Formulations for improving intestinal health

Abstract

The present application relates to formulations for improving intestinal health. Various benefits are created for intestinal health by delivering active agents to the colons of animals, including humans. The active ingredients comprise riboflavin, vitamin A, vitamin C, vitamin D, vitamin E, vitamin K, folic acid, beta-carotene, vitamin B1, nicotinic acid, vitamin B5, vitamin B6, biotin, vitamin B12, omega-3 fatty acid and a combination thereof. The benefits include increasing the concentration of at least one short-chain fatty acid and salts thereof in the intestine, increasing the diversity of microbiome in the intestine, increasing beneficial bacteria in the intestine, increasing the butyrate synthesis pathway in the intestine, improving the intestinal barrier function, reducing the redox potential of the intestine, and reducing the amount of gas generated in the intestine; and stimulating an intestinal immune response.

Description

[0001] This application is a divisional application of the Chinese patent application with application number 201980056670.6 and titled “Preparations for Improving Intestinal Health” filed by the applicant DSM Intellectual Property Asset Management Co., Ltd. on August 28, 2019. Technical Field

[0002] The present invention relates to preparations for improving intestinal health in animals and humans. In particular, the preparations of the present invention can be used to increase the concentration of short-chain fatty acids (SCFA) in the lower intestinal tract, and / or to increase the butyrate synthesis pathway in the lower intestinal tract. The preparations of the present invention can also be used to increase or regulate the microbiome diversity and / or abundance of beneficial bacteria (such as Bifidobacterium, Akkermansia and Faecalibacterium) in the lower intestinal tract. The present invention further relates to methods and compositions for improving intestinal barrier function and stimulating intestinal immune responses. Background Art

[0003] The microbiota of humans and animals is a collection of microorganisms living on and in the human / animal body, with the largest and most diverse microbial clusters living in the intestine. The intestinal microbiota co-evolves with the host, providing a stable environment for microorganisms, while microorganisms provide a wide range of functions for the host, such as digesting a large number of nutrients from a complex diet, producing nutrients and vitamins, resisting pathogens, and maintaining the immune system. Existing data show that abnormal composition of intestinal microbiota is associated with a variety of diseases, including decreased immunity, allergies, metabolic disorders (such as type 2 diabetes mellitus), and inflammatory bowel disease (IBD). One of the mechanisms by which microbiota affects human health and disease is its ability to produce harmful metabolites associated with disease development or beneficial metabolites to prevent disease. Dietary fiber, as well as proteins and peptides are metabolized by microbiota in the cecum and colon, and proteins and peptides cannot be digested by host enzymes in the upper digestive tract. The main products of microbial fermentation activities in the intestine are SCFA, especially acetate, propionate, and butyrate. There is increasing evidence that SCFAs in the human or animal intestine have beneficial effects on host metabolic properties such as glucose metabolism, the immune system, gastrointestinal integrity and barrier function, and overall intestinal health (including gastrointestinal motility).

[0004] WO 2007 / 058523 A1 describes the use of n-3 docosapentaenoic acid for the preparation of a pharmaceutical composition for improving intestinal barrier integrity, improving barrier function, stimulating intestinal maturation and / or reducing intestinal barrier permeability.

[0005] WO 2016 / 053085 A1 describes a composition comprising a uridine source and butyrate-producing fibers for preventing or treating constipation, transit time dysfunction or abnormal colon length.

[0006] US 2010 / 247489A1 discloses the use of a delayed release composition comprising specific minerals such as tungsten for reducing gas formation in the colon. The composition may further comprise vitamins or strains of acetogenic or butyric acid producing bacteria.

[0007] Tan et al. (2016) Cell Reports, Vol. 15, No. 12, 2809-2824 investigated the beneficial effects of dietary fiber on peanut allergy in mice. Tan et al. reported that this effect involved remodeling of the gut microbiota as well as increased levels of short-chain fatty acids and activity of the receptors GPR43 and GPR109a.

[0008] WO 2010 / 117274 A1 describes carbohydrates for enhancing the production of C5 and / or C6 short-chain fatty acids.

[0009] WO 2017 / 182347 A1 describes microcapsules comprising a core containing vitamin B3, characterized in that the coating layer system comprises two layers of shellac and a pH regulating substance provided between the two layers of shellac.

[0010] US 9,433,583 B2 describes a colon-targeted single dosage form comprising vitamin D and optionally other vitamins for the prevention of colorectal adenomatous polyps and colorectal cancer.

[0011] WO 2014 / 070014 describes the use of riboflavin (vitamin B2) for stimulating the population of Faecalibacterium prausnitzii. Summary of the invention

[0012] The inventors of the present application have found that in the presence of certain micronutrients, the intestinal microbiota produces increased amounts of SCFAs. In addition, an increase in the butyrate synthesis pathway was also observed. In further studies, it has been determined that micronutrients can also increase microbiome diversity and microbial abundance in the intestine, improve the barrier function of the intestine, and reduce gas production. Therefore, the present invention relates to the following items [1] to

[67] :

[0013] [1] An active agent for use as defined in claim 1; or

[0014] An active agent for use in improving intestinal health in animals, the active agent being selected from 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 comprises or consists of:

[0015] (i) increasing the concentration of at least one short-chain fatty acid or a salt thereof in the intestine,

[0016] (ii) increase the diversity of the microbiome in the intestine,

[0017] (iii) increase the abundance of microorganisms in the intestine,

[0018] (iv) promoting or increasing the butyrate synthesis pathway in the intestine,

[0019] (v) Improve intestinal barrier function,

[0020] (vi) reduce the redox potential of the intestine,

[0021] (vii) reduce the amount of gas produced in the intestines, and / or

[0022] (viii) stimulate intestinal immune response;

[0023] The use comprises administering to an animal a delayed release formulation comprising an effective dose of an active agent, wherein the release of the active agent is delayed until the intestine, preferably the large intestine.

[0024] [2] An active agent for use according to item [1], wherein the improvement includes or consists of: increasing the concentration of at least one short-chain fatty acid or a salt thereof in the intestine, and wherein the at least one short-chain fatty acid is selected from butyric acid, acetic acid, propionic acid and a combination thereof.

[0025] [3] The active agent for use according to item [2], wherein the at least one short-chain fatty acid is butyric acid.

[0026] [4] The active agent for use according to item [2], wherein the at least one short-chain fatty acid is acetic acid.

[0027] [5] The active agent for use according to item [2], wherein the at least one short-chain fatty acid is propionic acid.

[0028] [6] An active agent for use according to any of the preceding claims, wherein the use comprises or consists of: treating or preventing a condition associated with low concentrations of one or more SCFAs in the lower intestinal tract.

[0029] [7] An active agent for use according to item [6], wherein the disorder associated with low concentrations of one or more SCFAs in the lower intestinal tract is an inflammatory disorder.

[0030] [8] The active agent for use according to item [7], wherein the inflammatory disorder is characterized by chronic inflammation.

[0031] [9] The active agent for use according to item [6], wherein the condition associated with low concentrations of one or more SCFAs in the lower intestinal tract is atherogenesis.

[0032]

[10] An active agent for use according to item [6], wherein the condition associated with low concentrations of one or more SCFAs in the lower intestinal tract is a metabolic disorder.

[0033]

[11] The active agent for use according to item

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

[0034]

[12] An active agent for use according to item [1], wherein the improvement includes or consists of: increasing the diversity of the microbiome in the intestine.

