is an active substance for use in improving intestinal health, wherein the improvement comprises increasing the microbiota diversity in the intestinal tract and / or increasing the abundance of beneficial bacteria in the colon. The beneficial bacteria that are increased are: Acidaminococcus, Akkermansia sp., Bacteroides ovatus, Bifidobacterium spp., Blautia producta, Clostridium cocleatum, Collinsella aerofaciens, Dorea longicatena, Escherichia coli, Eubacterium spp., Faecalibacterium prausnitzii, Lachnospira pectinoschiza, and others. pectinoshiza, Lactobacillus spp., Parabacteroides distasonis, Raoultella spp., Roseburia spp., Ruminococcus spp., Streptococcus spp., and combinations thereof, with the proviso that, when riboflavin is present, it has an improving effect other than increasing the amount of Faecalibacterium prausnitzii. Preferably, the bacteria to be increased are selected from the group consisting of Bifidobacterium, Akkermansia, Faecalibacterium, and Bacteriodes.

[0183] The active agent may be used in animals, including humans, experiencing a disease selected from the group consisting of irritable bowel syndrome, metabolic disease, obesity, and inflammatory disease. Preferably, the active agent is selected from the group consisting of vitamin B1, vitamin B2, vitamin B5, vitamin B6, vitamin B12, β-carotene, biotin, vitamin C, vitamin E, vitamin D, folic acid, vitamin K, omega-3 fatty acids, and combinations thereof.

[0184] Stimulating the immune response Furthermore, it was observed that the production of certain markers and cytokines by the colonic epithelial cell line HT29 was increased after treating the cells with effluent from a 24 h intestinal batch fermentation upon administration of the active substances. The biomarkers and cytokines include GROa-CXCL1, IL-8 and MIP3A-CCL20.

[0185] GROa-CXCL1 is a chemokine that plays an important role in inflammation and acts as a chemoattractant for neutrophils. It activates the host immune response by recruiting and activating neutrophils to tissue sites for microbial killing. CXCL1 is also known to activate proteases and the release of reactive oxygen species (ROS) for defense against microorganisms.

[0186] Interleukin-8 (IL-8) is a chemotactic cytokine that has been shown to have neutrophil chemotactic activity and helps enhance chemotaxis, inducing chemotaxis of target cells and causing neutrophils and other granulocytes to migrate towards the site of infection.

[0187] MIP3A-CCL20 is also a chemokine that selectively attracts effector and memory T lymphocytes, immune DCs, and naive B cells expressing the receptor CCR6. CCL20 is constitutively expressed in immunological barriers such as the gastrointestinal tract and skin.

[0188] All of these biomarkers reflect the activation of the immune response.

[0189] Stimulating the immune response can benefit conditions involving chronic as well as acute inflammation.

[0190] Preferred substances are riboflavin, vitamin E, vitamin A, vitamin K, and combinations thereof.

[0191] Thus, another embodiment of the present invention is the use of a substance for stimulating an intestinal immune response, ameliorating symptoms of immunocompromise, and generating a systemic anti-inflammatory response by using and / or administering to the colon of an animal in need thereof an effective dose of an active substance, wherein said substance is selected from the group consisting of riboflavin, vitamin E, vitamin A, vitamin K, and combinations thereof. A further embodiment is a method of treating, preventing, or alleviating symptoms of chronic or acute inflammation, comprising administering to the colon of an animal an effective immunostimulatory amount of a substance, wherein said substance is selected from the group consisting of riboflavin, vitamin E, vitamin A, vitamin K, and combinations thereof.

[0192] Another embodiment of the present invention is an active substance for use in improving gut health, the improvement being the stimulation of an intestinal immune response. Preferably, said use is aimed at increasing immune response molecules selected from the group consisting of: GROa-CXCL1, MIP3A-CCL20, Interleukin 8, and combinations thereof. Preferably, the active substance is selected from the group consisting of Vitamin B2, Vitamin E, Vitamin D, Vitamin A, and Vitamin K, and mixtures thereof.

[0193] Another embodiment is a method of improving the intestinal immune response in an animal, including a human, experiencing acute or chronic inflammation, comprising administering to the intestinal tract an active agent selected from the group consisting of riboflavin, vitamin E, vitamin A, vitamin K, and combinations thereof.

[0194] [Oxidation-reduction potential] Although antioxidant compounds such as vitamins C and E do not normally reach the colon, colon-targeted delivery of antioxidant vitamins and compounds to the large intestine allows for a reduction in the redox potential of the intestinal tract. Such a low redox potential can alleviate symptoms associated with disturbances in the composition and activity of normal gut microbiota, particularly in situations where the gut microbiota environment faces the presence of more oxygen and / or oxygen radicals that impede the growth of obligate anaerobic beneficial bacteria, promote the growth of aerobic pathogenic organisms, and / or cause inflammation and cellular damage to host cells.

[0195] Therefore, another embodiment of the present invention is the use of active substances, preferably vitamins C and / or E, to lower the redox potential in the intestinal tract, thereby reducing the growth of aerotolerant pathogenic organisms, reducing intestinal inflammation and the resulting cellular damage, while promoting the growth of beneficial strict anaerobic bacteria.

[0196] Furthermore, another embodiment of the present invention is the use of active substances, preferably vitamins C and / or E, to reduce plasma free thiols, a measure of systemic redox status in various immune disorders such as inflammatory bowel disease.

[0197] Thus, another embodiment of the present invention is a method of decreasing the redox potential of the intestinal tract, thereby: Promoting the growth of obligate anaerobic beneficial bacteria in the colon; Reduction of aerotolerant pathogenic organisms in the intestinal tract; Reducing inflammation in the intestinal tract; and Reducing cell damage in the intestinal tract caused by inflammation in the intestinal tract and providing a benefit selected from the group consisting of: The method includes administering to an animal, including a human, in need thereof a substance selected from the group consisting of riboflavin, vitamin C, vitamin E, and mixtures thereof.

[0198] [Gas generation] Colonic incubation was carried out in a closed incubation system, which allowed the evaluation of gas accumulation in the head space, which can be measured using a manometer. Gas production is a measure of microbial activity and therefore of the rate of fermentation of potentially prebiotic substrates. It can be used as an indicator of fullness and digestive well-being upon prebiotic consumption.

[0199] As demonstrated in the examples, active substances, preferably vitamin B2 and / or vitamin C, can be used to reduce the amount of gas produced in the intestinal tract.

[0200] Thus, another embodiment of the present invention is a method of treating, preventing, or reducing the amount of gas produced in the intestinal tract, comprising delivering to the large intestine an active agent selected from the group consisting of vitamin B2, vitamin C, and combinations thereof.

[0201] [Strengthening the intestinal barrier] Co-cultures of Caco-2 and HT29-MTX-E12 cells were used to evaluate the protective effect of the supernatants before and after fermentation. Transepithelial electrical resistance (TEER) readings correlate with the density of the confluent monolayer (Srinivasan et al. 2015) and can be used as an indicator of intestinal barrier strength. Decreased intestinal barrier strength is observed in pathologies associated with several pathologies including irritable bowel disease, Crohn's disease, ulcerative colitis, leaky gut and malnutrition.

[0202] Preferred agents for improving the intestinal barrier are riboflavin, vitamin C, vitamin E, omega-3 fatty acids, vitamin D, vitamin A, folic acid, vitamin K, and mixtures thereof. Accordingly, another embodiment of the present invention is a method of treating, preventing or alleviating symptoms of a condition selected from the group consisting of irritable bowel disease, Crohn's disease, ulcerative colitis, leaky gut and malnutrition, comprising administering to an animal, including a human in need thereof, an agent selected from the group consisting of riboflavin, vitamin C, vitamin E, omega-3 fatty acids, vitamin D, vitamin A, folic acid, vitamin K, and mixtures thereof.

[0203] Another embodiment of the present invention is the use of an active substance for improving intestinal health, said improvement including improving the barrier function of the intestine. This use may be carried out in animals, including humans, experiencing a condition selected from the group consisting of irritable bowel disease, Crohn's disease, ulcerative colitis, leaky gut, and malnutrition. Preferably, the active substance is selected from the group consisting of vitamin B2, vitamin C, vitamin E, omega-3 fatty acids, vitamin D, vitamin A, folic acid, vitamin K1, and mixtures thereof.

[0204] [Dosage and concentration] The dosages used herein are intended to be added to the active ingredient that mammals take for general nutrition.Instead, these dosages act on the intestinal microflora environment as a whole, at the level of genera, species and strains of intestinal microorganisms.It is not intended that the active substance is directly metabolized by mammals, but rather that it is utilized by colonic bacterial populations.Therefore, the amounts listed below are consumed by animals in addition to normal food, but they are not directly available to animals due to delayed release.

[0205] [Table 1]

[0206] Preferably, riboflavin is administered in an amount such that its local concentration in the colon is at least 0.01 mg / ml, preferably at least 0.1 mg / ml, more preferably at least 0.5 mg / ml, most preferably at least 1.25 mg / ml, for example about 1.4 mg / ml. Preferred local concentrations in the colon are from about 0.01 mg / ml to about 5 mg / ml, or from about 0.1 mg / ml to about 4 mg / ml, or from about 0.5 mg / ml to about 3 mg / ml, or from about 1.25 mg / ml to about 2 mg / ml.

[0207] In one embodiment suitable for poultry, the supraphysiological amount may be at least about 10x or 20x the recommended dose, for example if the recommended daily dose is 5 mg then the amount administered in the feed to have riboflavin present in the colon is 50 mg or 100 mg.

[0208] Preferably, vitamin E is administered in an amount such that its local concentration in the colon is at least 0.01 mg / ml, preferably at least 0.05 mg / ml, most preferably at least 0.1 mg / ml. The preferred local concentration in the colon is from about 0.01 mg / ml to about 5 mg / ml, more preferably from about 0.03 mg / ml to about 4 mg / ml, most preferably from about 0.05 mg / ml to about 3 mg / ml.

[0209] Preferably, ascorbic acid is administered in an amount such that its local concentration in the colon is at least 0.05 mg / ml, preferably at least 0.1 mg / ml, most preferably at least 0.5 mg / ml. Preferred local concentrations in the colon are from about 0.05 mg / ml to about 50 mg / ml, more preferably from about 0.1 mg / ml to about 20 mg / ml, most preferably from about 1 mg / ml to about 10 mg / ml.

[0210] Preferably, vitamin D is administered in an amount such that its local concentration in the colon is at least 0.01 μg / ml, preferably at least 0.03 μg / ml, most preferably at least 0.04 μg / ml or at least 0.1 μg / ml. Preferred local concentrations in the colon are from about 0.01 μg / ml to about 20 μg / ml, more preferably from about 0.03 μg / ml to about 10 μg / ml, most preferably from about 0.04 μg / ml to about 2 μg / ml, or from about 0.1 μg / ml to about 2 μg / ml.

[0211] Preferably, vitamin A is administered in an amount such that its local concentration in the colon is at least 0.05 μg / ml, preferably at least 0.1 μg / ml, most preferably at least 0.2 μg / ml. Preferred local concentrations in the colon are from about 0.05 μg / ml to about 20 μg / ml, more preferably from about 0.1 μg / ml to about 10 μg / ml, more preferably from about 0.2 μg / ml to about 5 μg / ml.

[0212] Preferably, vitamin K is administered in an amount such that its local concentration in the colon is at least 0.01 mg / ml, preferably at least 0.03 mg / ml, most preferably at least 0.04 mg / ml. The preferred local concentration in the colon is from about 0.01 mg / ml to about 5 mg / ml, more preferably from about 0.03 mg / ml to about 1 mg / ml, most preferably from about 0.05 mg / ml to about 0.5 mg / ml.

[0213] Preferably, folic acid is administered in an amount such that its local concentration in the colon is at least 0.1 μg / ml, preferably at least 0.5 μg / ml, most preferably at least 1 μg / ml.Preferred local concentrations in the colon are from about 0.1 μg / ml to about 20 μg / ml, more preferably from about 0.5 μg / ml to about 15 μg / ml, most preferably from about 1 μg / ml to about 10 μg / ml.

[0214] Preferably, β-carotene is administered in an amount such that its local concentration in the colon is at least 5 μg / ml, preferably at least 40 μg / ml, most preferably at least 100 μg / ml. The preferred local concentration in the colon is from about 5 μg / ml to about 20 mg / ml, more preferably from about 40 μg / ml to about 10 mg / ml, most preferably from about 100 μg / ml to about 5 mg / ml.

[0215] Preferably, vitamin B1 is administered in an amount such that its local concentration in the colon is at least 1 μg / ml, preferably at least 10 μg / ml, most preferably at least 20 μg / ml. The preferred local concentration in the colon is about 1 μg / ml to about 10 mg / ml, more preferably about 10 μg / ml to about 5 mg / ml, most preferably about 20 μg / ml to about 1 mg / ml.

[0216] Preferably, niacin is administered in an amount such that its local concentration in the colon is at least 0.5 μg / ml, preferably at least 1 μg / ml, most preferably at least 2 μg / ml or at least 3 μg / ml. The preferred local concentration in the colon is from about 0.5 μg / ml to about 1 mg / ml, more preferably from about 1 μg / ml to about 500 μg / ml, most preferably from about 2 μg / ml to about 100 μg / ml. When niacin is administered, it is not the only active substance.

[0217] Preferably, vitamin B5 is administered in an amount such that its local concentration in the colon is at least 0.05 mg / ml, preferably at least 0.1 mg / ml, most preferably at least 0.4 mg / ml.Preferred local concentrations in the colon are from about 0.05 mg / ml to about 100 mg / ml, more preferably from about 0.1 mg / ml to about 50 mg / ml, most preferably from about 0.4 mg / ml to about 20 mg / ml.

[0218] Preferably, vitamin B6 is administered in an amount such that its local concentration in the colon is at least 1 μg / ml, preferably at least 5 μg / ml, most preferably at least 10 μg / ml.Preferred local concentrations in the colon are from about 1 μg / ml to about 3 mg / ml, more preferably from about 5 μg / ml to about 1 mg / ml, most preferably from about 10 μg / ml to about 500 μg / ml.

[0219] Preferably, biotin is administered in an amount such that its local concentration in the colon is at least 1 μg / ml, preferably at least 2 μg / ml, most preferably at least 4 μg / ml.Preferred local concentrations in the colon are from about 1 μg / ml to about 1 mg / ml, more preferably from about 2 μg / ml to about 500 μg / ml, most preferably from about 4 μg / ml to about 200 μg / ml.

[0220] Preferably, vitamin B12 is administered in an amount such that its local concentration in the colon is at least 0.01 μg / ml, preferably at least 0.03 μg / ml, most preferably at least 0.05 μg / ml. The preferred local concentration in the colon is from about 0.01 μg / ml to about 10 μg / ml, more preferably from about 0.03 μg / ml to about 5 μg / ml, most preferably from about 0.05 μg / ml to about 2 μg / ml.