[0035]

[13] An active agent for use according to item

[12] , wherein the use comprises or consists of: treating or preventing a condition associated with insufficient diversity of the microbiome in the intestine.

[0036]

[14] The active agent for use according to item [1], wherein the improvement comprises or consists of: increasing the abundance of at least one microorganism selected from Bifidobacterium, Akkermansia and Faecalibacterium in the human intestine.

[0037]

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

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

[0038]

[16] An active agent for use according to item [1], wherein the improvement comprises or consists of: increasing the abundance of at least one microorganism selected from Bacteroides, Alisebenia and Faecalibacterium.

[0039]

[17] An active agent for use according to item [1], wherein the improvement includes or consists of: promoting or increasing the butyrate synthesis pathway in the intestine.

[0040]

[18] An active agent for use according to item

[17] , wherein the use comprises or consists of the following: treating or preventing a condition associated with insufficient butyrate synthesis in the intestine.

[0041]

[19] An active agent for use in treating or preventing conditions associated with impaired intestinal barrier function in animals (preferably mammals), the active agent being selected from 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, omega-3 fatty acids and combinations thereof, wherein the use comprises administering to the animal a delayed-release formulation comprising an effective dose of the active agent, wherein the release of the active agent is delayed until the large intestine.

[0042]

[20] The active agent for use according to item

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

[0043]

[21] The active agent for use according to item

[19] or

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

[0044]

[22] The active agent for use according to item

[19] or

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

[0045]

[23] The active agent for use according to Item

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

[0046]

[24] The active agent for use according to Item

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

[0047]

[25] The active agent for use according to item

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

[0048]

[26] The active agent for use according to Item

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

[0049]

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

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

[0050]

[28] An active agent for use according to any one of items [1] to

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

[0051]

[29] An active agent for use according to any one of items [1] to

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

[0052]

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

[29] , wherein the active agent consists of riboflavin.

[0053]

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

[29] , wherein the active agent consists of vitamin A.

[0054]

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

[29] , wherein the active agent consists of vitamin C.

[0055]

[33] An active agent for use according to any one of items [1] to

[29] , wherein the active agent consists of vitamin D.

[0056]

[34] An active agent for use according to any one of items [1] to

[29] , wherein the active agent consists of vitamin E.

[0057]

[35] An active agent for use according to any one of items [1] to

[29] , wherein the active agent consists of vitamin K.

[0058]

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

[29] , wherein the active agent consists of folic acid.

[0059]

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

[29] , wherein the active agent consists of β-carotene.

[0060]

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

[29] , wherein the active agent consists of vitamin B1.

[0061]

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

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

[0062]

[40] An active agent for use according to any one of items [1] to

[29] , wherein the active agent consists of vitamin B5.

[0063]

[41] An active agent for use according to any one of items [1] to

[29] , wherein the active agent consists of vitamin B6.

[0064]

[42] An active agent for use according to any one of items [1] to

[29] , wherein the active agent consists of biotin.

[0065]

[43] An active agent for use according to any one of items [1] to

[29] , wherein the active agent consists of vitamin B12.

[0066]

[44] An active agent for use according to any one of items [1] to

[29] , wherein the active agent consists of at least one ω-3 fatty acid.

[0067]

[45] The active agent for use according to item

[44] , wherein the active agent consists of docosahexaenoic acid (DHA), eicosapentaenoic acid (EPA) or a combination thereof.

[0068]

[46] The active agent for use according to item

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

[0069]

[47] An active agent for use according to item

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

[0070]

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

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

[0071]

[49] An active agent for use according to any one of items [1] to

[29] , wherein the active agent is composed 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.

[0072]

[50] An active agent for use according to any of the preceding items, wherein the active agent is administered to an animal, preferably a mammal, at a daily dose of up to 2.9 mg riboflavin / kg body weight, up to 42.9 μg vitamin A / kg body weight, up to 3.6 μg vitamin D / kg body weight, up to 28.6 mg vitamin C / kg body weight, up to 14.3 mg vitamin E / kg body weight, up to 143 μg vitamin K / kg body weight, up to 14.3 μg folic acid / kg body weight, up to 0.4 mg β-carotene, 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 ω-3 fatty acids / kg body weight or a combination thereof, wherein preferably EPA is up to 25.7 mg / kg body weight and DHA is up to 14.3 mg / kg body weight.

[0073]

[51] A delayed release formulation for delivering an active agent to the intestine, the formulation comprising the active agent and an enteric layer or coating, wherein the active agent consists 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 a combination thereof, wherein preferably EPA is up to 1800 mg and DHA is up to 1000 mg, and wherein the release of the active agent is delayed until the intestine.

[0074]

[52] A delayed release formulation for delivering an active agent to the intestine, the formulation comprising an active agent and an enteric layer or coating, wherein the active agent is composed of 1-85 mg riboflavin, 0.2-0.4 mg vitamin A, 400-600 mg vitamin C, 5-80 μg vitamin D, 80-120 μg vitamin E, 80-140 μg vitamin K, 0.3-0.5 mg folic acid, 80-120 mg ω-3 fatty acids or a combination thereof, and wherein the release of the active agent is delayed until the intestine.

[0075]

[53] The delayed-release formulation of item

[51] or

[52] , wherein the active agent is composed 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 ω-3 fatty acids or a combination thereof.

[0076]

[54] The composition of any one of items

[51] to

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

[0077]

[55] Non-therapeutic use of an active agent selected from 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, omega-3 fatty acids and combinations thereof for improving the condition or health of the intestine of an animal (preferably a mammal, a monogastric animal or poultry not suffering from any intestinal disorder or pathology).

[0078]

[56] The non-therapeutic use of

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

[0079]

[57] The non-therapeutic use of

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

[0080]

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

[55] to

[57] , wherein the improvement includes or consists of: increasing the concentration of at least one short-chain fatty acid or a salt thereof in the intestine.

[0081]

[59] The non-therapeutic use of any one of

[55] to

[57] , wherein the improvement includes or consists of: increasing the diversity of the microbiome in the intestine.

[0082]

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

[55] to

[57] , wherein the improvement comprises or consists of: increasing the abundance of Bifidobacterium, Akkermansia and / or Faecalibacterium in the intestine.

[0083]

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

[55] to

[57] , wherein the improvement includes or consists of: promoting or increasing the butyrate synthesis pathway in the intestine.

[0084]

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

[55] to

[57] , wherein the improvement comprises or consists of: improving intestinal barrier function, wherein the active agent does not contain omega-3 fatty acids.

[0085]

[63] The non-therapeutic use of any one of

[55] to

[62] , wherein the active agent does not contain vitamin D3.

[0086]

[64] The non-therapeutic use of any one of

[55] to

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

[0087]

[65] Non-therapeutic use of an active agent for maintaining or improving intestinal function in a healthy mammal, wherein the active agent is selected from 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.

[0088]

[66] A method for improving intestinal health in an animal, wherein the improvement comprises or consists of:

[0089] (i) increasing the concentration of at least one short-chain fatty acid or a salt thereof in the intestine,

[0090] (ii) increasing the diversity of the microbiome in the intestine,

[0091] (iii) increasing the abundance of microorganisms in the intestine,

[0092] (iv) promoting or increasing the butyrate synthesis pathway in the intestine,

[0093] (v) Improve intestinal barrier function,

[0094] (vi) reduce the redox potential of the intestine, and / or

[0095] (vii) reduce the amount of gas produced in the intestines;

[0096] comprising administering to an animal a delayed release formulation comprising an effective dose of an active agent, wherein the release of the active agent is delayed until the intestine, or a feed comprising a supraphysiological dose of the active agent and present in the intestine in an effective amount;

[0097] And wherein the active agent is selected from 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.

[0098]

[67] A poultry or non-human feed comprising a supraphysiological dose of an active agent selected from 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 intestine.

[0099] The present invention also relates to the following embodiments:

[0100] (1) An active agent for use in increasing the concentration of at least one short-chain fatty acid (SCFA) or a salt thereof in the intestine of a mammal, the active agent being selected from 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, omega-3 fatty acids and combinations thereof, wherein the use comprises administering to the mammal a delayed-release formulation comprising an effective dose of the active agent, wherein the release of the active agent is delayed until the intestine.

[0101] (2) The active agent for use according to embodiment (1), wherein the at least one short-chain fatty acid is selected from butyric acid, acetic acid, propionic acid and a combination thereof.

[0102] (3) The active agent for use according to embodiment (2), wherein the at least one short-chain fatty acid is butyric acid.

[0103] (4) The active agent for use according to embodiment (2), wherein the at least one short-chain fatty acid is acetic acid.

[0104] (5) The active agent for use according to embodiment (2), wherein the at least one short-chain fatty acid is propionic acid.

[0105] (6) An active agent for use according to any one of the preceding embodiments, wherein the use comprises or consists of treating or preventing a condition associated with low concentrations of one or more SCFAs in the lower intestinal tract.

[0106] (7) The active agent for use according to embodiment (6), wherein the disorder associated with low concentrations of one or more SCFAs in the lower intestinal tract is an inflammatory disorder.

[0107] (8) The active agent for use according to embodiment (7), wherein the inflammatory disorder is characterized by chronic inflammation.

[0108] (9) The active agent for use according to embodiment (6), wherein the disorder associated with low concentrations of one or more SCFAs in the lower intestinal tract is atherogenesis.

[0109] (10) The active agent for use according to embodiment (6), wherein the disorder associated with low concentration of one or more SCFAs in the lower intestinal tract is a metabolic disorder.

[0110] (11) An active agent for use in treating or preventing a condition associated with impaired intestinal barrier function in a mammal, the active agent being selected from riboflavin, vitamin A, vitamin C, vitamin D, vitamin E, vitamin K, folic acid, omega-3 fatty acids, and combinations thereof, wherein the use comprises administering to the mammal a delayed-release formulation comprising an effective dose of the active agent, wherein the release of the active agent is delayed until the intestinal tract.

[0111] (12) The active agent for use according to embodiment (12), wherein the disorder associated with impaired intestinal barrier function in a mammal is characterized by leaky gut.

[0112] (13) The active agent for use according to embodiment (12) or (13), wherein the disorder associated with impaired intestinal barrier function is caused by malnutrition.

[0113] (14) The active agent for use according to embodiment (12) or (13), wherein the disorder associated with impaired intestinal barrier function in mammals is inflammatory bowel disease.

[0114] (15) The active agent for use according to embodiment (15), wherein the inflammatory bowel disease is Crohn's disease.

[0115] (16) The active agent for use according to embodiment (15), wherein the inflammatory bowel disease is ulcerative colitis.

[0116] (17) The active agent for use according to any one of embodiments (1) to (17), wherein the delayed-release preparation is a sustained-release preparation.

[0117] (18) The active agent for use according to any one of embodiments (1) to (17), wherein the release of the active agent is delayed until the large intestine.

[0118] (19) An active agent for use according to any one of embodiments (1) to (17), wherein the release of the active agent is delayed until the colon.

[0119] (20) An active agent for use according to any one of the preceding embodiments, wherein the active agent consists of riboflavin.

[0120] (21) An active agent for use according to any one of the preceding embodiments, wherein the active agent consists of vitamin A.

[0121] (22) An active agent for use according to any one of the preceding embodiments, wherein the active agent consists of vitamin C.

[0122] (23) An active agent for use according to any one of the preceding embodiments, wherein the active agent consists of vitamin D.

[0123] (24) An active agent for use according to any one of the preceding embodiments, wherein the active agent consists of vitamin E.

[0124] (25) An active agent for use according to any one of the preceding embodiments, wherein the active agent consists of vitamin K.

[0125] (26) An active agent for use according to any one of the preceding embodiments, wherein the active agent consists of folic acid.

[0126] (27) An active agent for use according to any one of the preceding embodiments, wherein the active agent consists of at least one ω-3 fatty acid.

[0127] (28) The active agent for use according to any one of the preceding embodiments, wherein the active agent consists of docosahexaenoic acid (DHA), eicosapentaenoic acid (EPA) or a combination thereof.

[0128] (29) An active agent for use according to any one of the preceding embodiments, wherein the active agent consists of DHA.

[0129] (30) An active agent for use according to any one of the preceding embodiments, wherein the active agent consists of EPA.

[0130] (31) An active agent for use according to any one of the preceding embodiments, wherein the active agent consists of a combination of DHA and EPA.

[0131] (32) An active agent for use according to any one of the preceding embodiments, wherein the active agent 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.

[0132] (33) An active agent for use according to any of the preceding embodiments, wherein the active agent is administered to a mammal at a daily dose of 0.01-1.2 mg riboflavin / kg body weight, 2.9-5.7 μg vitamin A / kg body weight, 0.1-1.1 μg vitamin D / kg body weight, 0.2-1.7 mg vitamin E / kg body weight, 1.1-2.0 μg vitamin K / kg body weight, 4.3-7.1 μg folic acid / kg body weight, 1.1-1.7 mg ω-3 fatty acids / kg body weight, or a combination thereof.

[0133] (34) An active agent for use according to any of the preceding embodiments, wherein the active agent is administered to a mammal at a daily dose of 1.0-1.1 mg riboflavin / kg body weight, 3.6-5.0 μg vitamin A / kg body weight, 0.2-0.4 μg vitamin D / kg body weight, 1.3-1.6 mg vitamin E / kg body weight, 1.4-1.7 μg vitamin K / kg body weight, 5.0-6.4 μg folic acid / kg body weight, 1.3-1.6 mg ω-3 fatty acids / kg body weight, or a combination thereof.

[0134] (35) A delayed-release formulation for delivering an active agent to the intestine, the formulation comprising an active agent and an enteric coating, wherein the active agent is composed 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 a combination thereof, and wherein the release of the active agent is delayed until the intestine.

[0135] (36) The delayed-release formulation of embodiment (36), wherein the active agent is composed 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 ω-3 fatty acids or a combination thereof.

[0136] (37) The delayed-release preparation of embodiment (36) or (37), wherein the delayed-release preparation is a sustained-release preparation.

[0137] (38) The delayed-release formulation of embodiment (36) or (37), wherein the release of the active agent is delayed until the large intestine, preferably in the colon.

[0138] (39) The delayed-release formulation of embodiment (36) or (37), wherein the release of the active agent is delayed until the colon.

[0139] (40) The delayed-release formulation of any one of embodiments (36) to (40), comprising riboflavin as the only active agent.

[0140] (41) The delayed-release formulation of any one of embodiments (36) to (40), comprising vitamin A as the only active agent.

[0141] (42) The delayed-release formulation of any one of embodiments (36) to (40), comprising vitamin C as the only active agent.

[0142] (43) The delayed-release formulation of any one of embodiments (36) to (40), comprising vitamin D as the only active agent.

[0143] (44) The delayed-release formulation of any one of embodiments (36) to (40), comprising vitamin E as the only active agent.