[0221] Preferably, the ω-3 fatty acid is administered in an amount such that its local concentration in the colon is at least 0.02 mg / ml, preferably at least 0.05 mg / ml, most preferably at least 0.1 mg / ml. The preferred local concentration in the colon is about 0.02 mg / ml to about 2 mg / ml, more preferably about 0.05 mg / ml to about 1.5 mg / ml, most preferably about 0.1 mg / ml to about 1 mg / ml. In another embodiment, the ω-3 fatty acid is administered in an amount such that its local concentration in the colon is at least 0.02 mg / ml, preferably at least 0.05 mg / ml, most preferably at least 0.1 mg / ml. The preferred local concentration in the colon is about 0.02 mg / ml to about 100 mg / ml, more preferably about 0.05 mg / ml to about 75 mg / ml, most preferably about 0.1 mg / ml to about 60 mg / ml. In another embodiment, the active agent is administered to the mammal in a daily amount of up to 2.9 mg riboflavin per kg body weight, up to 42.9 μg vitamin A per kg body weight, up to 3.6 μg vitamin D per kg body weight, up to 28.6 mg vitamin C per kg body weight, up to 14.3 mg vitamin E per kg body weight, up to 143 μg vitamin K per kg body weight, up to 14.3 μg folic acid per kg body weight, up to 0.4 mg beta-carotene per kg body weight, up to 1.4 mg vitamin B1, up to 21.4 mg niacin, up to 14.3 mg vitamin B5, up to 1.4 mg vitamin B6, up to 35.7 μg biotin, up to 43 μg vitamin B12, 71.4 mg omega-3 fatty acids or combinations thereof, wherein preferably EPA is at a maximum of 25.7 mg per kg body weight and DHA is at a maximum of 14.3 mg per kg body weight.

[0222] Another embodiment is a delayed release formulation for delivery of an active agent to the intestinal tract, comprising an active agent and an enteric layer or shell, wherein the active agent is comprised of up to 200 mg riboflavin, up to 3000 μg vitamin A, up to 2000 mg vitamin C, up to 250 μg vitamin D, up to 1000 mg vitamin E, up to 10 mg vitamin K, up to 1 mg folic acid, up to 25 mg beta-carotene, up to 100 mg vitamin B1, up to 1500 mg niacin, up to 1000 mg vitamin B5, up to 100 mg vitamin B6, up to 2500 μg biotin, up to 3000 μg vitamin B12, up to 5000 mg omega-3 fatty acids or combinations thereof, preferably up to 1800 mg EPA and up to 1000 mg DHA, and wherein release of the active agent is delayed to the intestinal tract.

[0223] Another embodiment is a delayed release formulation for delivery of an active agent to the intestinal tract, comprising an active agent and an enteric layer or shell, wherein the active agent is comprised of 1-85 mg riboflavin, 0.2-0.4 mg vitamin A, 400-600 mg vitamin C, 5-80 μg vitamin D, 80-120 mg vitamin E, 80-140 μg vitamin K, 0.3-0.5 mg folic acid, 80-120 mg omega-3 fatty acids or combinations thereof, and wherein release of the active agent is delayed to the intestinal tract.

[0224] Another embodiment is a delayed release formulation of the item, wherein the active substances consist of 70-80 mg riboflavin, 0.25-0.35 mg vitamin A, 450-550 mg vitamin C, 15-25 μg vitamin D, 90-110 mg vitamin E, 100-120 μg vitamin K, 0.35-0.45 mg folic acid, 90-110 mg omega-3 fatty acids, or combinations thereof.

[0225] In one embodiment, riboflavin is administered as the only active agent.

[0226] In another embodiment, vitamin A is administered as the only active agent.

[0227] In another embodiment, vitamin C is administered as the only active agent.

[0228] In another embodiment, vitamin D is administered as the only active agent.

[0229] In another embodiment, vitamin E is administered as the only active agent.

[0230] In another embodiment, vitamin K is administered as the only active agent.

[0231] In another embodiment, omega-3 fatty acids are administered as the only active agent.

[0232] In one embodiment, folic acid is administered as the only active agent.

[0233] In another embodiment, β-carotene is administered as the only active agent.

[0234] In another embodiment, vitamin B1 is administered as the only active substance.

[0235] In another embodiment, vitamin B5 is administered as the only active agent.

[0236] In another embodiment, vitamin B6 is administered as the only active agent.

[0237] In another embodiment, biotin is administered as the only active agent.

[0238] In another embodiment, vitamin B12 is administered as the only active substance.

[0239] In another embodiment, DHA is administered as the only active agent.

[0240] In another embodiment, EPA is administered as the only active agent.

[0241] In another embodiment, EPA and DHA are administered as the only active agents.

[0242] In another embodiment, the active agent is comprised of a combination of riboflavin, vitamin A, vitamin D, vitamin E, vitamin K, folic acid, EPA and DHA. In yet another embodiment, the active agent is comprised of a combination of riboflavin, vitamin A, vitamin C, vitamin D, vitamin E, vitamin K, folic acid, EPA and DHA. In yet another embodiment, the active agent is comprised of a combination of riboflavin, vitamin A, vitamin C, vitamin D, vitamin E, vitamin K, folic acid, beta-carotene, vitamin B1, niacin, vitamin B5, vitamin B6, biotin, vitamin B12, EPA and DHA.

[0243] In one embodiment, the delayed release formulation of the present invention is administered once daily, hi another embodiment, the formulation is administered twice daily or every other day.

[0244] In one embodiment, the active agent is administered to a mammal. The mammal may be a horse, cow, pig, dog, or cat. More preferably, the mammal is a human, e.g., an adult. In one embodiment, the adult has a body mass index (BMI) of at least 20, or at least 25, or at least 30. For example, the BMI may be in the range of 25-35, or 26-34, or 27-33, or 28-32.

[0245] [Condition to be treated or prevented] According to the first aspect of the present invention, the active substance is administered to increase the concentration of one or more SCFAs.Therefore, any condition associated with impaired SCFA levels in the colon can be treated or prevented.The level of SCFA is impaired if it is significantly lower than that of healthy individuals, for example at least 10%, or at least 20%, or at least 30% lower.Fecal concentration can be used as a measure of the concentration in the colon.

[0246] The conditions to be treated or prevented according to the present invention include inflammatory diseases, disorders characterized by inflammation, especially chronic inflammation, and atherogenesis.In addition, metabolic disorders such as type 2 diabetes or obesity can be treated or prevented or improved.In addition, immunosuppression or allergy can be treated.

[0247] According to another aspect of the invention, the active agent is administered to improve or enhance the barrier function of the intestinal tract. In one embodiment, the barrier function of the small intestine is improved or enhanced. In another embodiment, the barrier function of the large intestine is improved or enhanced. In another embodiment, the barrier function in the intestine is improved or enhanced. Thus, conditions or disorders associated with increased intestinal permeability can be treated, ameliorated or prevented. Conditions to be treated include, but are not limited to, disorders characterized by increased intestinal permeability, intestinal disease (e.g., environmental enteropathy, malnutrition-induced enteropathy), inflammatory bowel disease (e.g., Crohn's disease or ulcerative colitis), and irritable bowel syndrome.

[0248] The integrity of the intestinal barrier function can be determined by measuring the initial transepithelial electrical resistance (TEER) in a model system (see Examples). Alternatively, the integrity of the intestinal barrier function can be determined by measuring the transport of Lucifer Yellow from the apical to the basolateral compartment (see Examples). In humans, intestinal permeability can be measured indirectly by measuring the circulating concentrations of bacterial lipopolysaccharide or antibodies to the lipopolysaccharide core, or directly by a dual sugar absorption test (Dorshow et al, Scientific Reports volume 7, Article number: 10888 (2017)). For example, the lactulose:mannitose ratio in urine after dual sugar ingestion can be determined. Alternatively, blood alpha-1 antitrypsin:myeloperoxidase; and / or REG1B protein can be determined.

[0249] Another aspect of the invention aims to reduce gas production during colonic fermentation, which leads to improved gut health, less bloating and greater compatibility with fiber and prebiotic treatments.

[0250] [formulation] As used herein, "delayed release" refers to the release of an active agent at a time later than immediately after administration. Preferably, "delayed release" means that upon oral administration, the active agent is delivered to the large intestine at a later time compared to an immediate release formulation.

[0251] An "enteric layer" is a layer surrounding a core that contains an active substance, which layer confers resistance to gastric juices. An "enteric shell" is a shell or matrix that surrounds or encapsulates an active substance, which shell confers resistance to gastric juices. Alternatively, a matrix-based delivery system can be used. In a matrix-based system, there is no discrete layer of coating material, but the active substance is more or less homogeneously distributed within the matrix. In addition, there are colonic release systems that embed an active substance within a fiber matrix (enzyme-triggered type) and an enteric coating thereon.

[0252] In a preferred embodiment for humans, the formulations of the invention are solid dosage forms for oral administration. The formulations may be in the form of capsules, pellets, beads, spheres, minispheres, tablets, minitablets, or granules, optionally coated with a delayed release coating or shell that prevents release of the active agent before the small intestine, preferably before the colon.

[0253] Coating, shell or matrix materials for delayed release of active substances, especially targeted release in the ileum or large intestine, upon oral administration, are known in the art. These are further classified into coating materials that disintegrate above a certain pH, coating materials that disintegrate after a certain residence time in the gastrointestinal tract, and coating materials that disintegrate due to enzyme triggers specific to the microflora in a certain region of the intestinal tract. Generally, coating or shell materials from different categories are used in combination. These three different categories of coating or shell materials for targeting the large intestine are reviewed, for example, in Bansal et al. (Pollim.Med.2014,44,2,109-118). In one embodiment of the present invention, the delayed release coating comprises at least one component selected from coating materials that disintegrate in a pH-dependent manner, coating materials that disintegrate in a time-dependent manner, coating materials that disintegrate due to enzyme triggers in the intestinal environment (e.g., the intestinal environment of the ileum and large intestine), and combinations thereof.

[0254] Coating materials that disintegrate in a pH-dependent manner include polyvinyl acetate phthalate, cellulose acetate trimellitate, hydroxypropyl methylcellulose phthalate HP-50, HP-55 or HP-55S, cellulose acetate phthalate, shellac, hydroxypropyl methylcellulose acetate succinate (HPMCAS), poly(methacrylic acid, ethyl acrylate) 1:1 (Eudragit® L100-55, Eudragit® L30D-55), poly(methacrylic acid, methyl methacrylate) 1:1 (Eudragit® L-100, Eudragit® L12.5), poly(methacrylic acid, methyl methacrylate) 1:2 (Eudragit® S-100, Eudragit® S12.5, and Eudragit® FS30D).

[0255] Coating materials that disintegrate in a time-dependent manner include Eudragit® RL, Eudragit® RS, and ethylcellulose.

[0256] Coating materials that are degraded by enzyme triggers in the large intestine include chondroitin sulfate, pectin, guar gum, chitosan, inulin, lactulose, raffinose, stachyose, alginate, dextran, xanthan gum, locust bean gum, arabinogalactan, cyclodextrin, pullulan, carrageenan, scleroglucan, chitin, curdulan, levan, amylopectin, starch, amylose, resistant starch, and azo compounds that are degraded by azo bond-degrading bacteria.

[0257] In one embodiment, the formulation comprises an enteric capsule filled with a composition containing an active substance. The enteric capsule provides resistance to the acidic environment of the stomach. For example, a softgel formulation can deliver an active substance in solution and also provide the advantages of a solid dosage form. Softgel capsules are particularly suitable for hydrophobic active substances that are poorly soluble in water. Vitamin K and omega-3 fatty acids are preferably formulated into softgel capsules.

[0258] In another embodiment, the formulation is a tablet comprising (i) a core containing the active agent and (ii) a delayed release coating, such as an enteric coating. It may also be a hard gel capsule.

[0259] Release of the drug may be delayed until the small intestine. In another embodiment, release of the drug is delayed until the terminal small intestine. In yet another embodiment, release of the drug is delayed until the colon.

[0260] For example, one formulation may comprise an active agent and an enteric layer or shell, the active agent being selected from the group consisting of up to 200 mg riboflavin, up to 3000 μg vitamin A, up to 2000 mg vitamin C, up to 250 μg vitamin D, up to 1000 mg vitamin E, up to 10 mg vitamin K, up to 1 mg folic acid, up to 25 mg beta-carotene, up to 100 mg vitamin B1, up to 1500 mg niacin, up to 1000 mg vitamin B5, up to 100 mg vitamin B6, up to 2500 μg biotin, up to 3000 μg vitamin B12, up to 5000 mg omega-3 fatty acids or combinations thereof, where preferably, EPA is at most 1800 mg and DHA is at most 1000 mg, and release of the active agent is delayed until the intestinal tract.

[0261] Another aspect of the invention is the non-therapeutic use of an active agent selected from the group consisting of riboflavin, vitamin A, vitamin C, vitamin D, vitamin E, vitamin K, folic acid, beta-carotene, vitamin B1, niacin, vitamin B5, vitamin B6, biotin, vitamin B12, omega-3 fatty acids and combinations thereof for increasing the concentration of at least one short chain fatty acid or its salt in the intestinal tract of a mammal not suffering from an intestinal disorder or pathology, with the proviso that, where niacin is the active agent, it is not the only active agent. The mammal is preferably a healthy human.

[0262] Yet another aspect of the invention is the non-therapeutic use of an active agent selected from the group consisting of riboflavin, vitamin A, vitamin C, vitamin D, vitamin E, vitamin K, folic acid, beta-carotene, vitamin B1, niacin, vitamin B5, vitamin B6, biotin, vitamin B12, omega-3 fatty acids and combinations thereof for the maintenance or improvement of intestinal function in a healthy mammal, preferably a healthy human, provided that, where niacin is the active agent, it is not the only active agent.

[0263] One consideration of the present invention is that the active substances used are not intended to provide nutrition to mammalian recipients; rather, they are intended to directly benefit the microbial population in the large intestine, thereby indirectly benefiting the mammal.Therefore, the dosage is intended to be in addition to the normal daily consumption of vitamins, fatty acids, and other nutrients that are part of mammalian diet.Therefore, by avoiding absorption in the stomach and preferably in the small intestine, the microbial population can be provided with nutrition and maintain optimal ability to keep the intestinal barrier intact.In this way, the formulation of the present invention is useful for maintaining a healthy intestine.

[0264] The inventors also observed a beneficial effect on digestion in general. As shown in the examples, pigs supplemented with vitamin B2 showed higher DM digestibility of substrates (feed or pig stomach contents) incubated in pepsin-HCl compared to non-supplemented. This was unexpected and clearly a positive effect on gut functionality. Furthermore, the inventors observed a beneficial effect on colonic fermentation using cecal contents from pigs, namely reduced methane production. Without intending to be bound by theory, the inventors believe that this can be explained by vitamin B2 being able to suppress the activity of methanogen or archaeal populations, thus positively impacting the environment.

[0265] [Example] [Example 1] Materials and Methods [Donors and sample preparation] In the first set of in vitro experiments at the start of the enteric batch fermentation incubation, all test materials were added from stock solutions to a modified nutrient medium containing the following (g / l): 2.5 K2HPO4, 10.9 KH2PO4, 2 NaHCO3, 2 enzyme extract, 2 peptone, 1 mucin, 0.5 cysteine, 2 Tween 80, 2 glucose, 2 starch, 2 cellobiose, 0.1 NaCl, 0.01 MgSO4, 7 H2O, 0.01 CaCl2, 6 H2O, 0.05 hemin, 0.5 bile salts.