[0144] (45) The delayed-release formulation of any one of embodiments (36) to (40), comprising vitamin K as the only active agent.

[0145] (46) The delayed-release formulation of any one of embodiments (36) to (40), comprising folic acid as the only active agent.

[0146] (47) The delayed-release formulation of any one of embodiments (36) to (40), comprising β-carotene as the only active agent.

[0147] (48) The delayed-release formulation of any one of embodiments (36) to (40), comprising vitamin B1 as the only active agent.

[0148] (49) The delayed-release formulation of any one of embodiments (36) to (40), comprising niacin as the only active agent.

[0149] (50) The delayed-release formulation of any one of embodiments (36) to (40), comprising vitamin B5 as the only active agent.

[0150] (51) The delayed-release formulation of any one of embodiments (36) to (40), comprising vitamin B6 as the only active agent.

[0151] (52) The delayed-release formulation of any one of embodiments (36) to (40), comprising biotin as the only active agent.

[0152] (53) The delayed-release formulation of any one of embodiments (36) to (40), comprising vitamin B12 as the only active agent.

[0153] (54) The delayed-release formulation of any one of embodiments (36) to (40), comprising one or more ω-3 fatty acids as the only active agent.

[0154] (55) Non-therapeutic use of an active agent selected from 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, for increasing the concentration of at least one short-chain fatty acid or a salt thereof in the intestine of a mammal not suffering from any disorder or pathological condition.

[0155] (56) The non-therapeutic use according to embodiment (49), wherein the mammal is a healthy human.

[0156] (57) Non-therapeutic use of an active agent for maintaining or improving intestinal function in a healthy mammal, wherein the active agent is selected from 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, omega-3 fatty acids, and combinations thereof.

[0157] (58) The non-therapeutic use of embodiment (51), wherein the mammal is a human. BRIEF DESCRIPTION OF THE DRAWINGS

[0158] Figure 1 Acetate production after 48 hours of fermentation after administration of different test products as aqueous solutions (upper graph) or dissolved in ethanol (lower graph). Negative controls for each stock solution are also included. RTG = Riboflavin Table Grade, R5P = Riboflavin-5-Phosphate, AA = Ascorbic Acid, RTG+AA = Riboflavin Table Grade + Ascorbic Acid, FA = Folic Acid, VKl = Vitamin K1, 0080EE = DHA, 4535EE = DHA + EPA, 8000EE = EPA. Each compound was tested at 3 doses (0.2x, 1x and 5x) (Table 1).

[0159] Figure 2Propionate production after 48 hours of fermentation after administration of different test products as aqueous solutions (upper graph) or dissolved in ethanol (lower graph). Negative controls for each stock solution are also included. RTG = Riboflavin grade, R5P = Riboflavin-5-phosphate, AA = Ascorbic acid, RTG+AA = Riboflavin grade + Ascorbic acid, FA = Folic acid, VK1 = Vitamin K1, 0080EE = DHA, 4535EE = DHA + EPA, 8000EE = EPA. Each compound was tested at 3 doses (0.2x, 1x and 5x) (Table 1).

[0160] Figure 3 Butyrate production after 48 hours of fermentation after administration of different test products as aqueous solutions (upper graph) or dissolved in ethanol (lower graph). Negative controls for each stock solution are also included. RTG = Riboflavin grade, R5P = Riboflavin-5-phosphate, AA = Ascorbic acid, RTG + AA = Riboflavin grade + Ascorbic acid, FA = Folic acid, VK1 = Vitamin K1, 0080EE = DHA, 4535EE = DHA + EPA, 8000EE = EPA. Each compound was tested at 3 doses (0.2x, 1x and 5x) (Table 1).

[0161] Figure 4 Total SCFA production after 48 h of fermentation after administration of different test products as aqueous solutions (upper graph) or dissolved in ethanol (lower graph). Negative controls for each stock solution were also included. RTG = Riboflavin grade, R5P = Riboflavin-5-phosphate, AA = Ascorbic acid, RTG + AA = Riboflavin grade + Ascorbic acid, FA = Folic acid, VKl = Vitamin K1, 0080EE = DHA, 4535EE = DHA + EPA, 8000EE = EPA. Each compound was tested at 3 doses (0.2x, 1x and 5x) (Table 1).

[0162] Figure 5 Schematic representation of Caco-2 and goblet cells co-cultured in a transwell plate. Measurement of transepithelial electrical resistance (TEER) provides information about the integrity of tight junctions formed between polarized cells. A "leaky gut" is characterized by increased intestinal permeability and can be obtained in vitro by stressing cells. Addition of apical or basolateral stressors results in a significant, time-dependent decrease in TEER and an increase in the rate of luciferin yellow cell paracellular flux compared to control cells.

[0163] Figure 6Effects of vitamins and ω-3-fatty acids on intestinal integrity in a healthy intestinal cell model. Values ​​are described as the difference between the transepithelial electrical resistance (TEER) of cells treated with 48h fermentation samples and the TEER of cells treated with 0h fermentation samples. Each compound was tested at 3 doses (0.2x, 1x and 5x) (Table 1).

[0164] Figure 7 Effects of vitamins and ω-3-fatty acids on intestinal integrity in the basolateral induction of a 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 3 doses (0.2x, 1x and 5x) (Table 1).

[0165] Figure 8 Effects of vitamins and ω-3-fatty acids on intestinal integrity in the apical induction of a 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 3 doses (0.2x, 1x and 5x) (Table 1).

[0166] Fig. 9 Shannon diversity index after 24 hours of fermentation after application of different test products as aqueous solutions (upper graph) or dissolved in ethanol (lower graph). Negative controls for each stock solution were also included. Each compound was tested at 3 doses (0.2x, 1x and 5x) (Table 1).

[0167] Fig.10 Relative abundance (%) of Bifidobacteria after 24 hours of fermentation after administration of different test products as aqueous solutions (upper graph) or dissolved in ethanol (lower graph). A negative control for each stock solution was also included. Each compound was tested at 3 doses (0.2x, 1x and 5x) (Table 1).

[0168] Fig.11 Relative abundance (%) of genes involved in the acetyl-CoA fermentation to butyrate pathway after 24 h of fermentation after administration of different test products as aqueous solutions (upper graph) or dissolved in ethanol (lower graph). A negative control for each stock solution was also included. Each compound was tested at 3 doses (0.2x, 1x and 5x) (Table 1).

[0169] Fig.12 Effects of vitamins and ω-3 fatty acids on GROa-CXCL1 production in HT29 cells. Data are expressed as pg / ml.

[0170] Fig.13 Effects of vitamins and ω-3 fatty acids on IL-8 production by HT29 cells. Data are expressed as pg / ml.

[0171] Fig.14 Effects of vitamins and ω-3 fatty acids on the production of MIP3A-CCL20 in HT29 cells. Data are expressed as pg / ml.

[0172] Fig.15 Air pressure (kPa) at different time intervals (0-6h, 6-24h and 24-48h) after application of different test products as aqueous solutions (upper graph) or dissolved in ethanol (lower graph). A negative control for each stock solution was also included. Each compound was tested at 3 doses (0.2x, 1x and 5x) (Table 1).

[0173] Fig.16 Total SCFA production at different time intervals (0-6h, 6-24h and 24-48h) after administration of different test products for donor C (upper graph) or donor D (lower graph). A negative control (blank) was also included. Each compound was tested at 3 doses (0.2x, 1x and 5x) (Table 2).