[0266] The following compounds were added from stock solutions and prepared in water: Riboflavin table grade (abbreviation RTG), Riboflavin-5-phosphate (R5P), Ascorbic acid granular powder (AA), Dry Vitamin E 50CWS-S (E50 CWS-S), Dry Vitamin D3 100 CWS (D3 100 CWS), Dry Vitamin A Acetate 325CWS / A, Riboflavin Table Grade + Ascorbic Acid Folic acid,

[0267] Other compounds were prepared in ethanol: Vitamin K1 (VK1), ·MEG-3 0080 EE oil (MEG-3 0080 EE), ·MEG-3 4535 EE oil (MEG-3 4535 EE), ·MEG-3 8000 EE oil (MEG-3 8000 EE).

[0268] Each compound was tested at three different concentrations; an overview is given in Table 1. Two blanks of each type of stock solution were added, since ethanol is known to have antibacterial activity. Freshly prepared fecal suspensions from selected donors were added to the reactors as a source of microbial population. Each reactor had a volume of 70 ml. All tests, except the blanks, were performed in a single replicate, resulting in 40 independent incubations, as detailed in Table 1. The incubation conditions for the prescreening and final experiments were 48 h at 37 °C under shaking (90 rpm) and anaerobic conditions. DNA was extracted from effluent samples collected from each fermentation flask before (0 h fermentation) and after (24 h fermentation) fermentation and used for microbiota composition analysis by shotgun sequencing. Outflow samples were collected from each fermentation flask before (0 h fermentation) and after (48 h fermentation), sterilized by filtration through a 0.22 μm filter, and then used for quantification of short-chain fatty acids (SCFAs), immunological biomarkers in the cell culture system, and in vitro analysis.

[0269] [Table 2]

[0270] [Table 3]

[0271] In a second set of in vitro experiments, the test materials were added to a nutrient medium containing basic nutrients present in the colon (e.g. host-derived glycans such as mucins) at the beginning of the short-term colonic incubation. Three doses per formulation were tested. The nutrient medium used for this particular project was again supplemented with NaCl, MgSO4, CaCl2, hemin and bile salts, as well as the carbon sources glucose, starch and cellobiose. A blank was added for each donor containing the same nutrient medium used for the formulation incubation, but without the addition of the formulation. In that way, the evaluation could consist of the background activity of the bacterial population resulting from the fermentation of the compounds in the nutrient medium. As a source of colonic microbiota, fecal inocula of two healthy adults were added. The incubation was carried out for 48 h at 37 °C, protected from light, under shaking (90 rpm) and anaerobic conditions. The following compounds were tested: Thiamine hydrochloride ·β-Carotene 10% CWS / S Niacinamide PC Calcium D-pantothenate Pyridoxine hydrochloride D-Biotin Vitamin B12 crystals

[0272] Each compound was tested at three different concentrations; as outlined in Table 2 below:

[0273] [Table 4]

[0274] [Short-chain fatty acids] Short chain fatty acids (SCFAs) are an assessment of microbial carbohydrate metabolism (acetate, propionate and butyrate) or protein metabolism (branched SCFAs) and can be compared to typical fermentation patterns for normal GI microbiota. Samples for SCFA analysis were analyzed after 0, 6, 24 and 48 h of incubation for the two sets of in vitro experiments described above.

[0275] After addition of 2-methylhexanoic acid as an internal standard, SCFAs were extracted from the samples with diethyl ether. Extracts were analyzed using a capillary fatty acid-free EC-1000 Econo-Cap column (size: 25 mm × 0.53 mm, film thickness 1.2 mM; Alltech, Laarne, Belgium), a GC-2014 gas chromatograph (Shimazu,'s-Hertogenbosch, the Netherlands) equipped with a flame ionization detector and a split injector. The injection volume was 1 mL, the temperature profile was set from 110 to 160 °C, with a temperature increase of 6 °C / min. The carrier gas was nitrogen, and the injector and detector temperatures were 100 and 220 °C, respectively. The formation of unbranched and branched SCFAs was calculated by summing the molar concentrations of acetate, propionate, butyrate, valerate and caproate and summing the molar concentrations of isobutyrate, isovalerate and isocaproate, respectively. Total SCFA production was defined as the sum of unbranched and branched SCFAs.

[0276] [Cell culture] Co-cultures of Caco-2 and HT29-MTX-E12 cells were used to evaluate the protective effect of pre-fermentation (0 h) and post-fermentation (48 h) supernatants from the two sets of in vitro experiments described above.

[0277] Both Caco-2 ECACC 86010202 and HT29-MTX-E12 ECACC 12040401 cells (both from the European Collection of Cell Cultures, Salisbury, UK) were cultured in DMEM medium supplemented with 4.5 g / L D-glucose, 4 mM L-glutamine, 1 mM sodium pyruvate, 1% MEM non-essential amino acids, 50 μg / mL gentamicin (Life Technologies Europe BV, Zug, Switzerland) and 10% heat-inactivated FBS (Sigma-Aldrich, Buchs, Switzerland) at 37 °C in an atmosphere of 5% CO2. Subconfluent cells were trypsinized with 0.25% trypsin / EDTA (Life Technologies Europe BV, Zug, Switzerland) and then passaged twice a week at a ratio of 1:5 to 1:10. Cells between passages 55 and 85 were used in this study.

[0278] Cells were seeded at a density of 20,000 cells / well in a 7:3 ratio (Caco-2:HT29-MTX-E12) in a Corning HTS Transwell-24 system PET membrane, 0.4 μM pore size, cell growth area 0.33 cm2 / well (Corning BV, Amsterdam, Netherlands) and cultured as described above. The medium was changed every 2 to 3 days.

[0279] [Measuring barrier integrity in a healthy intestinal model] After culturing the cells for 13 days, initial transepithelial electrical resistance (TEER) readings were obtained for both sets of in vitro experiments using an EVOM2 Voltohmmeter equipped with STX100 electrodes (EVOM, World Precision Instruments, Berlin). TEER values ​​correlate with the tightness of confluent monolayers (Srinivasan et al. 2015).

[0280] The background TEER (insert) was subtracted from the total TEER (cell monolayer plus insert) to obtain the monolayer resistance, which was then normalized to the surface area by multiplying with the area of ​​the insert (Srinivasan et al. 2015). The health of the monolayer was determined at a basal level by measuring the TEER. A TEER of 300 Ωcm2 was considered as a confluent monolayer (see Results section). The cells were then treated with sterile filtered fermentation supernatant or fermentation medium as a control (diluted 1:20 in growth medium). After 24 h of incubation, the resistance across each cell monolayer was measured again and the percentage change in TEER compared to the initial TEER was calculated for each insert, presenting a model of a healthy intestine. All TEER measurements were performed by placing the plate containing the insert on a 37 °C warming plate and were performed for the same duration.

[0281] [Measurement of barrier integrity in a leaky gut model] For both sets of in vitro experiments, the integrity of the cell monolayer after stressor testing was assessed by both TEER values ​​and by measuring the transport of Lucifer Yellow from the apical to the basolateral compartment (leaky gut model). Prewarmed HBSS transport buffer, consisting of Hanks' balanced salt solution, Ca2+ and Mg2+, pH 7.4, containing 5.5 mM D-(+)-glucose, sodium bicarbonate, and supplemented with 4 mM L-glutamine, 20 mM HEPES (Life Technologies Europe BV, Zug, Switzerland), was used for all permeability experiments. Using a reservoir tray, inserts were rinsed twice with HBSS, and then 100 μl of HBSS was added to the apical side of the inserts, transferred to a new well containing fresh HBSS, and equilibrated for 60 min at 37°C in an incubator before performing TEER measurements to obtain baseline resistance readings.

[0282] Lucifer Yellow CH dilithium salt (Sigma-Aldrich, Buchs, Switzerland) was dissolved in HBSS at a final concentration of 100 μg / ml. The basolateral stress factor EtOH (Merck, KgaA, Darmstadt, Germany) was diluted to 5% in HBSS. The apical stress factor rhamnolipid R90 (AGAE Technologies, Corvallis, USA) was dissolved in HBSS containing Lucifer Yellow at a final concentration of 350 μg / ml. All solutions were prepared fresh each time.

[0283] The HBSS transport buffer from the basolateral chamber was first aspirated, and then the HBSS from the apical chamber was replaced with Lucifer Yellow solution. For apical stressor testing, Lucifer Yellow solution containing 350 μg / ml rhamnolipid or Lucifer Yellow solution without stressor as a control was loaded into the apical compartment, followed by blank HBSS being added to the basolateral chamber. For basolateral stressor testing, Lucifer Yellow solution was added to the apical side of all wells, and either 5% EtOH in HBSS or blank HBSS for control wells was introduced into the basolateral compartment.

[0284] After 3 h of incubation at 37° C., the permeability of the cell monolayer was assessed by measuring the TEER values. TEER data were expressed as a percentage of the initial value.

[0285] After TEER measurements, 100 μl aliquot samples were removed from the basolateral side in duplicate to quantify the amount of Lucifer Yellow transported. Fluorescence levels (excitation at 420 nm and emission at 530 nm) were measured in a 96-well fluorescence plate reader (Spectramax M5 Series Multi-Mode Microplate Reader, Molecular Devices, Sunnyvale, CA, USA).

[0286] [Analysis of microbiota composition by shotgun sequencing] For the first set of in vitro experiments, total DNA was extracted from all samples collected throughout the study using the QIAamp DNA stool minikit (Qiagen, Crawley, United Kingdom) according to the manufacturer's instructions, except for the addition of a bead-beating step and an increase in the melting temperature to 95°C, as previously described (McCormack et al., 2018).

[0287] After DNA isolation, DNA was quantified using Qubit High Sensitivity DNA assay (Thermo Fisher). Total metagenomic libraries were then prepared using the Illumina Nextera XT kit (Illumina) according to the manufacturer's instructions with the following modifications: first, the tagmentation time was increased to 7 min; second, after index incorporation and Ampure purification of the products, samples were each individually sized by running on an Agilent High Sensitivity Chip (Agilent) and then quantified using Qubit High Sensitivity DNA assay (Thermo Fisher) according to the Teagasc Sequencing Platform SOP. Samples were then pooled equimolarly and sequenced (300 cycles) on an Illumina NextSeq500 equipped with the NextSeq500 / 550v2 High Output Reagent Kit. All sequencing was performed at the Teagasc sequencing facility following standard Illumina sequencing protocols.

[0288] Delivered raw FASTQ sequence files were quality checked as follows: removal of poor quality and duplicate reads and trimming were performed using a combination of SAM and Picard tools. Taxonomy was assigned to reads using Kraken software and functional analysis was performed using Superfocus.

[0289] For the second set of in vitro experiments, we followed Vandeputte et al. (2017) to map the community shifts induced by the different treatments in great detail by combining two techniques: 1) 16S targeted Illumina sequencing, a PCR-based method that amplifies microbial sequences to saturation to obtain the abundance percentages of different taxa at various phylogenetic levels (microbial phylum, family and OTU levels); and 2) precise quantification of total bacterial cells in the samples by flow cytometry. By combining the high-resolution phylogenetic information of 16S targeted Illumina with precise quantification of cell numbers by flow cytometry, we achieved a quantitative count of the different taxa populations inside the reactors.

[0290] [HT29 cell culture and quantification of immunological biomarkers] For both sets of in vitro experiments, HT29 cells were obtained from the American Type Culture Collection (Manassas, VA). Cells were cultured in DMEM supplemented with 10% fetal bovine serum (FBS), 50 units / ml penicillin, 50 μg / ml streptomycin, L-glutamine, and non-essential amino acids (Life Technologies Europe BV, Zug, Switzerland). Cells were seeded in 12-well plates at 8 × 105 cells per well and used after 2 days of preculture. They were starved in DMEM containing 0.25% FBS for 18 h before treatment. Cells were treated for 24 h with 100 ng / mL TNF-α or sterile-filtered fermentation supernatant diluted 1:20 in DMEM containing 0.25% FBS. All treatments were performed in triplicate. Consequently, cytokines, chemokines and interleukins were quantified in HT29 supernatants using Luminex Technology (LiquiChip Workstation IS 200, Qiagen, Hilden, Germany) with Bio-Plex Pro Human Cytokine Panel kit (Bio-Rad, Hercules, CA) or Luminex Screening Assay kit (R&D Systems, Inc., Minneapolis, MN) according to the manufacturer's instructions. Data were acquired using Luminex IS 2.3 software and evaluated using LiquiChip Analyser software (Qiagen, Hilden, Germany).

[0291] [Gas generation] In both sets of in vitro experiments, colonic incubations were performed in a closed incubation system as previously described. This allows the assessment of gas accumulation in the head space, which can be measured using a manometer. Gas production is a measure of microbial activity and therefore of the fermentation rate of potentially prebiotic substrates.

[0292] [result] [The first set of experiments depicted in Figures 1-15] The following observations were made regarding acetate production after 48 h fermentation following administration of vitamins and omega-3 fatty acids: Compared with the corresponding controls, - Vitamin E supplementation at 1x and 5x doses increases acetate production - 1x dose of vitamin A supplementation increases acetate production - Supplementation with 1x dose of vitamin B2+C increases acetate production - Folic acid supplementation at 0.2x, 1x, and 5x doses increases acetate production - Vitamin K1 supplementation at 0.2x, 1x and 5x doses increases acetate production - Supplementation with DHA at 0.2x, 1x, and 5x doses increases acetate production - Supplementation with 0.2x and 1x doses of DHA+EPA increases acetate production - Supplementation with EPA at 0.2x and 1x doses increases acetate production.

[0293] The following observations were made regarding propionate production after 48 h fermentation following administration of vitamins and omega-3 fatty acids: Compared with the corresponding controls, - Supplementation with 1x dose of vitamin E increases propionate production - Vitamin D supplementation at 1x and 5x doses increases propionate production - Vitamin A supplementation at 0.2x, 1x and 5x doses increases propionate production - Supplementation with 0.2x the dose of vitamin B2+C increases propionate production - Folic acid supplementation at 0.2x, 1x, and 5x doses increases propionate production - Supplementation with vitamin K1 at 0.2x, 1x and 5x doses increases propionate production - Supplementation with DHA at 0.2x, 1x, and 5x doses increases propionate production - Supplementation with 0.2x, 1x and 5x doses of DHA+EPA increases propionate production - Supplementation with EPA at 0.2x, 1x and 5x doses increases propionate production.

[0294] The following observations were made regarding butyrate production after 48 h fermentation following administration of vitamins and omega-3 fatty acids: Compared with the corresponding controls, - Supplementation with 5x the dose of vitamin B2 increases butyrate production - Supplementation with 5x the dose of vitamin B2-5-phosphate increases butyrate production - Vitamin D supplementation at 0.2x and 1x doses increases butyrate production - Vitamin A supplementation at 0.2x, 1x and 5x doses increases butyrate production - Folic acid supplementation at 0.2x, 1x, and 5x doses increases butyrate production - Vitamin K1 supplementation at 0.2x, 1x and 5x doses increases butyrate production - Supplementation with DHA at 0.2x, 1x, and 5x doses increases butyrate production - Supplementation with 0.2x and 1x doses of DHA+EPA increases butyrate production - EPA supplementation at 0.2x, 1x and 5x doses increases butyrate production.

[0295] The following observations were made regarding total SCFA production after 48 h fermentation following administration of vitamins and omega-3 fatty acids: Compared with the corresponding controls, - Supplementation with 1x the dose of vitamin E increases total SCFA production - Vitamin D supplementation at 1x and 5x doses increases total SCFA production - Vitamin A supplementation at 0.2x and 5x doses increases total SCFA production - Folic acid supplementation at 0.2x, 1x, and 5x doses increases total SCFA production - Vitamin K1 supplementation at 0.2x, 1x and 5x doses increases total SCFA production - Supplementation with 0.2x, 1x, and 5x doses of DHA increases total SCFA production - Supplementation with 0.2x and 1x doses of DHA+EPA increases total SCFA production - Supplementation with 0.2x and 1x doses of EPA increases total SCFA production.

[0296] Regarding the TEER measurements of cells treated with 48 h fermented samples after subtracting the TEER of cells treated with 0 h fermented samples in the healthy intestinal cell model (Figure 6), the following observations were made: Compared with the corresponding controls, - Cells treated with samples from fermentation of 0.2x the dose of vitamin B2-5-phosphate showed an increase in TEER - Cells treated with samples from fermentation at a 1x dose of vitamin C showed an increase in TEER - Cells treated with samples from fermentation of 1x and 5x doses of vitamin E showed increased TEER - Cells treated with samples from fermentation of DHA+EPA at 0.2x, 1x and 5x doses showed increased TEER - Cells treated with samples from fermentation of EPA at 0.2x, 1x and 5x doses showed increased TEER

[0297] Regarding TEER measurements of cells treated with 48 h fermentation samples after subtracting the TEER of cells treated with 0 h fermentation samples in the basolateral induction of the leaky cell model (FIG. 7), the following observations were made: Compared with the corresponding controls, - Cells treated with samples from fermentation of 0.2x the dose of vitamin B2 showed an increase in TEER - Cells treated with samples from fermentation of 0.2x the dose of vitamin B2 + vitamin C showed an increase in TEER - Cells treated with samples from fermentation at 0.2x the dose of vitamin E showed an increase in TEER - Cells treated with samples from fermentation of 0.2x and 1x doses of DHA+EPA show increased TEER - Cells treated with samples from fermentation of 0.2x and 1x doses of EPA show an increase in TEER.

[0298] Regarding Lucifer Yellow measurements of cells treated with 48 h fermented samples after subtracting the TEER of cells treated with 48 h fermented samples in the basolateral induction of the leaky cell model (FIG. 7), the following observations were made: Compared with the corresponding controls, - Cells treated with samples from fermentation containing 0.2x the dose of vitamin B2 showed a decrease in Lucifer Yellow. - Cells treated with a sample from a fermentation containing 0.2x the dose of vitamin B2 + vitamin C showed a decrease in Lucifer Yellow. - Cells treated with samples from fermentation at a 0.2x dose of vitamin E showed a decrease in Lucifer Yellow. - Cells treated with samples from fermentation of 0.2x the dose of DHA+EPA show a reduction in Lucifer Yellow.

[0299] Regarding the TEER measurements of cells treated with the 48 h fermentation sample after subtracting the TEER of cells treated with the 0 h fermentation sample in the apical induction of the leaky cell model (FIG. 8), the following observations were made: Compared with the corresponding controls, - Cells treated with samples from fermentation of 0.2x the dose of vitamin B2 showed an increase in TEER - Cells treated with samples from fermentation of 0.2x the dose of vitamin B2-5-phosphate showed an increase in TEER - Cells treated with samples from fermentation of 0.2x and 1x doses of vitamin C showed increased TEER - Cells treated with samples from fermentation of 0.2x the dose of vitamin B2 + vitamin C showed an increase in TEER - Cells treated with samples from fermentation of 0.2x, 1x and 5x doses of vitamin E showed increased TEER - Cells treated with samples from fermentation at a 5x dose of vitamin D showed an increase in TEER - Cells treated with samples from fermentation of 0.2x, 1x and 5x doses of vitamin A showed increased TEER - Cells treated with samples from fermentation of 0.2x and 1x doses of folic acid showed an increase in TEER - Cells treated with samples from fermentation of 0.2x the dose of DHA+EPA showed an increase in TEER - Cells treated with samples from fermentation of 0.2x, 1x and 5x doses of EPA show an increase in TEER.

[0300] Regarding Lucifer Yellow measurements of cells treated with 48h fermented samples after subtracting the TEER of cells treated with 48h fermented samples in the apical induction of the leaky cell model (FIG. 8), the following observations were made: Compared with the corresponding controls, - Cells treated with samples from fermentation containing 0.2x the dose of vitamin B2 showed a decrease in Lucifer Yellow. - Cells treated with a sample from the fermentation of 0.2x the dose of vitamin B2-5-phosphate showed a decrease in Lucifer Yellow. - Cells treated with samples from fermentation at 0.2x and 1x doses of vitamin C showed a decrease in Lucifer Yellow. - Cells treated with a sample from a fermentation containing 0.2x the dose of vitamin B2 + vitamin C showed a decrease in Lucifer Yellow. - Cells treated with samples from fermentation of 1x and 5x doses of vitamin E showed a decrease in Lucifer Yellow. - Cells treated with samples from fermentation at 1x and 5x doses of vitamin A showed a decrease in Lucifer Yellow. - Cells treated with samples from fermentation at a 0.2x dose of vitamin K1 show a decrease in Lucifer Yellow. - Cells treated with samples from fermentation of 0.2x the dose of DHA+EPA show a reduction in Lucifer Yellow.

[0301] The following observations were made regarding the microbiota α diversity after 24 h fermentation following administration of vitamins and omega-3 fatty acids: Compared with the corresponding controls, - Vitamin B2 supplementation at 1x and 5x doses increases microbiota diversity - Supplementation with vitamin B2-5-phosphate at 0.2x and 1x doses increases microbiota diversity - 5x doses of vitamin C supplementation increase microbiome diversity - Vitamin E supplementation at 0.2x, 1x and 5x doses increases microbiota diversity - Vitamin D supplementation at 0.2x and 5x doses increases microbiota diversity - Vitamin A supplementation at 1x and 5x doses increases microbiota diversity - Supplementation with 1x and 5x doses of vitamin B2 + vitamin C increases microbiota diversity - Folic acid supplementation at 0.2x and 1x doses increases microbiota diversity - Vitamin K1 supplementation at 0.2x, 1x and 5x doses increases microbiota diversity - Supplementation with 0.2x, 1x and 5x doses of DHA increases microbiota diversity - Supplementation with 1x and 5x doses of DHA+EPA increases microbiota diversity - Supplementation with EPA at 0.2x, 1x and 5x doses increases microbiota diversity.

[0302] Regarding the relative abundance of Bifidobacterium after 24 h fermentation following administration of vitamins and omega-3 fatty acids, the following observations were made: Compared with the corresponding controls, - Vitamin B2 supplementation at 1x and 5x doses increases the relative abundance of Bifidobacterium - Vitamin B2-5-phosphate supplementation at 0.2x and 1x doses increases the relative abundance of Bifidobacterium - Vitamin C supplementation at a 1x dose increases the relative abundance of Bifidobacterium - Vitamin E supplementation at 0.2x, 1x and 5x doses increases the relative abundance of Bifidobacterium - Vitamin D supplementation at 0.2x and 5x doses increases the relative abundance of Bifidobacterium - Vitamin A supplementation at a 1x dose increases the relative abundance of Bifidobacterium - Supplementation with 1x and 5x doses of vitamin B2 + vitamin C increases the relative abundance of Bifidobacterium - Folic acid supplementation at 0.2x and 1x doses increases the relative abundance of Bifidobacterium - Supplementation with 0.2x the dose of vitamin K1 increases the relative abundance of Bifidobacterium - Supplementation with 0.2x and 5x doses of DHA increases the relative abundance of Bifidobacterium - Supplementation with 5x the dose of DHA+EPA increases the relative abundance of Bifidobacterium - EPA supplementation at 0.2x, 1x and 5x doses increases the relative abundance of Bifidobacterium.

[0303] Similar observations were made for other beneficial bacteria.

[0304] Regarding the relative abundance of genes involved in acetyl-CoA fermentation to the butyrate synthesis pathway after 24 h of fermentation following vitamin and omega-3 fatty acid administration, the following observations were made: Compared with the corresponding controls, - Supplementation with 1x and 5x doses of vitamin B2 increases the relative abundance of genes involved in acetyl-CoA fermentation to the butyrate synthesis pathway - Vitamin B2-5-phosphate supplementation at 0.2x and 1x doses increases the relative abundance of genes involved in acetyl-CoA fermentation to the butyrate synthesis pathway - 5x doses of vitamin C supplementation increase the relative abundance of genes involved in acetyl-CoA fermentation to the butyrate synthesis pathway - Vitamin E supplementation at a 0.2x dose increases the relative abundance of genes involved in acetyl-CoA fermentation to the butyrate synthesis pathway - Vitamin D supplementation at a 0.2x dose increases the relative abundance of genes involved in acetyl-CoA fermentation to the butyrate synthesis pathway - Vitamin A supplementation at 1x and 5x doses increases the relative abundance of genes involved in acetyl-CoA fermentation to the butyrate synthesis pathway - Supplementation with 5x the dose of vitamin B2 + vitamin C increases the relative abundance of genes involved in acetyl-CoA fermentation to the butyrate synthesis pathway - Folic acid supplementation at a 0.2x dose increases the relative abundance of genes involved in acetyl-CoA fermentation to the butyrate synthesis pathway - Vitamin K1 supplementation at 0.2x, 1x and 5x doses increases the relative abundance of genes involved in acetyl-CoA fermentation to the butyrate synthesis pathway - Supplementation with DHA at 0.2x, 1x, and 5x doses increases the relative abundance of genes involved in acetyl-CoA fermentation to the butyrate synthesis pathway - Supplementation with 0.2x, 1x and 5x doses of DHA+EPA increases the relative abundance of genes involved in acetyl-CoA fermentation to the butyrate synthesis pathway - EPA supplementation at 0.2×, 1× and 5× doses increases the relative abundance of genes involved in acetyl-CoA fermentation to the butyrate synthesis pathway.

[0305] Regarding GROa-CXCL1 production by HT29 cells after treatment with effluent from the fermentation 24 h after administration of vitamins and omega-3 fatty acids, the following observations were made: Compared with the corresponding controls, - Vitamin B2 supplementation at 1x and 5x doses increases GROa-CXCL1 production - 5x doses of vitamin E supplementation increase GROa-CXCL1 production - Supplementation with 1x dose of vitamin B2 + vitamin C increases GROa-CXCL1 production - Supplementation with DHA at 0.2x, 1x and 5x doses increases GROa-CXCL1 production - Supplementation with 0.2x, 1x and 5x doses of DHA+EPA increases GROa-CXCL1 production - EPA supplementation at 1x and 5x doses increases GROa-CXCL1 production.

[0306] Regarding IL-8 production by HT29 cells after treatment with effluent from the fermentation 24 h after administration of vitamins and omega-3 fatty acids, the following observations were made: Compared with the corresponding controls, - Vitamin B2 supplementation at 1x and 5x doses increases IL-8 production - Supplementation with 5x the dose of vitamin B2-5-phosphate increases IL-8 production - Vitamin E supplementation at 0.2x, 1x and 5x doses increases IL-8 production - Vitamin A supplementation at 0.2x and 5x doses increases IL-8 production - Supplementation with 1x dose of vitamin B2 + vitamin C increases GIL-8 production - 0.2x and 5x doses of folic acid supplementation increase IL-8 production - Supplementation with 0.2x the dose of vitamin K1 increases IL-8 production - Supplementation with DHA at 0.2x, 1x and 5x doses increases IL-8 production - Supplementation with DHA+EPA at 0.2x, 1x and 5x doses increases IL-8 production - EPA supplementation at 0.2x, 1x and 5x doses increases IL-8 production.

[0307] Regarding MIP3A-CCL20 production by HT29 cells after treatment with effluent from the fermentation 24 h after administration of vitamins and omega-3 fatty acids, the following observations were made: Compared with the corresponding controls, - Vitamin E supplementation at 0.2x, 1x and 5x doses increases MIP3A-CCL20 production - Vitamin D supplementation at 0.2x, 1x and 5x doses increases MIP3A-CCL20 production - Vitamin A supplementation at 0.2x, 1x and 5x doses increases MIP3A-CCL20 production - Vitamin K1 supplementation at 0.2x, 1x and 5x doses increases MIP3A-CCL20 production - Supplementation with DHA at 0.2x, 1x and 5x doses increases MIP3A-CCL20 production - Supplementation with 0.2x, 1x and 5x doses of DHA+EPA increases MIP3A-CCL20 production - EPA supplementation at 0.2x, 1x and 5x doses increases MIP3A-CCL20 production.

[0308] The following observations were made regarding gas production after 48 h of fermentation following administration of vitamins and omega-3 fatty acids: Compared with the corresponding controls, Supplementation with 1x and 5x doses of vitamin C reduces gas production Supplementation with vitamin E at 1x and 5x doses reduces gas production Supplementation with 1x and 5x doses of vitamins C and B2 reduces gas production.

[0309] [Second set of in vitro experiments shown in Figures 16-19] Regarding acetate production after 48 h fermentation following administration of vitamins and β-carotene, the following observations were made: -Acetate production increases with 1x and 5x doses of β-carotene supplementation in both donors compared to matched controls.

[0310] The following observations were made regarding propionate production after 48 h of fermentation following vitamin administration: - In one of the two donors (Donor C), supplementation with 1x and 5x doses of β-carotene increased propionate production - Supplementation with 0.2x the dose of vitamin B5 in one of the two donors (Donor C) increases propionate production compared to matched controls.

[0311] The following observations were made regarding butyrate production after 48 h of fermentation following vitamin administration: - Supplementation with 1x dose of β-carotene increased butyrate production in both donors - butyrate production increases with 0.2x dose of β-carotene supplementation in one of the two donors (donor C) compared to the matched control.

[0312] Regarding total SCFA production after 48 h fermentation following administration of vitamins and β-carotene, the following observations were made: - Supplementation with 1x and 5x doses of β-carotene increased total SCFA production in both donors compared to matched controls - Supplementation with 5x the dose of vitamin B5 reduced branched-SCFA production in both donors compared to matched controls.

[0313] Regarding the relative abundance of Bifidobacteriaceae after 24 h fermentation following vitamin administration, the following observations were made: Compared with the corresponding controls, - For both donors, β-carotene supplementation at 1× and 5× doses increased the relative abundance of Bifidobacteriaceae - Supplementation with 5x the dose of vitamin B5 in both donors increased the relative abundance of Bifidobacteriaceae - For one of the two donors (Donor D), supplementation with biotin at 0.2x, 1x and 5x doses increased the relative abundance of Bifidobacteriaceae. - For one of the two donors (Donor D), supplementation with 1x and 5x doses of pyridoxine increases the relative abundance of Bifidobacteriaceae.

[0314] Regarding the relative abundance of Eggerthellaceae after 24 h fermentation following vitamin treatment, the following observations were made: - Eggerthellaceae relative abundance increases with 5x dose of β-carotene supplementation for both donors compared to matched controls.

[0315] Regarding the relative abundance of Desulfovibrionaceae after 24 h fermentation following vitamin administration, the following observations were made: Compared with the corresponding controls, - For both donors, 5x doses of β-carotene supplementation reduced the relative abundance of Desulfovibrionaceae. - For one of the two donors (Donor C), supplementation with a 5x dose of vitamin B5 reduces the relative abundance of Desulfovibrionaceae.