[0174] Fig.17 Abundance of Bifidobacteria (log(cells / ml)) in control cultures and in cultures with test products after 24 h of incubation using fecal inoculum of donor C and donor D.

[0175] Fig.18 Bray-Curtis distance between water as control and supplemented vitamins (beta diversity). The dashed line shows the Bray-Curtis distance between the two controls at 24 h, which served as a reference point.

[0176] Fig.19 Shannon Diversity Index (alpha diversity) in water and vitamin supplements as controls. The dotted line shows the alpha diversity value in the water control at 24 h. Diversity index values ​​greater than this reference control show an increase in diversity.

[0177] Fig. 20 A and B Principal coordinate analysis (PCoA) based on weighted (A) and unweighted (B) Unifrac distances of cecal microbiota profiles in broiler chickens in groups A (green shading), B (gold shading), and C (pink shading). Samples are depicted as points filled with different shades of color, from light (earlier samples) to darker (later samples), according to the color legend provided. The primary and secondary axes are reported in each figure; the percentage of variation in the data set explained by each axis is reported.

[0178] Fig.21 AF principal coordinates analysis (PCoA) based on weighted (AC) and unweighted (DE) Unifrac distances of cecal microbiota profiles of broiler chickens obtained at time points T1 (A, D), T2 (B, E) and T3 (C, F). Samples are depicted as Figure 1 The points shown are shaded in different colors, green for group A, gold for group B, and pink for group B. The primary and secondary axes are reported in each figure; the percentage of variation in the data set explained by each axis is reported.

[0179] Fig. 22A-F Alpha diversity as measured by the FaithPD index (AC) and number of observed OTUs (DF) in the cecal microbiota of broiler chickens obtained at time points T1 (A, D), T2 (B, E), and T3 (C, F). Box and whisker distributions of diversity indices in all samples are depicted. Green, gold, and pink shading are used to distinguish chickens in groups A, B, and C, respectively. *, statistically significant difference (P < 0.0001).

[0180] Fig.23 Hyphal-level microbiota of the cecal ecosystem of broiler chickens. The mean phylogenetic distribution of the cecal microbiome of broiler chickens at each available time point (T1, T2, T3) in groups A, B, and C is provided as a pie chart at the phylum level; bacterial taxa were filtered as >0.1% in at least 1% of all available samples and graphically represented.

[0181] Fig.24 Family-level microbiota distribution of broiler chicken cecal ecosystem. The mean phylogenetic distribution of the cecal microbiome of broiler chickens from groups A, B, and C for each available time point (T1, T2, T3) is provided as a pie chart at the family level; bacterial taxa were filtered as >0.5% in at least 1% of all available samples, and the graphs are presented.

[0182] Fig.25 Relative abundance distribution of bacterial families in the cecal microbiota of broiler chickens. Box and whisker distributions of relative abundance (%) in all samples at three time points (from left to right) are depicted for these families, indicating significant differences between the three groups (A, B, C) at at least 1 time point. Green, gold, and pink shading are used to distinguish chickens in groups A, B, and C, respectively. When statistical significance (P<0.05) was reached, Bejamini-Hocherg corrected P values ​​are reported.

[0183] Fig.26Relative abundance distribution of bacterial genera in the cecal microbiota of broiler chickens. Box and whisker distributions of relative abundance (° / o) in all samples at three time points (from left to right) are depicted for those genera, indicating significant differences between the three groups (A, B, C) in at least 1 time point. Green, gold, and pink shading are used to distinguish chickens in groups A, B, and C, respectively. When statistical significance (P<0.05) was reached, Bejamini-Hocherg corrected P values ​​are reported.

[0184] Fig. 27 Principal coordinates analysis (PCoA) based on weighted (A) and unweighted (B) UniFrac distances of ileal microbiota profiles of broiler chickens in groups A (green shading), B (gold shading), and C (pink shading). Samples are depicted as triangles filled with different shades of color, from light (earlier samples) to dark (later samples), according to the color legend provided. The primary and secondary axes are reported in each figure; the percentage of variation in the data set explained by each axis is reported.

[0185] Fig.28 Principal coordinates analysis (PCoA) based on weighted (AC) and unweighted (DF) Unifrac distances of ileal microbiota profiles in broiler chickens obtained at time points T1 (A, D), T2 (B, E) and T3 (C, F). Samples are depicted as triangles filled with different color shading, as shown in Figure 2. Fig. 27 As shown, group A is green, group B is gold, and group B is pink. In each figure, primary and secondary axes are reported; the percentage of variation in the data set explained by each axis is reported.

[0186] Fig.29 Alpha diversity in the ileal microbiota of broiler chickens obtained at time points T1 (A, D), T2 (B, E) and T3 (C, F), measured as Faith PD index (AC) and number of observed OTUs (DF). Box and whisker distributions of diversity indices in all samples are depicted. Green, gold and pink shading are used to distinguish chickens in groups A, B and C, respectively. Statistically significant differences (Kruskall Wallis test) are indicated with corresponding P values.

[0187] Fig.30 Phylum-level microbiota distribution of broiler chicken ileal ecosystem. The mean phylogenetic distribution of the broiler chicken ileal microbiome for each available time point (T1, T2, T3) in groups A, B, and C is provided in the form of pie charts under phylum-level bacteria; taxa were filtered and graphically represented as >0.1% in at least 1 of all available samples.

[0188] Fig.31Family-level microbiota distribution of broiler chicken ileal ecosystem. The mean phylogenetic distribution of the broiler chicken ileal microbiome for each available time point (T1, T2, T3) in groups A, B, and C is provided in the form of pie charts under family-level bacteria; taxa were filtered and graphically represented as >0.5% in at least 1 of all available samples.

[0189] Fig.32 Relative abundance distribution of Peptostreptococcusaceae in ileal microbiota of broiler chickens. Box and whisker distribution of relative abundance in all samples is depicted under three time points (from left to right). Chickens in groups A, B, and C are distinguished by green, gold, and pink shading, respectively. When statistical significance is reached (P < 0.05), Bejamini Hocherg-corrected P values ​​are reported.

[0190] Fig.33 Principal coordinates analysis PCoA based on weighted (A) and unweighted (B) UniFrac distances of litter microbiota distribution in broilers in groups A (green shading), B (gold shading) and C (pink shading). Samples are depicted as squares filled with different color shades, from light (earlier samples) to dark (later samples). The first and second coordinate axes are reported in each figure; the percentage of variation in the data set explained by each axis is reported.

[0191] Fig.34 Phylum-level microbiota distribution of the litter ecosystem. The mean phylogenetic distribution of the litter microbiome of broiler chickens at each available time point (T1, T2, T3) in groups A, B, and C is provided in the form of pie charts under phylum-level bacteria; taxa were filtered as >0.1% in at least 1 of all available samples and graphically represented.

[0192] Fig.35 Family-level microbiota distribution of the litter ecosystem. The mean phylogenetic distribution of the litter microbiome of broiler chickens at each available time point (T1, T2, T3) in groups A, B, and C is provided in the form of a pie chart under family-level bacteria; taxa were filtered and graphically represented as >0.5% in at least 1 of all available samples.

[0193] Fig.36 DSM cecal sample C281 pH 7.13 aqueous extract 1 H-NMR spectra. The vertical scale in A is enlarged to better appreciate the small signal in the downfield region (aromatic region).

[0194] Fig.37 All samples were graded and normalized 1A) PCA score plot of H-NMR spectra, and B) PCA scores of samples without outliers (graphs were made using R software).