[0316] Regarding the relative abundance of Akkermansiaceae after 24 h fermentation following vitamin treatment, the following observations were made: Compared with the corresponding controls, - For both donors, 5x doses of β-carotene supplementation increased the relative abundance of Akkermansiaceae. - For one of the two donors (donor C), supplementation with 1x dose of thiamine increased the relative abundance of Akkermansiaceae. - For one of the two donors (Donor C), supplementation with 0.2x the dose of niacinamide increased the relative abundance of Akkermansiaceae. - For one of the two donors (donor D), supplementation with 0.2x the dose of vitamin B5 increased the relative abundance of Akkermansiaceae. - For one of the two donors (donor D), supplementation with pyridoxine at doses of 0.2x, 1x, and 5x increased the relative abundance of Akkermansiaceae. - For one of the two donors (donor D), supplementation with 0.2x the dose of biotin increased the relative abundance of Akkermansiaceae. - For one of the two donors (donor D), supplementation with vitamin B12 at 0.2x and 1x doses increases the relative abundance of Akkermansiaceae.

[0317] Regarding the relative abundance of Prevotellaceae after 24 h fermentation following vitamin treatment, the following observations were made: Compared with the corresponding controls, - For one of the two donors (Donor C), supplementation with 1x dose of β-carotene increased the relative abundance of Prevotellaceae. - For one of the two donors (donor C), supplementation with a 5x dose of pyridoxine increases the relative abundance of Prevotellaceae.

[0318] Regarding the relative abundance of Clostridiaceae after 24 h fermentation following vitamin administration, the following observations were made: -Compared to the matched control, supplementation with 1x dose of β-carotene increases the relative abundance of Clostridiaceae for one of the two donors (Donor C).

[0319] Regarding the relative abundance of Erysipelotrichaceae after 24 h fermentation following vitamin treatment, the following observations were made: Compared with the corresponding controls, - For one of the two donors (Donor C), supplementation with 5x the dose of thiamine increased the relative abundance of Erysipelotrichaceae. - For one of the two donors (Donor C), supplementation with 1x and 5x doses of niacinamide increased the relative abundance of Erysipelotrichaceae. - For one of the two donors (donor C), supplementation with 1x dose of pyridoxine increased the relative abundance of Erysipelotrichaceae. - For one of the two donors (donor C), supplementation with biotin at 0.2x and 1x doses increased the relative abundance of Erysipelotrichaceae. - For one of the two donors (Donor C), supplementation with 1x and 5x doses of vitamin B12 increased the relative abundance of Erysipelotrichaceae. - For one of the two donors (donor D), supplementation with a 1x dose of vitamin B5 increases the relative abundance of Erysipelotrichaceae.

[0320] Regarding the relative abundance of Coriobacteriaceae after 24 h fermentation following vitamin administration, the following observations were made: - In one of the two donors (donor D), the relative abundance of Coriobacteriaceae increases with 1x and 5x doses of β-carotene supplementation compared to the corresponding control.

[0321] Regarding the relative abundance of Barnesiellaceae after 24 h fermentation following vitamin treatment, the following observations were made: Compared with the corresponding controls, - For one of the two donors (donor D), supplementation with 5x the dose of β-carotene increased the relative abundance of Barnesiellaceae. - For one of the two donors (donor D), supplementation with a 1x dose of biotin increases the relative abundance of Barnesiellaceae.

[0322] Regarding the relative abundance of Faecalibacterium prausnitzii after 24 h fermentation following vitamin administration, the following observations were made: Compared with the corresponding controls, - For one of the two donors (donor D), supplementation with 1x and 5x doses of β-carotene increased the relative abundance of Faecalibacterium prausnitzii. - For one of the two donors (donor D), supplementation with vitamin B5 at 0.2x, 1x and 5x doses increased the relative abundance of Faecalibacterium prausnitzii - For one of the two donors (donor D), supplementation with pyridoxine at doses of 0.2x, 1x, and 5x increased the relative abundance of Faecalibacterium prausnitzii. - For one of the two donors (donor D), supplementation with biotin at 0.2×, 1×, and 5× doses increased the relative abundance of Faecalibacterium prausnitzii. - For one of the two donors (donor D), supplementation with vitamin B12 at 0.2x, 1x and 5x doses increased the relative abundance of Faecalibacterium prausnitzii.

[0323] Regarding the relative abundance of Veillonellaceae after 24 h fermentation following vitamin treatment, the following observations were made: Compared with the corresponding controls, - For one of the two donors (donor D), supplementation with 1x and 5x doses of β-carotene increased the relative abundance of Veillonellaceae. - For one of the two donors (donor D), supplementation with 0.2x the dose of vitamin B5 increased the relative abundance of Veillonellaceae. - For one of the two donors (donor D), supplementation with a 5x dose of pyridoxine increases the relative abundance of Veillonellaceae.

[0324] Regarding microbiota diversity, the following observations were made: Supplementation with 1× and 5× doses of β-carotene increases β-diversity in both donors. Supplementation with 0.2× dose of β-carotene increases β-diversity in one of the two donors (donor D). Supplementation with 5× the dose of vitamin B increased β-diversity in both donors. Supplementation with 0.2× and 1× doses of β-carotene increases β-diversity in one of the two donors (donor D). Supplementation with vitamin B6 at 0.2×, 1× and 5× doses increases β-diversity in one of the two donors (donor D). Supplementation with vitamin B12 at 0.2×, 1× and 5× doses increases β-diversity in one of the two donors (donor D). Supplementation with a 1× dose of vitamin B7 increases β-diversity in both donors. Supplementation with a 5× dose of vitamin B7 increases β-diversity in one of the two donors (donor D). Supplementation with a 1x dose of vitamin B1 increases β-diversity in one of two donors (donor C). Supplementation with a 5x dose of vitamin B1 increases β-diversity in one of two donors (donor D). Supplementation with 0.2× and 5× doses of vitamin B3 increases β-diversity in one of the two donors (donor D). Supplementation with a 1× dose of vitamin B1 increases β-diversity in one of the two donors (donor C) compared to matched controls. Supplementation with 0.2× and 5× doses of vitamin B5 increases α-diversity in one of the two donors (donor D). Supplementation with a 5× dose of vitamin B6 increases β-diversity in one of the two donors (donor D). Supplementation with 0.2× and 1× doses of vitamin B12 increases α-diversity in one of the two donors (donor D). Supplementation with 0.2× dose of vitamin B7 increases α-diversity in both donors. Supplementation with a 5× dose of vitamin B7 increases α-diversity in one of the two donors (donor D). Supplementation with 0.2× and 1× doses of vitamin B1 increases α-diversity in one of the two donors (donor C). Supplementation with a 1× dose of vitamin B3 increases α-diversity in one of the two donors (donor C).

[0325] [Example 2] [Formulations for human use] The following formulations, each containing a single active ingredient, were prepared and loaded into enteric coated hard gelatin capsules (Capsugel) coated with Eudragit® S 100 polymer.

[0326] [Table 5]

[0327] The formulations are suitable for administration once daily, preferably for at least 28 consecutive days.

[0328] [Example 3] [Poultry model] Three groups of 120 chickens (all females) with the same genetic characteristics were housed in three separate rooms each characterized by the same environmental conditions and fed three different diets: (A) control diet (5 mg / kg riboflavin); (B) control diet + 50 mg / kg vitamin riboflavin; (C) control diet + 100 mg / kg vitamin riboflavin. Each group was sampled at three time points on specific days corresponding to the time when the diet was changed according to the feeding program (i.e. S1=14 days, S2=28 days, S3=38 / 42 days).

[0329] The 42-day broilers (at the end of the trial) weigh 2.6 kg and consume approximately 205 g feed per bird per day. The three test doses therefore correspond to riboflavin intakes of 0.38; 3.82 and 7.65 mg / kg body weight at 5; 50 and 100 mg / kg of diet, respectively, with the intake per kg body weight being much higher at the lower weights. Table 4 below shows the theoretical growth rates of the broilers and the amount of riboflavin to be added.

[0330] [Table 6]

[0331] During each sampling (ie, S1, S2 and S3), a total of 40 birds / group were humanely euthanized and 9 liter samples were collected (ie, 3 samples / group).

[0332] [Microbiota analysis of cecal samples] A total of 741 caecal samples have been analysed by the Unit of Microbial Ecology of Health, including 120 samples from time point 1, 119 from time point 2 (1 bird from group C missing) and 118 from time point 3 (1 bird from groups B and 1 bird from groups C missing) and 118 from time point 3 (1 bird from groups B and 1 bird from groups C missing).

[0333] Materials and Methods DNA extraction from cecal samples: A commercial kit: DNeasy PowerSoil kit (Qiagen, Hilden, Germany) was selected and applied to all available cecal samples according to the manufacturer's instructions. DNA yields were measured using a Nanodrop instrument.

[0334] 16S rRNA gene PCR amplification and sequencing. The V3-V4 hypervariable region of the 16S rRNA gene was PCR amplified using 341F and 785R primers with Illumina overhang adapter sequences. Amplicon purification was performed by using AMPure XP Beads magnetic beads (Beckman Coulter, Brea, CA). For indexed library preparation, the Nextera XT DNA Library Prep Kit (Illumina, San Diego, CA) was used. An additional magnetic bead purification step was performed and libraries were quantified using a Qubit 3.0 fluorometer (Invitrogen) before pooling at 4 nM. The library pool was denatured with NaOH 0.2 N and then diluted to 6 pM. Sequencing was performed on an Illumina MiSeq platform using a 2 × 250 bp paired-end protocol according to the manufacturer's instructions (Illumina).

[0335] Bioinformatics and statistics. Raw sequences were processed using a pipeline combining PANDAseq (Masella et al., 2012) and QIIME2 (Caporaso et al., 2010; https: / / qiime2.org). After removal of chimeric sequences, high-quality reads were filtered and binned into high-resolution operational taxonomic units (OTUs) according to a 99% taxonomic threshold by the open reference strategy implemented in dada2 (Callahan et al., 2016). Taxonomy was assigned using the vsearch classifier (Rognes et al., 2016) and the SILVA database (Quast et al., 2013) as a reference. Alpha diversity was measured using the Faith PC index and the number of observed OTUs. Statistics were performed using R Studio software version 1.0.136 running on R software 3.1.3 (https: / / www.r-project.org / ) implementing the libraries vegan, made4, and PMCMR. Beta diversity was estimated by calculating weighted UniFrac distances and visualized by Principal Coordinates Analyses (PCoAs). The significance of the separation between sample groups was tested by permutational multivariate analysis of variance using the function adonis from the vegan package. Bacterial phylogenetic groups (genera, families, phyla) showing a minimum relative abundance of 0.5% in at least 1% of the samples were retained for further analysis and graphical representation. Compositional differences between sample groups were tested using the Krustal-Wallis test. P values ​​were corrected for multiple comparisons using the Benjamini-Hochberg method.

[0336] [result] Sequencing of 16S rRNA amplicons from 741 DNA samples yielded 4,986,865 high-quality sequences, ranging from 1,099 to 15,182 with a mean of 6,490 ± 2715 sequences per sample. Reads were clustered into 20,950 operational taxonomic units (OTUs) based on 99% similarity.

[0337] Firstly, the β diversity analysis based on all available samples of chickens from group B (Figure 20) shows different longitudinal trajectories regarding the microbiota structure in the cecum for groups A and C.

[0338] This trend is particularly evident when plotting the entire sample set using weighted UniFrac distances (Figure 20A), whereas the PCoA obtained using unweighted UniFrac distances shows more overlap between the various groups (Figure 20B), indicating that differences in the microbiota of broilers in group B likely reside in the most abundant bacterial species rather than in subdominant ones.

[0339] The same analysis was performed separately using samples from time points T1, T2, and T3 (Figure 21). At T1, samples from groups A, B, and C largely overlap (Figures 21A and 21D), while group B samples begin to cluster separately at T2 and T3, especially when using the Weighted UniFrac assay (Figures 21B and 21C).

[0340] Conversely, with regard to biodiversity (α diversity, FIG. 18), the nutritional regime administered to chickens in group C induces a significant increase (P<0.0001) in the species richness calculated by the “Observed OTU” measurement method at time points T2 and T3 (FIGS. 22E and 22F).

[0341] The visualization of the average microbiota profiles notably showed that the compositional differences between the three experimental groups could be assessed at the phylum level (Figure 23). In group B, the relative abundance of Bacteroidetes (Figure 23, blue) increases significantly at T2, whereas in group A, the increase in Bacteroidetes abundance occurs only at T3. The diet administered to group C seems to partially inhibit this increase. The compositional analysis at the family level (Figure 24) shows that only the diet administered to group B promotes a progressive increase in Bacteroidaceae (Figure 24, dark blue), whereas in groups A and C, the Bacteroidetes phylum is, on average, mostly composed of bacteria belonging to the family Rikenellaceae (Figure 24, light blue). This observation was statistically confirmed at both the family (Figure 25) and genus levels (Figure 25). The family Bacteroidaceae and the genus Bacteroides show significantly higher abundance in group B at all available time points. In contrast, the family Rikenellaceae and its genus Alistipes show higher abundance in groups A and C, but only in T3.

[0342] In contrast, the average family level profile obtained for broilers in group C showed a progressive increase (T1 to T3) in the abundance of Bifidobacteriaceae (Figure 24, yellow) that was not observed in groups A and B (Figure 24). The significance of this difference was confirmed at T2 and T3 at both family (Figure 25) and genus levels (Bifidobacterium, Figure 26). It was also demonstrated that at the family level, the increase in Bifidobacteriaceae led to the impairment of Coriobacteriaceae, another family belonging to the phylum Actinobacteria (Figure 25).

[0343] Most interestingly, genus-level analysis showed that both dietary regimens administered to groups B and C appeared to accelerate the increase in the relative abundance of Faecalibacterium, which was significantly higher in groups B and C versus control group A at T1 (Figure 26). At subsequent time points, the relative abundance of Faecalibacterium in the three groups did not differ significantly. This longitudinal trend mirrors that observed for the family Ruminococcaceae (Figure 25), to which Faecalibacterium belongs. At time point T2, another genus belonging to the family Ruminococcaceae, the group [Eubacterium]·coprostanoligenes, shows higher abundance in groups B and C compared to control group A (Figure 26).

[0344] [Description of the ileal microbiota in three broiler flocks (groups A, B, C) along the production cycle (times T1, T2, and T3)] In a first observation, the β-diversity analysis on all available ileal samples (Figure 27) showed that samples taken from the three different chicken groups (A, B, and C) did not follow separate longitudinal trajectories in terms of microbiota structure, unlike what was observed in the case of the cecal microbiota. Indeed, samples from the three groups overlap in the case of both the Weighted and Unweighted UniFrac measurements used for sample distance calculation (Figures 27A and B).

[0345] Sequencing of 16S rRNA amplicons from 741 DNA samples yielded 4,986,865 high-quality sequences, ranging from 1,099 to 15,182 with a mean of 6,490 ± 2715 sequences per sample. Reads were clustered into 20,950 operational taxonomic units (OTUs) based on 99% similarity.

[0346] This was confirmed by plotting samples taken at different time points for separate PCoA (Figure 28).

[0347] Regarding biodiversity (alpha diversity, FIG. 29), the nutritional regime administered to chickens of groups B and C induces a significant decrease in the diversity of the ileal ecosystem (calculated as Faith's PD index) compared to group A at the first time point (T1, P<0.001, FIG. 29A). On the contrary, the same diet (groups B and C) induces a significant increase in terms of species richness calculated by the "Observed OTU" measurement method at time points T2 and T3 (significant only at time point T2, P<0.0001) (FIGS. 29E and 29F).

[0348] Visualization of the average microbiota profile revealed that at the phylum level, the ileal microbiota was similar to that reported for the cecum, with the majority of bacteria belonging to the phylum Firmicutes (Figure 30, green). However, at lower phylogenetic levels (families), the specificity of the ileal microbiota was evident, with a predominance of Lactobacillaceae (a family belonging to the phylum Firmicutes) at all available time points (Figure 31, lavender).

[0349] The longitudinal analysis clearly showed a progressive decrease of Enterobacteriaceae (Figure 31, red) from T1 to T3, independent of the nutritional regime (groups A, B and C). Furthermore, a progressive decrease of Clostridiaceae (Figure 31, dark green) can be observed from T1 to T3 in favor of Peptostreptococcaceae (Figure 31, light green). This is especially evident at T3 in samples taken from chickens of groups A and C, as confirmed by the Kruskall-Wallis test (P<0.0001, Figure 32). Peptostreptococcaceae is a bacterial taxon with unknown function in the intestinal tract of broilers. Although studies focused on the cecal microbiota of chickens have shown significant variations in the abundance of this taxon in response to changes in diet, supplements and antibiotic treatments (Munyaka et al., 2016; Costa et al., 2017; Zeits et al., 2019), studies on the ileal microbiota are still too scarce to hypothesize a possible functional role for Peptostreptococcaceae variations.

[0350] [Description of the waste microbiota in three broiler flocks (Flocks A, B, C) along the production cycle (times T1, T2 and T3)] In a first observation, the beta diversity analysis on all available waste samples (Figure 33) revealed that samples taken from boxes with chickens from group C clustered separately from the other samples in terms of microbiota structure when the distance between samples was calculated using the Unweighted Unifrac metric (Adonis test). Visualization of the mean microbiota profile revealed that at the phylum level, the ileal microbiota was similar to that reported for the cecum, with the majority of bacteria belonging to the phylum Firmicutes (Figure 30, green). However, at lower phylogenetic levels (families), the specificity of the ileal microbiota was evident, dominated at all available time points mainly by Lactobacillaceae (a family belonging to the phylum Firmicutes) (Figure 31, lavender).

[0351] The longitudinal analysis clearly showed a progressive decrease of Enterobacteriaceae (Figure 31, red) from T1 to T3, independent of the nutritional regime (groups A, B and C). Furthermore, a progressive decrease of Clostridiaceae (Figure 31, dark green) can be observed from T1 to T3 in favor of Peptostreptococcaceae (Figure 31, light green). This is especially evident at T3 in samples taken from chickens of groups A and C, as confirmed by the Kruskall-Wallis test (P<0.0001, Figure 32). Peptostreptococcaceae is a bacterial taxon with unknown function in the intestinal tract of broilers. Studies focused on the cecal microbiota of chickens have shown significant variations in the abundance of this taxon in response to changes in diet, supplements and antibiotic treatments (Munyaka et al., 2016; Costa et al., 2017; Zeits et al., 2019), but studies on the ileal microbiota are still very scarce, making it difficult to hypothesize a possible functional role for Peptostreptococcaceae variations.

[0352] [Description of the waste microbiota in three broiler flocks (Flocks A, B, C) along the production cycle (times T1, T2 and T3)] In a first observation, the beta diversity analysis (Figure 33) on all available waste samples revealed that samples from boxes with chickens from group C clustered separately from the other samples in terms of microbiota structure when the distance between samples was calculated using the Unweighted Unifrac metric (Adonis test). Visualization of the mean microbiota profile revealed that at the phylum level, the ileal microbiota was similar to that reported for the cecum, with the majority of bacteria belonging to the phylum Firmicutes (Figure 30, green). However, at lower phylogenetic levels (families), the specificity of the ileal microbiota was evident, dominated mainly by Lactobacillaceae (a family belonging to the phylum Firmicutes) at all available time points (Figure 31, lavender; P=0.006, Figure 33B).

[0353] This was not confirmed by the analysis based on weighted Unifrac distance (Figure 33A), indicating that the difference between the group C waste microbiota and the other wastes may lie in subdominant bacterial taxa. Also, the separation of the group C waste from the other waste samples in the PCoA based on unweighted Unifrac distance (Figure 33B) was located along the second ordinate axis (MDS2), which only explains 9.4% of the total variability of the dataset.

[0354] Visualization of the average microbiota profiles (Figures 34 and 35) clearly demonstrated the absence of a single major dominant taxon at both the phylum and family level. However, no significant differences in bacterial composition (family or genus level) and species richness / diversity (data not shown) of the waste microbiota were revealed in the analyzed datasets.

[0355] [Microbiome analysis] [Sample to be analyzed] A total of 63 samples were provided to the Bio-NMR Group of Department of Agricultural and Food Science and the Interdepartmental Center for Industrial Agri-Food Research (CIRI) of University of Bologna (Italy), including 21 available cecal samples from the first time point, 21 from the second time point, and 21 from the third time point.

[0356] [Sample preparation.] Preliminary analysis was performed on 63 samples further classified as follows: control sample supplied with 10 mg / kg vitamin B2 (A), treated sample supplied with 50 mg / kg vitamin B2 (B), treated sample supplied with 100 mg / kg vitamin B2 (C). Samples were prepared for nuclear magnetic resonance (NMR) analysis by vortex mixing 1 mL of deionized water and feces for 5 min, followed by centrifugation for 10 min at 14,000 rpm and 4°C. Approximately 540 mL of the supernatant was added to 100 μL of D2O 1.5 M phosphate buffer containing 0.1% TSP (3-(trimethylsilyl)propionic acid-d4), NaN3 2 mM, and set to pH 7.40. Prior to analysis, samples were centrifuged again for 10 min and 590 uL was transferred to an NMR tube.

[0357] [NMR spectrum obtained.] Proton NMR (1H-NMR) spectra were recorded at 298 K using an AVANCE III spectrometer (Bruker, Milan, Italy) operating at a frequency of 600.13 MHz. Hydrogen-deuterium-oxide (HOD) residual signals were suppressed by presaturation, and broad signals from slowly tumbling molecules were removed by introducing a Carr-Purcell-Meiboom-Gill filter27 for a free induction decay sequence. The filter consisted of a series of 400 echoes spaced 800 μs apart for a total time of 328 ms. Each spectrum was acquired by summing 256 transients with 32K data points over a 7211.54-Hz spectrum (2.27 s observation time). The recycle delay was set to 8 s, taking into account the longitudinal relaxation time of the protons under investigation. Spectra were processed in Top Spin 3.0 (Bruker) using the automated command apk0.noe to perform baseline and phase correction in one go, and applying a line broadening factor of 1 Hz. Peaks were assigned by comparing their chemical shifts and multiplicities with the literature and using the Chenomx NMR suit 8.1 software.

[0358] [1H-NMR spectrum pretreatment] After Fourier transformation, phase and baseline correction, the spectra were calibrated with respect to a chemical shift of 0.00 ppm assigned to the internal standard TSP; the spectral periphery region together with the water signal was removed. The spectra were then normalized using the Probabilistic Exponential Algorithm (PQN) (Regional Scaling) separately for the two different regions (as this worked best for this type of sample). After normalization and before any possible statistical analysis, the spectra were binned into 100 data point intervals of 0.0183 ppm each. The resulting new spectral profile consisted of 410 binned data, which were saved as a matrix in a text file and imported into both R and Python spaces for Multivariate Statistical Analysis (MvSA).

[0359] [Statistical analysis] MvSA has been applied to the binned dataset matrix. Specifically, Principal Component Analysis (PCA) and Partial Least Square Discriminant Analysis (PLS-DA) have been employed. PCA is used to search only one type of omics data to identify the largest sources of variation, whereas the multilevel approach aims to highlight within-subject treatment effects (in this case samples belonging to groups B and C treated with 50 and 100 mg / kg vitamin B2, respectively) apart from the biological variation between biological samples.

[0360] [result] [NMR spectrum] FIG. 36 records the 1H-NMR spectrum of a sample designated C281 at pH 7.13.

[0361] Several metabolites were assigned by comparing their chemical shifts and multiplicities with the literature and by using the Chenomx NMR suit 8.1 software (abbreviations for multiplicities are as follows: s = singlet, dd = doublet of doublets, d = doublet, t = triplet and m = multiplet (indicating a complex pattern). 1) butyrate (t: 0.899 ppm, m: 1.562 ppm, t: 2.165 ppm); 2) leucine (t: 0.959 ppm, m: 1.702 ppm, m: 3.734 ppm); 3) valine (d: 0.978 ppm, m: 1.562 ppm, m: 2.165 ppm); 4) cyclohexane (t: 0.978 ppm, m: 1.562 ppm, m: 3.734 ppm); 5) cyclohexane (t: 0.978 ppm, m: 1.562 ppm, m: 2.165 ppm); 6) cyclohexane (t: 0.978 ppm, m: 1.562 ppm, m: 3.734 ppm); 7) cyclohexane (t: 0.978 ppm, m: 1.562 ppm, m: 2.165 ppm); 8) cyclohexane (t: 0.978 ppm, m: 1.562 ppm, m: 3.734 ppm); 9) cyclohexane (t: 0.978 ppm, m: 1.562 ppm, m: 2.165 ppm); 10) cyclohexane (t: 0.978 ppm, m: 1.562 ppm, m: 2.165 ppm); 11) cyclohexane (t: 0.978 ppm, m: 1.562 ppm, m: 2.165 ppm); 12) cyclohexane (t: pm, d: 1.030, m: 2.261 ppm, d: 3.583 ppm; 4) propionate (t: 1.060 ppm, q: 2.188 ppm); 5) ethanol (t: 1.189 ppm, q: 3.664 ppm); 6) lactate (d: 1.335 ppm, q: 4.124 ppm); 7) alanine (d: 1.485 ppm, q: 3.792 ppm); 8) acetate (s: 1.925 ppm); 9) glutamate (m: 2 .047ppm, m:2.134ppm, t:2.339ppm, m:2.371, q:3.768ppm;10) Succinate (s:2.409ppm);11) Aspartate (q:2.692ppm, dd:2.815ppm, q:3.893ppm);12) Dimethylamine (s:2.714ppm);13) Trimethylamine (s:2.878ppm);14) Creatine (s:3.011ppm, s:3 .918ppm);15) Formate (s:8.461ppm);16) Uracil (d:5.810ppm, d:7.550ppm);17) Phenylalanine (m:7.434ppm, m:7.382ppm, d:7.337ppm);18) Tyrosine (d:7.198ppm, d:6.907ppm);19) Fumarate (s:6.525ppm);20) Taurine (t:3.429ppm, t:3.273ppm).

[0362] [Multivariate statistical analysis] First, we combined different unsupervised methods of size reduction (PCA, Factor Analytis, Independent Component Analysis, Kernel Driven PCA) and classifiers (Random Forest, ADABoosted Decision Tree, Support Vector Machine) to find the best dimensionality reduction model that allows separation between groups and identifies any outlier samples. Figure 37 shows the results from an unscaled PCA performed on the binned and regional scaled dataset. The PCA in Figure 37A shows the presence of two outliers (samples 44 and 46, T3-A-C250 and T3-A-C261, respectively). The results of the new PCA after removing these outliers are shown in Figure 37B. The first two principal components account for 84% of the total variance, but do not show any visible trends or separations, and do not show any further outliers.

[0363] As the unsupervised methods do not reveal any separation, it became necessary to apply a supervised approach (Figure 38). After filtering of outliers, 61 subjects with 437 spectral features each were used for the final PLS-DA (Szymanska, E., et al., 2012). In combination with the first 3-PLS directions, especially the first direction, which was the most informative in identifying the temporal group separation, a 5-component PLS-DA model was trained on the standard scaled data (Figure 38A). Thus, the best prediction accuracy was obtained by feeding the first 3-PLS directions to the Linear Kernel Support Vector Classifier, which achieved an average prediction accuracy of about 89%. The ADABoosting Tree classifier was also evaluated for comparison, which yielded a slightly worse average performance (Valdes, G., et al., 2016). This result is consistent with the 2d-PLS-DA spatial projection, where the three groups are found to be linearly separable when considering time as a variable; however, no significant separation is found when considering control versus treated samples (Figure 38B). To avoid overfitting, all steps of the pipeline used are internally cross-validated, with special attention to the classification and prediction steps, where stratified 10-fold cross-validation is performed. To increase the signal-to-noise ratio and filter uninformative spectral signals, the PLS-DA average weights associated with the most informative signals in the spectra were extracted after smoothing and baseline correction.

[0364] The main metabolites involved in the separation are: uracil (Figure 39), glutamate (Figure 40), alanine (Figure 41), creatine (Figure 42), butyrate (Figure 43), propionate (Figure 44) and acetate (Figure 45). In these figures, the integrated area of ​​each metabolite was compared between samples at different time points (T0-T1-T2 corresponding to days 14, 28 and 38 / 42).

[0365] The integrated area of ​​uracil (Figure 39) appears to increase its concentration by day 42 (T2) for all treatments, especially for sample C, whose concentration is always higher than the others. Glutamate (Figure 40) shows a tendency to increase its concentration after 28 days (T1), especially for samples A and B, which remain constant at T3. The integrated area of ​​alanine (Figure 41) also increases at T1, especially for sample C. In contrast, creatine shows a tendency to increase only for samples A and B at T2, but then decrease their concentration at T3 (Figure 42).

[0366] Figures 43 and 44 relate to the organic acids identified in the spectra. As before, the integrals of the peak intervals are compared among the three groups along the time points. Figures 43 and 44 show the results for butyrate and propionate.

[0367] Butyrate (Figure 43) does not seem to be affected by either treatment or time. In fact, the integrated areas at T0, T1 and T2 in all samples are more or less the same. The same trend occurs for acetate (Figure 45). In the case of propionate, an increasing trend is seen (Figure 44).

[0368] It must be emphasized that the results are affected by a paucity of data and therefore no significant trends emerge from them. For that reason, further analyses were performed to fit trends for the four main metabolites: Figure 46 shows how the integrated areas of the assigned organic acids change along the time points in each group.

[0369] [Metagenomics] [Sample to be analyzed] A total of 63 samples were provided to the Food Safety Laboratory of the Department of Agricultural and Food Sciences-University of Bologna (UNIBO), including 21 cecal samples taken from the first time point, 21 from the second time point, and 21 from the third time point.

[0370] [Metagenomic analysis method] [DNA extraction for metagenomic sequencing] DNA is extracted from each sample of cecal contents using the bead-beating method. Briefly, 0.25 g of cecal contents are suspended in 1 ml of lysis buffer (500 mM NaCl, 50 mM Tris-Cl, pH 8.0, 50 mM EDTA, 4% SDS) with MagNA Lyser Green Beads (Roche, Milan, Italy) and homogenized with a MagNA Lyser (Roche) at 6500 rpm for 25 s. Samples were then heated at 70°C for 15 min and then centrifuged to separate DNA from bacterial cell debris. This process is repeated with a second 300 μl aliquot of lysis buffer. Samples were then subjected to 10 Mv / v ammonium acetate (Sigma, Milan, Italy) precipitation, followed by isopropanol (Sigma) precipitation and 70% ethanol (Carlo Erba, Milan, Italy) washing, before being resuspended in 100 ul of 1× Tris-EDTA (Sigma). Samples were then treated with DNase-free RNase (Roche) and then incubated overnight at 4°C before being processed with the QIAmp® DNA Stool Mini Kit (Qiagen, Milan, Italy) according to the manufacturer's instructions with some modifications. Samples were measured with a BioSpectrometer® (Eppendorf, Milan, Italy) to assess DNA quantity and quality. All samples meeting the DNA quality score (i.e., A260 / 280 nm ratio of 1.8 to 2.0) will be subjected to library preparation using the Nextera XT DNA Library Preparation Kit (Illumina, San Diego, CA). Then, all libraries with an average size of 300-500 bp and a minimum concentration of 4 pM are sequenced using a NexSeq500 (Illumina) at 150 pM in paired-end mode.