[0195] Fig.38 A) Binned and normalized 61 samples 1 PLS-DA score plot of H NMR spectra, where the three groups appear to be linearly separable according to time points. B) PLS-DA score plot considering the control group [A] and the treatment group [B+C] (Graphics were generated using Python software).

[0196] Fig.39 Box plots of the integration of the uracil signal at 5.793 ppm (d); samples are compared according to the following time points: A) at T0, corresponding to 14 days; B) at T1, corresponding to 28 days, and C) T2, corresponding to 42 days.

[0197] Fig.40 Box plot of the integration of the glutamate signal at 2.0447 ppm (m); samples were compared according to the following time points: A) at T0, corresponding to 14 days; B) at T1, corresponding to 28 days, and C) T2, corresponding to 42 days. The concentration of uracil appears to increase to 42 days in all treatments, especially for sample C.

[0198] Fig.41 Box plots of the integration of the alanine signal at 1.470 ppm (d); samples are compared according to the following time points: A) at T0, corresponding to 14 days; B) at T1, corresponding to 28 days, and C) T2, corresponding to 42 days.

[0199] Fig.42 Box plots of the integration of the creatine signal at 3.920 ppm(s); samples are compared according to the following time points: A) at T0, corresponding to 14 days; B) at T1, corresponding to 28 days, and C) T2, corresponding to 42 days.

[0200] Fig.43 Box plot of the integration of the butyrate signal at 0.883 ppm (t); samples are compared according to the following time points: A) at T0, corresponding to 14 days; B) at T1, corresponding to 28 days, and C) T2, corresponding to 42 days.

[0201] Fig.44 Box plot of the integration of the propionate signal at 1.0446 ppm(m); samples are compared according to the following time points: A) at T0, corresponding to 14 days; B) at T1, corresponding to 28 days, and C) T2, corresponding to 42 days.

[0202] Fig.45Box plots of the integration of the acetate signal at 1.0909 ppm(s); samples are compared according to the following time points: A) at T0, corresponding to 14 days; B) at T1, corresponding to 28 days, and C) T2, corresponding to 42 days.

[0203] Fig.46 Lognormal fit of the integral of the mean metabolite signal versus time. Comparisons are grouped with standard deviation as error bars. Semi-transparent areas define the 95% confidence intervals of the fit.

[0204] Fig.47 PCA score plot of all normalized functional features (6361 data) for all samples (63 samples).

[0205] Fig.48 PCA score plots of normalized functional features for the treatment groups (Groups B and C) compared to the control group (Group A) for each time point.

[0206] Fig.49 Major KEGG metabolic pathways in the cecum of broiler chickens from three different groups A, B and C for each available time point (T1, T2, T3).

[0207] Fig.50 Relative abundance of Environmental Information Processing and Metabolism pathways (KEGG database) in the cecum of broiler chickens from three different groups A, B and C for each available time point (T1, T2, T3) that were statistically significant. The green and red boxes are associated with groups B and C, respectively, while the blue box is associated with group A. P values ​​are shown in the upper right corner of each graph.

[0208] Fig.51 Secondary KEGG metabolic pathways with relative abundance ≥ 0.5% in the ceca of broiler chickens of three different groups A, B and C for each available time point (T1, T2, T3).

[0209] Fig.52Four different pathways for butyrate synthesis and the corresponding genes (protein names) are shown. The main substrates are shown. The terminal genes are emphasized in red. L2Hgdh, 2-hydroxyglutarate dehydrogenase; Gct (Get), glutaconate CoA transferase (α, β subunits); HgCoAd, 2-hydroxy-glutaryl-CoA dehydrogenase (α, β, γ subunits); Gcd, glutaconyl-CoA decarboxylase (α, β subunits); Th1, thiolase; hbd, β-hydroxybutyryl-CoA dehydrogenase; Cro, enoyl hydratase (crotonase); Bcd (Bed), butyryl-CoA dehydrogenase (including 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 lyase; Kal, 3-aminobutyryl-CoA deaminase; AbfH, 4-hydroxybutyrate dehydrogenase; AbfD, 4-hydroxybutyryl-CoA dehydrogenase; Isom, vinylacetyl-CoA 3,2-isomerase (same as AbfD protein): 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 of each butyryl-CoA transferase are shown. (Vital et al., 2014)

[0210] definition

[0211] The term "beneficial bacteria" used throughout the specification and claims includes at least one bacterial group selected from the following genera: Acidaminococcus, Akkermansia, Bacteroides, Bifidobacterium, Blautia, Clostridium, Collinsella, Dorea, Escherichia coli, Eubacterium, Faecalibacterium, Lachnospira, Lactobacillus, Parabacteroides, La-Ure, Roseburia, Ruminococcus, Prevotella and Christensenella. In addition, the following Erysipelothrixaceae, Red Stinkbugaceae, Barnesellaceae can include beneficial bacterial groups.

[0212] As used throughout the specification and claims, when reference is made to "niacin", it is not the sole active agent. If present, it is in combination with at least one other active agent, and niacin is preferably absent.

[0213] A "supraphysiological" dose is the same as a "supra-nutritional" dose, i.e., a dose that is higher than the recommended amount of the active ingredient for the animal, such that at least an effective amount of the active ingredient is present in the intestine of the animal, preferably in its lower intestine and / or colon, and is available to directly nourish the bacterial population.

[0214] By "only active agent" is meant the only active agent present in the intestine, preferably the colon.

[0215] As used herein, the term "intestine" refers to the portion of the gastrointestinal tract consisting of the small intestine and the large intestine.

[0216] As used herein, the term "vitamin A" refers to a compound selected from retinol, retinal, retinoic acid, and provitamin A carotenoids (most notably beta-carotene). In a preferred embodiment, vitamin A is retinol. Any concentration or amount of vitamin A used herein refers to retinol equivalents.

[0217] The term "vitamin E" refers to a compound having alpha-tocopherol activity. In a preferred embodiment, the vitamin E is alpha-tocopherol and its esters, in particular tocopheryl acetate. Any concentration or amount of vitamin E mentioned herein relates to alpha-tocopherol equivalents.

[0218] As used herein, the term "vitamin D" refers to vitamin D3. 25-hydroxyvitamin D3 can be used instead of vitamin D3 or in addition to vitamin D3, preferably for non-human species. The relative strength of 25-hydroxyvitamin D3 to vitamin D3 is about 40:1, so the dose of 25-hydroxyvitamin D3 should be adjusted accordingly.

[0219] The term "riboflavin" includes riboflavin and its esters, particularly riboflavin-5'-phosphate.

[0220] The term "folic acid" refers to folic acid and pharmaceutically acceptable salts thereof.

[0221] The term "vitamin C" refers to ascorbic acid, its pharmaceutically acceptable salts (eg, sodium ascorbate and calcium ascorbate), and its pharmaceutically acceptable esters (particularly ascorbyl palmitate).

[0222] The term "vitamin K" includes vitamin K1 and vitamin K2.

[0223] The term "beta-carotene" refers to beta-carotene or provitamin A.

[0224] The term "vitamin B1" refers to thiamine, anti-neurotropic and pharmaceutically acceptable salts thereof (eg, thiamine mononitrate).

[0225] The term "niacin" refers to nicotinic acid, vitamin B3, pyridine-3-carboxylic acid, and includes nicotinamide or niacinamide. It may be present in combination with at least one other active ingredient, but the present invention specifically excludes it when it is the only active ingredient.

[0226] The term "vitamin B5" refers to pantothenic acid and its pharmaceutically acceptable salts (eg, calcium pantothenate), and includes its derivatives pantothenol or panthenol.

[0227] The term "vitamin B6" refers to pyridoxal phosphate and includes pyridoxal 5' phosphate.

[0228] The term "biotin" refers to vitamin B7, vitamin H, coenzyme R, biotin.

[0229] The term "vitamin B12" refers to cobalamin and includes all forms of vitamin B12 (eg, cyanocobalamin and methylcobalamin).

[0230] "Omega-3 fatty acids" are polyunsaturated fatty acids having 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. DETAILED DESCRIPTION

[0231] In one aspect, the invention relates to an active agent for use in improving intestinal health in an animal, the active agent being selected from 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, wherein the improvement comprises or consists of:

[0232] (i) increasing the concentration of at least one short-chain fatty acid or a salt thereof in the intestine,

[0233] (ii) increasing the diversity of the microbiome in the intestine,

[0234] (iii) increasing the microbial abundance of beneficial bacteria in the intestine,

[0235] (iv) promoting or increasing the butyrate synthesis pathway in the intestine,

[0236] (v) Improve intestinal barrier function;

[0237] (vi) reducing the redox potential of the intestine;

[0238] (vii) reduce the amount of gas produced in the intestines, and

[0239] (vii) stimulate intestinal immune response;

[0240] The use comprises administering to an animal

[0241] a) a formulation comprising an effective dose of an active agent, wherein the active agent is delivered to the large intestine; or

[0242] b) supraphysiological doses of the active ingredient so that at least an effective amount of the active ingredient is present in the intestine of the animal;

[0243] The premise is that if niacin is the active agent, it is not the only active agent; and if riboflavin is present, it has an ameliorative effect other than increasing the amount of F. prausnitzii.

[0244] Typically, the use comprises administering a delayed release formulation comprising an effective dose of an active agent to an animal, which may be a mammal, wherein the release of the active agent is delayed until the large intestine or until the colon, or by a non-oral route, wherein the agent is delivered directly to the colon. Delayed release formulations and their preparation are explained in more detail below.

[0245] When the animal is a non-human animal, such as a monogastric animal or poultry, it can be administered through a delayed release formulation to delay the release of the active agent until the large intestine, or by administering it using a supraphysiological dose in the feed so that an effective amount of the active agent is present in the intestine. When the animal is a human, it can be administered in a variety of ways as detailed in the instructions, including administration of a delayed release formulation, administration by a non-oral route and / or administration by a supraphysiological dose.

[0246] In some embodiments, niacin is not considered as one of the active agents of the present invention.

[0247] Increase short-chain fatty acids / promote the synthesis of butyrate

[0248] As used herein, the term "short-chain fatty acids" (SCFA) refers to fatty acids having two to six carbon atoms. SCFA include formic acid, acetic acid, propionic acid, butyric acid, 2-methylpropionic acid, 3-methylbutyric acid and caproic acid. The most important SCFA are acetic acid, propionic acid and butyric acid.

[0249] The "increase" in the concentration of one or more SCFAs in the colon of an animal is relative to the SCFA concentration of an animal to which the active agent is not administered. Animals to which the active agent is not administered are referred to herein as "control animals". Preferably, the "increase" in the concentration of one or more SCFAs in the colon of an animal is relative to the SCFA concentration of the same animal to which the active agent is not administered. That is, the control animal in this case is typically the same animal before the active agent is started. 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 poultry.

[0250] 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.

[0251] In one embodiment, the concentration of one or more SCFA increases after a single application of the active agent. In another embodiment, the concentration of one or more SCFA increases after two applications of the active agent (applied for two consecutive days). In another embodiment, the concentration of one or more SCFA increases after seven applications of the active agent (applied for seven consecutive days). In another embodiment, the concentration of one or more SCFA increases after 14 applications of the active agent (applied for 14 consecutive days). Preferably, the concentration of one or more SCFA (e.g., acetic acid, propionic acid and / or butyric acid) increases after 21 applications of the active agent (applied for 21 consecutive days). Most preferably, the concentration of one or more SCFA (e.g., acetic acid, propionic acid and / or butyric acid) increases after 28 applications of the active agent (applied for 28 consecutive days).

[0252] 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% relative to the concentration of SCFAs in the colon of a control.In the control no active agent is administered.

[0253] The concentration of SCFAs in the colon can be determined by obtaining a fecal sample from a mammal to which an active agent is administered and measuring the concentration of one or more SCFAs.

[0254] Methods for measuring the concentration of commonly known SCFAs are known to the person skilled in the art, for example by gas chromatography. For example, De Weirdt et al. (2010) (DOI: 10.1111 / j.1574-6941.2010.00974.x) describes suitable methods.

[0255] Alternatively, the reactor and fecal suspension can be used as an in vitro model to determine the increase in one or more SCFAs following administration of an active agent, as described in the Examples of this application.

[0256] It was further found that the administration of the active agent resulted in an increase in the relative abundance of genes involved in the acetyl-CoA fermentation to butyrate pathway. Thus, the present invention relates to the use of an active agent for activating or increasing the butyrate synthesis pathway. The relative abundance of genes involved in the butyrate pathway associated with acetyl-CoA fermentation can be determined as described in the Examples.

[0257] 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 intestinal barrier function. Vitamin A, vitamin C, riboflavin, vitamin D, vitamin E, vitamin K, folic acid, and omega-3 fatty acids have been found to increase the concentration of at least one SCFA in the colon of a mammal when administered in certain circumstances.

[0258] 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 can be treated, improved, or prevented by this action include metabolic disorders, type 2 diabetes, obesity, chronic inflammation, and arterogenesis.

[0259] Therefore, another embodiment of the present invention is a method of increasing at least one short-chain fatty acid and / or increasing the activity of the butyrate synthesis pathway in an animal, including a human, suffering from a condition selected from metabolic disorders, type 2 diabetes, obesity, chronic inflammation and arteriogenesis, the method comprising administering at least one active agent selected from vitamin A, vitamin C, riboflavin, vitamin E, folic acid, beta-carotene, omega 3 fatty acids and combinations thereof.

[0260] Another embodiment of the present invention is an active agent for use in improving intestinal health in animals (including humans), wherein the improvement comprises increasing the concentration of at least one short-chain fatty acid or a salt thereof in the intestine; or promoting or increasing the butyrate synthesis pathway in the intestine; wherein the short-chain fatty acid is selected from acetic acid, propionic acid and butyric acid or a salt thereof.

[0261] Another embodiment of the present invention is an active agent for use in increasing the concentration of at least one short-chain fatty acid or its salt in the intestine, wherein the active agent is selected from vitamin A, β-carotene, vitamin C, riboflavin, vitamin D, vitamin E, vitamin K, folic acid, ω3 fatty acids and combinations thereof.

[0262] Another embodiment of the present invention is an active agent for improving intestinal health in animals including humans, wherein the improvement comprises increasing the concentration of at least one short-chain fatty acid or a salt thereof in the intestine; or promoting or increasing the butyrate synthesis pathway in the intestine, wherein the animal (including humans) suffers from a condition selected from the group consisting of: metabolic disorders, type 2 diabetes, obesity, chronic inflammation and atherogenesis.

[0263] Microbiome diversity and enhancing beneficial bacteria

[0264] It was further observed that the administration of an active agent increases the diversity of the microbiome in the intestine. Therefore, the present invention relates to the use of an active agent for increasing the diversity of the intestinal microbiome, preferably the diversity of the colonic microbiome. The diversity of the microbiome can be determined as described in the examples for humans, pigs and poultry.