[0371] [Sequencing strategy and platform] All samples were sequenced paired-end. The average read length was 300 pb. Sequencing runs were performed using an Illumina NexSeq500. Submitted metagenomes were characterized by outputs ranging from 20 Mbp to 12 Gbp.

[0372] [Bioinformatics analysis] Raw reads were filtered and trimmed using the MGRast (MR) (https: / / www.mg-rast.org) (Keegan et al., 2016) bioinformatics pipeline. Furthermore, functional annotation was performed with the MG-RAST v3.6 metagenomic analysis server (Meyer et al., 2008) using default setting parameters (e-value cutoff: <1e-5, minimum identity % cutoff: 60%, and minimum alignment length cutoff: 15). Genes were mapped to SEED and KEGG (Kyoto Encyclopedia of Genes and Genomes) classifications using SEED and KEGG identifiers for functional classification, respectively.

[0373] [Statistical analysis] Comparative functional profiling was performed using STAMP v2.0.8 software for statistical analysis (Parks and Beiko, 2010; Parks et al., 2014). To remove bias due to differences in sequencing effort, gene counts were normalized by dividing the number of gene hits for each individual function by the total number of gene hits in each metagenomic dataset. To identify SEED and KEGG functions with different abundances in the metagenomes, statistical tests of relative gene abundance compared to other metagenomes were performed by applying the two-sided Welch's exact test with the Benjamini-Hochberg False Discovery Rate (FDR) multiple testing correction method and a p-value < 0.05.

[0374] [result] First, a Principal Component Analysis (PCA) based on the "functional characteristics data" aimed to find the best dimensionality reduction model allowing the separation of the groups and to identify any outlier samples. In Figure 47 the results from the PCA carried out on 63 subjects with 6361 functional characteristics are recorded. The first two principal components account for 81% of the total variance: a clearly visible separation is displayed between the treatment groups (groups B and C) and the control group (group A). ​​With regard to the time points, no separation could be found.

[0375] [Description of the primary KEGG metabolic pathways of three broiler groups (groups A, B, C) along the production cycle (time points T1, T2, and T3)] Visualization of the average functional gene profiles at level 1 based on the KEGG database of the broiler ceca of the three different groups A, B and C for each of the available time points (T1, T2, T3) clearly demonstrated that the compositional differences between the three experimental groups could be assessed at level 1 (Figure 49). In particular, in group A, the relative abundance of gene information processing (Figure 49, orange) was statistically significantly increased at each time point compared to the treatment group (Table 5). In contrast, the relative abundance of environmental information processing (Figure 50A) was statistically significantly increased at each time point in groups B and C compared to group A (Table 5). Furthermore, the relative abundance of metabolites (Figure 50B) was statistically increased in groups B and C compared to group A at 28 days of age, and Organismal Systems was statistically increased in group A compared to groups B and C (Table 5).

[0376] [Table 7]

[0377] [Description of secondary KEGG metabolic pathways of three broiler groups (groups A, B, C) along the production cycle (time points T1, T2, and T3)] Visualization of secondary KEGG metabolic pathways with relative abundance > 0.5% demonstrated that compositional differences between the three experimental groups at each time point were appreciable (Figure 51) (i.e., translation, transcription at 14 days; membrane transport, translation, replication and repair at 28 days; membrane transport, translation, replication and repair, nucleotide metabolism at 42 days).

[0378] In particular, the relative abundance of metabolites of cofactors and vitamins at each time point in groups B and C was statistically significantly increased compared to group A (Tables 6, 7 and 8, light grey). Conversely, the relative abundance of translation, transcription and environmental adaptation was statistically significantly increased at each time point in group A compared to groups B and C (Table 5, white).

[0379] Turning to other pathways, the relative abundances of membrane transport, replication and repair, biosynthesis of other secondary metabolites, metabolism of terpenoids and polyketides, and metabolism of other amino acids were statistically increased in groups B and C compared to groups A at 28 and 42 days of age (Tables 7 and 8, orange). In contrast, the relative abundances of translation, energy metabolism, signal transduction, transcription, infection, and environmental adaptation were statistically increased in groups A compared to groups B and C at 28 and 42 days of age (Tables 7 and 8, white). Finally, the relative abundances of lipid metabolism were statistically significantly increased in groups B and C compared to groups A at 28 days of age (Tables 6 and 7, light gray), whereas folding, sorting and degradation, cell communication and signaling molecules and interactions were statistically increased in groups A compared to groups B and C at 28 days of age (Table 7, white).

[0380] [Table 8]

[0381] [Table 9]

[0382] [Table 10]

[0383] [Description of tertiary KEGG metabolic pathways of three broiler groups (groups A, B, C) along the production cycle (time points T1, T2, and T3)] Analysis of tertiary KEGG metabolic pathways showed no reported differences between experimental groups at 14 days of age (data not recorded), but a number of pathways showed significant differences at 28 and 42 days of age (Tables 9 and 10 below).

[0384] In particular, at 28 and 42 days of age, 19 pathways with a relative abundance of ≥0.1% (ABC transporters, DNA replication, pyrimidine metabolism, homologous recombination, pentose phosphate pathway, phenylalanine, tyrosine and tryptophan biosynthesis, nicotinic acid and nicotinamide metabolism, phenylpropanoid biosynthesis, sulfur transport mechanism, glycerophospholipid metabolism, methane metabolism, pantothenic acid and CoA biosynthesis, selenium compound metabolism, folate biosynthesis, inositol phosphate metabolism, lysosomes, butanoic acid metabolism, biotin metabolism, phosphonic acid and phosphinic acid metabolism) were statistically significantly increased in groups B and C compared to group A (Tables 9 and 10, light gray). Conversely, nine pathways with relative abundances ≥ 0.1% (alanine, aspartate and glutamate metabolism, purine metabolism, RNA polymerase, RNA degradation, two-component systems, pyruvate metabolism, nucleotide excision repair, valine, leucine and isoleucine degradation, lysine degradation) were statistically significantly increased in group A compared to groups B and C (Tables 9 and 10, white).

[0385] Furthermore, 27 pathways (aminoacyl-tRNA biosynthesis, glycolysis / gluconeogenesis, oxidative phosphorylation, starch and sucrose metabolism, arginine and proline metabolism, galactose metabolism, amino and nucleotide sugar metabolism, peptidoglycan biosynthesis, histidine metabolism, phosphotransferase system (PTS), terpenoid backbone biosynthesis, fructose and mannose metabolism, fatty acid biosynthesis, citric acid cycle (TCA cycle), base excision repair, mismatch repair, tyrosine metabolism, glyoxylate and dicarboxylate metabolism, bacterial chemotaxis, peroxisomes, other glycan degradation, glutathione metabolism, vitamin B6 metabolism, zeatin biosynthesis, lipopolysaccharide biosynthesis, tryptophan metabolism, calcium signaling pathway) showed significant increases at 28 days of age in groups B and C compared to group A (Table 9, light grey).

[0386] Of particular interest are metabolic pathways such as fatty acid biosynthesis, the phosphotransferase system (PTS) and butanoic acid metabolism that were increased in the treatment groups and are associated with the production of SCFAs. Furthermore, statistically significant increases in the treatment groups (groups B and C) were reported for B vitamin pathways such as nicotinic acid and nicotinamide metabolism, pantothenic acid biosynthesis, folic acid biosynthesis, biotin metabolism and vitamin B6 metabolism.

[0387] [Table 11]

[0388] [Table 12]

[0389] [Table 13]

[0390] [Table 14]

[0391] [Table 15]

[0392] [Table 16]

[0393] [Evaluation of the relative abundance of genes related to SCFAs and glucosidases] Since short chain fatty acids (SCFAs) play an important role in intestinal physiology, we specifically analyzed the metabolic pathways leading to butyrate and propionate synthesis. As described in the literature, there are four potential pathways leading to butyrate production. The first major one was based on acetyl-CoA acetyltransferase (Figure 39).

[0394] Turning to the relative abundance of genes related to our butyrate production, we found that at least seven metabolic functions at 28 days of age, and five at 42 days of age, showed a statistically significant increase in the treated groups (groups B and C) compared to group A (Table 11 below). This evidence could be correlated with the symbiotic activity of different bacterial genera, such as Faecalibacterium and Eubacterium (Polansky et al., 2016; Rowland et al., 2018), and Bifidobacteria, as reported by Reviere et al., 2016. All these genera were identified in our study by microbiota analysis, and these results could predict the potential for butyrate production in animals treated with vitamin B2.

[0395] [Table 17]

[0396] Of great interest was the evidence of a statistically significant increase in the relative abundance of functional genes related to β-galactosidase in the ceca of broilers in the treatment groups (groups b and C) compared to group A (Figure 12, below). This result could be explained by an increase in Bifidobacteria and even Lactobacillus in the treatment groups, as reported in the microbiota analysis (Shaima et al., 2017). In humans, this enzyme is abundant in the colon where it promotes lactose fermentation, and measuring its activity gives an indication of the ability of the colonic microbiota to ferment intestinal lactose.

[0397] [Table 18]

[0398] [Conclusion] Both dietary regimes administered to groups B and C induced modifications in the cecal microbiota, including an increase in known anti-inflammatory and health-promoting bacteria belonging to the genus Faecalibacterium during the first period (T1) of the broiler production cycle compared to the control diet. During the second and third periods (T2 and T3) of the production cycle, the relative abundance of Faecalibacterium did not show significant differences among the three experimental groups. Taken together, our results seem to demonstrate a natural increase in Faecalibacterium during the broiler production cycle (control diet, group A) and that the dietary regimes administered to groups B and C broilers seem to hasten this increase. The diet administered to group C also induced a progressive increase in the abundance of another health-promoting group, Bifidobacterium, as well as an increase in species richness compared to the control diet (administered to group A). ​​Conversely, the diet administered to group B promoted an increase in the abundance of Bacteroides throughout the production cycle. · Increases in Bacteroidetes cause damage to another member of the phylum Bacteroides, the family Rikenellaceae (genus Alistipes). Both diets administered to broilers in groups B and C had negligible effects on the ileal microbiota composition and diversity, and no significant effects on the microbiota were detected in waste samples. As mentioned, the paucity of samples selected for metabolic studies affected the quality of the data and results. It is therefore important to have a more robust data set including analysis of all samples. However, it seems clear that metabolites such as acetate, glutamate, propionate, and butyrate are affected by the treatment. PCA performed on 63 subjects with 6361 functional characteristics showed a clear effect of vitamin B2, which caused a separation between the treatment groups (groups B and C) and the control group (group A). ​​This result was not evident as a function of the analysis time. Considering the primary KEGG metabolic pathway, the relative abundance of Genetic Information Processing and Organismal Systems increases in group A (5 ppm vitamin B2), whereas Environmental Information Processing and Metabolism increases in the treatment groups (50 and 100 ppm vitamin B2). Turning to the secondary KEGG metabolic pathways with relative abundance ≥ 0.5%, the results notably show a sizable compositional difference between the three experimental groups for each time point. In particular, the relative abundances of cofactor and vitamin metabolism, membrane transport, replication and repair, biosynthesis of other secondary metabolites, terpenoid and polyketide metabolism, and metabolism of other amino acids are increased in the treatment groups with 50 and 100 ppm vitamin B2. Conversely, translation, energy metabolism, signal transduction, transcription, infection, and environmental adaptation were statistically increased in the control group with 5 ppm vitamin B2. Turning to the tertiary KEGG metabolic pathways, no differences were reported between the experimental groups at 14 days of age, but a number of pathways showed significant differences at 28 and 42 days of age. Of particular interest, metabolic pathways such as fatty acid biosynthesis, phosphotransferase system (PTS) and butanoic acid metabolism were increased in the treatment groups (50 and 100 ppm vitamin B2) and were associated with the production of SCFAs. Furthermore, statistically significant increases in the treatment groups were also reported for B vitamin pathways, including nicotinic acid and nicotinamide metabolism, pantothenic acid biosynthesis, folic acid biosynthesis, biotin metabolism and vitamin B6 metabolism. Regarding butyrate production, at least seven metabolic functions at 28 days of age and five at 42 days of age were statistically increased in the treatment groups (50 and 100 ppm vitamin B2) compared to the control group (5 ppm vitamin B2). This evidence can be correlated with the syntrophic activity of different bacterial genera such as Faecalibacterium and Eubacterium (Zam et al., 2016; Rowland et al., 2018) and Bifidobacteria as reported by Reviere et al., 2016. All these genera were identified in our study by microbiota analysis and these results allow to predict the potential for butyrate production in animals treated with vitamin B2.

[0399] [Example 4] [Pig model] Materials and Methods Three pigs were fed a commercial diet for 4 weeks. The diet contained barley, wheat, and soybean meal as main components, and its main chemical composition was (g / kg): 151 crude protein, 34 crude fat, 40 crude fiber, 55 and ash. The animals were weighed individually at the beginning (average: 23.9 ± 1.01 Kg) and end (average: 43.3 ± 3.56 Kg) of the experimental period. After 30 days of acclimation, the animals were sacrificed. The pH of the stomach, small intestine, and hindgut contents was measured directly with a glass electrode pH meter (CRISON 507, CRISON, Barcelona, ​​Spain). The stomach and small intestine contents were immediately filtered, sampled, and then stored at -20 °C for use as digesta inoculum for the purpose of evaluation of ex vivo digestibility. As for the cecal contents, a portion was immediately sampled, dispersed in 20 ml tubes, frozen in liquid nitrogen, and then stored at −80°C for use as frozen inoculum for in vitro gas production studies, while the other portion was used directly as fresh fermentation inoculum.

[0400] [In vitro enzymatic digestion] To determine the enzymatic digestibility in the stomach and small intestine of pigs, two experiments were performed:

[0401] [A-Using exoenzymes:] Erlenmeyer flasks (100 mL total volume) containing 0.5 g of substrate alone or supplemented with the three eubiotics were incubated in duplicate for each treatment to determine in vitro enzymatic digestibility following the three-step method of Boisen and Fernandez (1997). Digestibility was as follows: [1] Pepsin [2] Pepsin + Pancreatin [3] Pepsin + Pancreatin + Viscozyme was determined using

[0402] [B. Using stomach contents and small intestine contents] A similar procedure for enzymatic digestibility was followed, except in this case stomach and small intestine contents of each animal were used instead of pepsin and pancreatin solutions, and the third step of Boisen and Fernandez (1997) was omitted. Digestibility was calculated as follows: Stomach contents (9mL) Stomach contents + small intestine contents (9mL) was determined using

[0403] [In vitro gas generation] The microbial fermentation pattern of the cecal contents was studied in vitro by the gas production method (Theodorou et al., 1994. Anim Feed Sci Technol 48:185-1-97). To simulate the digestive process occurring before the cecum, a 0.8 g amount of predigested feed (successively incubated in pepsin-HCl and pancreatin incubations according to the method of Boisen and Fernandez, 1997 Anim Feed Sci Technol 51:29-43) was used as substrate, alone or supplemented with vitamin B2. First, three identical glass bottles (total volume 110 mL) for each treatment were filled with 56 mL of incubation solution. The cecal contents of each of the three pigs for each treatment were mixed 2:1 with the incubation solution under a CO2 atmosphere and homogenized for a few minutes with a Waring blender (cecal contents contain 0.20 of the total incubation solution). Then, 24 mL of the mixture was added to each bottle to achieve a total liquid volume of 80 mL. In addition, three identical bottles without substrate were added as blanks. The bottles were sealed under CO2 flow and incubated at 39 °C for 12 h. Pressure was recorded every 2 h throughout the entire incubation period using a HD2124.02 manometer (Delta Ohm, Caselle di Selvazzano, Italy) fitted with a TP804 manometer. Readings were converted to volume by a linear regression equation previously established between pressures recorded in the same bottle under the same conditions and known air volumes (n = 103; R2 = 0.996). Gas volumes recorded over each incubation time were expressed per unit of organic matter (OM) incubated. Methane (CH4) concentrations were measured every 6 h. At 6 and 12 h incubation, samples were withdrawn and either acidified 1:1 with 0.1 N HCl or added to 0.5 ml of a deproteinized mixture of 0.5 M PO4H3 containing 2 mg / ml 4-methylvaleric acid, which were stored at -20°C for determination of ammonia and volatile fatty acid (VFA) concentrations, respectively. The procedure for in vitro gas production was the same for both fresh and frozen inocula, with the only difference being that frozen samples of cecal contents were kept at 38°C for 5 min prior to thawing.

[0404] For gas production, results were analyzed for each incubation time as a split-plot, considering donor pigs as blocks, contents (fresh vs. frozen) as main plots and treatments as subplots. Using the same split-plot method, results obtained from digesta contents (stomach vs. small intestine) were used as main plots. In vitro enzyme results were not statistically analyzed as there was no experimental source of variation. Differences were considered significant when P<0.05, trends towards significance were considered when 0.05 £P<0.10 and, if significant, differences were compared by Tukey's t-test at P<0.05.

[0405] [result] [Ex vivo and enzymatic digestion] In the case of gastric contents, substrate digestibility was found to be highest for vitamin B2 compared to the control (P<0.05). These differences were minimal after digestion of small intestinal contents, and no differences were recorded between treatments (P>0.05).

[0406] The substrate digestibility results observed with exogenous enzymes were consistent with those recorded for digesta contents, although the extent of pepsin-HCl digestion was somewhat lower than that for stomach contents. Thus, the digestibility recorded for the substrate supplemented with vitamin B2 when incubated in pepsin-HCl was higher than that of the other treatments. However, no differences were observed between the treatments when they were incubated in (P+pancreatin) and / or (P+P+viscozyme). The results indicate that the two methods are valid for assessing enzymatic digestion in pigs.

[0407] [In vitro gas generation] In general, the gas production patterns from treatments incubated with fresh cecal contents did not differ from those recorded for frozen cecal contents (P>0.05); however, after 4 h of incubation, the gas volumes recorded with fresh contents tended to be higher (P=0.081). In general, the gas volumes recorded for vitamin B2 were higher than the control after 4, 10, and 12 h of incubation (P<0.05).

[0408] The interaction between storage type and treatment throughout the incubation period did not reach significance (P<0.05), indicating that the treatments behaved similarly despite freezing of the cecal contents.

[0409] [Methane production] With regard to CH4 production, no differences were recorded between fresh and frozen inocula over the 12-h incubation (P>0.05). By 12-h incubation, no differences were recorded between treatments with regard to CH4 production of total gases (P>0.05), although numerically the lowest CH4 was recorded for vitamin B2. Similarly, when CH4 production was expressed as volume per unit of incubation substrate, no significant differences were recorded between treatments, although ranking of the treatments revealed that vitamin B2 recorded the lowest CH4 accumulation after 6 and 12-h incubation.

[0410] As vitamin B2 is known to play an important role in the production of energy and aids in carbohydrate breakdown, the results observed with vitamin B2 imply that this vitamin may have a positive effect on the activity of hindgut bacterial species. In addition, the reduction in methane production may also be explained by vitamin B2 being able to suppress the activity of methanogen or archaeal populations.

[0411] [Total volatile fatty acid concentration (TVFA), molar ratio of VFA, and ammonia production] No differences were recorded for the preservation of the cecal inoculum or between treatments, either in terms of total VFAs, molar ratios of VFAs, or ammonia concentrations, indicating that there was no significant promotion of the cecal fermentation profile by the experimental treatments tested.

[0412] In conclusion, vitamin B2 clearly exerted a positive effect both at the gastric level (probably stimulating pepsin activity) and at the hindgut level (enhancing microbial activity).

Claims

1. 1. An active agent selected from the group consisting of riboflavin, vitamin A, vitamin C, vitamin D, vitamin E, vitamin K, folic acid, beta-carotene, vitamin B1, niacin, vitamin B5, vitamin B6, biotin, vitamin B12, omega-3 fatty acids and combinations thereof for use in improving intestinal health in an animal, including a human, said improvement comprising: i. increasing the concentration of at least one short chain fatty acid or salt thereof in the intestinal tract; ii. increasing the microbiota diversity in the intestinal tract; iii. increasing the abundance of beneficial bacteria in the intestinal tract; iv. promotion or increase of the butyric acid synthesis pathway in the intestinal tract; v. improving the barrier function of the intestinal tract; vi. A decrease in the redox potential of the intestinal tract; vii. reducing the amount of gas produced in the intestinal tract; and / or viii. Stimulation of the intestinal immune response comprising or consisting of wherein said use comprises administering to said animal a) a formulation comprising an effective amount of an active agent, the active agent being delivered to the large intestine; or b) a supraphysiological dose of the active ingredient such that at least an effective amount of the active agent is present in the intestinal tract of the animal; administering However, when niacin is present, it is not the only active substance; when riboflavin is present, it is an active substance that has ameliorative effects other than increasing the amount of Faecalibacterium prausnitzii.

2. The active agent of claim 1 , wherein the animal is a human and the effective amount of the active agent is delivered in a delayed release formulation.

3. 3. The active substance according to claim 1 or 2, wherein the improvement comprises an increase in the concentration of at least one short chain fatty acid or salt thereof in the intestinal tract; or a promotion or increase in the butyric acid synthesis pathway in the intestinal tract; wherein the short chain fatty acid is selected from the group consisting of acetic acid, propionic acid and butyric acid or salts thereof.

4. 4. The active of claim 3, wherein the active is selected from the group consisting of vitamin A, beta-carotene, vitamin C, riboflavin, vitamin D, vitamin E, vitamin K, folic acid, omega-3 fatty acids, and combinations thereof.

5. 5. The active substance according to claim 3 or 4, wherein the animal, including a human, is experiencing a condition selected from the group consisting of metabolic disorders, type 2 diabetes, obesity, chronic inflammation, and atherogenesis.

6. 3. The active substance according to claim 1 or 2, wherein the improvement comprises an increase in the microbiota diversity in the intestinal tract.

7. The active substance according to claim 6 , wherein the microbiota diversity is increased and / or the abundance of beneficial bacteria is increased in the colon.

8. The beneficial bacteria to be increased are selected from the group consisting of Acidaminococcus, Akkermansia, Bacteroides ovatus, Bifidobacterium, Blautia producta, Clostridium cocleatum, Collinsella aerofaciens, Dorea longicatena, Escherichia coli, and Eubacterium. spp.), Faecalibacterium prausnitzii, Lachnospira pectinoshiza, Lactobacillus spp., Parabacteroides distasonis, Raoultella spp., Roseburia spp., Ruminococcus spp., Streptococcus 8. The active substance according to claim 6 or 7, which is selected from the group consisting of:

9. The active substance according to any one of claims 6 to 8, wherein the bacteria to be increased are selected from the group consisting of Bifidobacterium, Akkermansia, Faecalibacterium and Bacteroides.

10. 10. The active substance according to any one of claims 6 to 9, wherein the animal, including a human, is experiencing a condition selected from the group consisting of irritable bowel syndrome, metabolic disorders, obesity, and inflammatory disorders.

11. 11. The active substance according to any one of claims 6 to 10, wherein the substance is selected from the group consisting of vitamin B1, vitamin B2, vitamin B5, vitamin B6, vitamin B12, β-carotene, biotin, vitamin C, vitamin E, vitamin D, folic acid, vitamin K, omega-3 fatty acids, and combinations thereof.

12. 3. The active substance according to claim 1 or 2, wherein the improvement comprises an improvement in the barrier function of the intestinal tract.

13. 13. The active agent of claim 12, wherein the animal, including a human, is experiencing a condition selected from the group consisting of irritable bowel syndrome, Crohn's disease, ulcerative colitis, leaky gut, and malnutrition.

14. 14. The active substance according to claim 12 or 13, wherein the active substance is selected from the group consisting of vitamin B2, vitamin C, vitamin E, omega-3 fatty acids, vitamin D, vitamin A, folic acid, vitamin K and mixtures thereof.

15. 3. The method of claim 1, wherein the improvement comprises a decrease in the redox potential of the intestinal tract.

16. The active substance of claim 15, wherein the reduction in the redox potential results in a benefit selected from the group consisting of promoting the growth of obligate anaerobic beneficial bacteria in the colon; reducing aerobic pathogenic organisms in the intestinal tract; reducing plasma free thiols; reducing inflammation in the intestinal tract; and reducing cellular damage in the intestinal tract caused by inflammation in the intestinal tract.

17. 17. The active substance according to claim 15 or 16, wherein the active substance is selected from the group consisting of vitamin C, vitamin E and mixtures thereof.

18. 3. The method of claim 1, wherein the improvement is a reduction in the amount of gas produced in the intestinal tract.

19. 20. The active agent of claim 18, wherein the active agent is vitamin B2, vitamin C, vitamin E, and combinations thereof.

20. 3. The method according to claim 1, wherein the improvement is a stimulation of the immune response of the intestinal tract.

21. 21. The method of claim 20, wherein the immune response is an increase in an immune response molecule selected from the group consisting of GROa-CXCL1, MIP3A-CCL20, interleukin 8, and combinations thereof.

22. 22. The active substance according to claim 20 or 21, wherein the active substance is selected from the group consisting of vitamin B2, vitamin E, vitamin D, vitamin A, and vitamin K, and mixtures thereof.

23. 1. A method for improving intestinal health in an animal, including a human, comprising: i. increasing the concentration of at least one short chain fatty acid or salt thereof in the intestinal tract; ii. increasing the microbiota diversity in the intestinal tract; iii. increasing the abundance of beneficial bacteria in the intestinal tract; iv. promotion or increase of the butyric acid synthesis pathway in the intestinal tract; v. improving the barrier function of the intestinal tract; vi. A decrease in the redox potential of the intestinal tract; vii. reducing the amount of gas produced in the intestinal tract; and / or viii. Stimulation of the intestinal immune response comprising or consisting of This includes administering to the animal an active agent selected from the group consisting of riboflavin, vitamin A, vitamin C, vitamin D, vitamin E, vitamin K, folic acid, beta-carotene, vitamin B1, niacin, vitamin B5, vitamin B6, biotin, vitamin B12, omega-3 fatty acids, and combinations thereof, wherein the active agent is a) a formulation comprising an effective amount of the active agent, wherein the active agent is delivered to the large intestine; or b) a supraphysiological dose of the active ingredient such that at least an effective amount of the active agent is present in the intestinal tract of the animal. Present in; A method wherein, however, niacin, when present, is not the only active substance; and riboflavin, when present, has an improving effect other than increasing the amount of Faecalibacterium prausnitzii.

24. 24. The method of claim 23, wherein the animal is a human and the active agent is delivered to the intestinal tract.

25. 24. The method of claim 23, wherein the active agent is present in a supraphysiological dose.

26. 24. The method of claim 23, wherein the improvement is an increase in at least one short chain fatty acid and / or an increase in the butyrate synthesis pathway activity in the animal.

27. 27. The method of claim 26, wherein the animal is experiencing a condition selected from the group consisting of metabolic disease, type 2 diabetes, obesity, chronic inflammation, and atherogenesis.

28. 27. The method of claim 26, comprising administering at least one active agent selected from the group consisting of vitamin A, vitamin C, riboflavin, vitamin E, folic acid, beta-carotene, omega-3 fatty acids, and combinations thereof.

29. 24. The method of claim 23, wherein the improvement is an increase in microbiota diversity in the intestinal tract or an increase in the abundance of beneficial bacteria in the intestinal tract.

30. 30. The method of claim 29, wherein the active agent is selected from the group consisting of vitamin B1, riboflavin, vitamin B5, vitamin B12, beta-carotene, biotin, vitamin C, vitamin E, vitamin D, folic acid, vitamin K, omega-3 fatty acids, and combinations thereof; with the proviso that when riboflavin is the active agent, it is present for a purpose other than increasing the abundance of Faecalibacterium prausnitzii.

31. 30. The method of claim 29, wherein the active agent is selected from the group consisting of vitamin B1, riboflavin, vitamin B5, vitamin B12, beta-carotene, biotin, vitamin C, vitamin E, vitamin D, folic acid, vitamin K, omega-3 fatty acids, and combinations thereof; with the proviso that when riboflavin is the active agent, it is present for a purpose other than increasing the abundance of F. prausnitzii.

32. 24. The method of claim 23, wherein the improvement is stimulation of an immune response in the intestinal tract.

33. 33. The method of claim 32, wherein the animal, including a human, is experiencing acute or chronic inflammation.

34. 34. The method of claim 33, wherein the active agent is selected from the group consisting of riboflavin, vitamin E, vitamin A, vitamin K, and combinations thereof.

35. The improvement is a decrease in the redox potential of the intestinal tract, thereby promoting the growth of obligate anaerobic beneficial bacteria in the colon; reduction of aerotolerant pathogenic organisms in the intestinal tract; reducing inflammation in the intestinal tract; and Reducing cellular damage in the intestinal tract caused by inflammation in the intestinal tract 33. The method of claim 32, wherein the method provides a benefit selected from the group consisting of:

36. 36. The method of claim 35, wherein the substance is selected from the group consisting of riboflavin, vitamin C, vitamin E, and mixtures thereof.

37. 24. The method of claim 23, wherein the amelioration is treatment, prevention, or reduction in the amount of gas produced in the intestinal tract.

38. 38. The method of claim 23 or 37, wherein the active agent is selected from the group consisting of vitamin B2, vitamin C, and combinations thereof.

39. 24. The method of claim 23, wherein the improvement is an improvement in intestinal barrier function.

40. 40. The method of claim 39, wherein the improved barrier function is a method of treating, preventing or alleviating the symptoms of a condition selected from the group consisting of irritable bowel syndrome, Crohn's disease, ulcerative colitis, leaky gut and malnutrition.

41. 41. The method of claim 40, wherein the substance is selected from the group consisting of riboflavin, vitamin C, vitamin E, omega-3 fatty acids, vitamin D, vitamin A, folic acid, vitamin K, and mixtures thereof.

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