[0265] In particular, the inventors have found that supplementation with an active agent increases the relative abundance of beneficial bacteria in humans, preferably lactobacilli, bifidobacteria, Akkermansia and Faecalibacterium. Therefore, the present invention relates to the use of an active agent for increasing the relative abundance of beneficial bacteria in the intestine, preferably in the colon, and the beneficial bacteria preferably include at least one of lactobacilli, bifidobacteria and / or Faecalibacterium.

[0266] For example, in poultry, Bacteroides and Faecalibacterium are increased. Thus, another embodiment is a method of increasing beneficial bacteria in an animal, the method comprising administering to the animal a supraphysiological dose of an active agent.

[0267] Increased abundance of beneficial bacteria may treat, prevent or alleviate symptoms of irritable bowel syndrome, metabolic diseases, obesity and inflammatory disorders.

[0268] Preferred agents are 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, but if riboflavin is the active agent, it is present for purposes other than increasing the abundance of Faecalibacterium prausnitzii.

[0269] Therefore, another embodiment of the present invention is a method for treating, preventing or reducing the symptoms of irritable bowel syndrome, metabolic diseases, obesity or inflammatory disorders in animals, including humans, in need thereof, the method comprising: administering to the colon of the animal a beneficial bacteria enhancing amount of an active agent; 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; provided that if riboflavin is the active agent, it is present for purposes other than increasing the abundance of Faecalibacterium prausnitzii.

[0270] Another embodiment of the invention is an active agent for improving intestinal health, wherein said improvement comprises increasing the diversity of the microbiome in said intestine and / or increasing the abundance of increased beneficial bacteria in the colon. The increased beneficial bacteria are selected from the group consisting of Acidaminococcus, Akkermansia sp., Bacteroides ovatus, Bifidobacterium spp., Blautiaproducta, Clostridium cocleatum, Collinsellaaerofaciens, Dorea longicatena, Escherichia coli, Eubacterium spp., Faecalibacterium prausnitzii, Lachnospira pectinoshiza, Lactobacillus spp., Parabacteroides distasonis, Raoultella spp., Roseburia spp., Ruminococcus spp.), Streptococcus spp., and combinations thereof, provided that if riboflavin is present, it has an ameliorative effect in addition to increasing the number of Faecalibacterium prausnitzii. Preferably, the increased bacteria are selected from Bifidobacterium, Akkermansia, Faecalibacterium and Bacteriodes.

[0271] The active agent can be used in animals (including humans) suffering from a condition selected from the group consisting of irritable bowel disease, metabolic disease, obesity and inflammatory conditions. Preferably, the active agent is selected from the group consisting of vitamin B1, vitamin B2, vitamin B5, vitamin B6, vitamin B12, beta-carotene, biotin, vitamin C, vitamin E, vitamin D, folic acid, vitamin K, omega 3 fatty acids and combinations thereof.

[0272] Stimulates immune response

[0273] It was further observed that certain markers and cytokines produced by the colonic epithelial cell line HT29 were increased after treatment of the cells with effluent from a 24h intestinal batch fermentation following administration of the active agent. The biomarkers and cytokines included GROa-CXCL1, IL-8, and MIP3A-CCL20.

[0274] GROa-CXCL1 is a chemokine that plays an important role in inflammation and acts as a chemoattractant for neutrophils. It activates the host's immune response by recruiting and activating neutrophils at tissue sites to kill microorganisms. CXCL1 is also known to activate the release of proteases and reactive oxygen species (ROS) for microbial defense.

[0275] Interleukin 8 (IL-8) is a chemoattractant cytokine known to have neutrophil migratory capacity, which contributes to enhanced chemotaxis. It induces chemotaxis in target cells, resulting in the migration of neutrophils and other granulocytes toward the site of infection.

[0276] MIP3A-CCL20 is also a chemokine that selectively attracts effector and memory T lymphocytes, immature DCs, and naive B cells expressing the receptor CCR6. CCL20 is constitutively expressed in immune barriers such as the gastrointestinal tract and skin.

[0277] All of these biomarkers reflect the activation of the immune response.

[0278] Stimulating an immune response can be beneficial in conditions involving chronic inflammation as well as acute inflammation.

[0279] Preferred agents are riboflavin, vitamin E, vitamin A, vitamin K, and combinations thereof.

[0280] Therefore, another embodiment of the present invention is the use of an agent that stimulates intestinal immune response, improves immune weakening symptoms, and produces systemic anti-inflammatory response by using and / or applying an effective amount of an active agent to the colon of an animal in need, wherein the agent is selected from riboflavin, vitamin E, vitamin A, vitamin K, and combinations thereof. Another embodiment is a method for treating, preventing, or alleviating chronic or acute inflammatory symptoms, the method comprising administering an immunostimulatory effective amount of an agent to the colon of an animal, wherein the agent is selected from riboflavin, vitamin E, vitamin A, vitamin K, and combinations thereof.

[0281] Another embodiment of the present invention is an active agent for improving intestinal health, wherein the improvement is stimulating intestinal immune response. Preferably, the application is to increase immune response molecules selected from the group consisting of: GROa-CXCL1, MIP3A-CCL20, interleukin 8 and combinations thereof. Preferably, the active agent is selected from the group consisting of: vitamin B2, vitamin E, vitamin D, vitamin A and vitamin K and mixtures thereof.

[0282] Another embodiment is a method of improving intestinal immune responses in animals (including humans) suffering from acute or chronic inflammation, the method comprising administering to the intestine an active agent selected from riboflavin, vitamin E, vitamin A, vitamin K, and combinations thereof.

[0283] Redox potential

[0284] Although antioxidant compounds (e.g., vitamins C and E) generally do not reach the colon, colon-targeted delivery of antioxidant vitamins and compounds to the large intestine can lower the redox potential in the intestine. This lower redox potential can counteract symptoms associated with disturbances in the composition and activity of normal intestinal microflora, particularly in situations where the intestinal microbiome environment is faced with increased presence of oxygen and / or oxygen free radicals, which can hinder the growth of strictly anaerobic beneficial microorganisms, promote the growth of aerotolerant pathogenic bacteria, and / or cause inflammation and cellular damage to host cells.

[0285] Therefore, another embodiment of the present invention is the use of an active agent, preferably vitamin C and / or E, to reduce the redox potential in the intestine, thereby reducing the growth of aerotolerant pathogenic bacteria, reducing intestinal inflammation and the resulting cell damage, and promoting the growth of beneficial strictly anaerobic bacteria.

[0286] Furthermore, another embodiment of the present invention is the use of an active agent, preferably vitamin C and / or E, for lowering plasma free thiols, a measure of the systemic redox state in various inflammatory conditions, such as inflammatory bowel disease.

[0287] Therefore, another embodiment of the present invention is a method of reducing the redox potential of the intestine, said method resulting in a benefit selected from the group consisting of:

[0288] Promotes the growth of strictly anaerobic beneficial bacteria in the colon;

[0289] Reduce aerobic pathogenic bacteria in the intestine;

[0290] Reduce inflammation in the gut; and

[0291] Reduces intestinal cell damage caused by inflammation in the intestine;

[0292] It comprises administering to an animal (including a human) in need thereof an agent selected from the group consisting of riboflavin, vitamin C, vitamin E, and mixtures thereof.

[0293] Gas production

[0294] Colonic incubations were performed in a closed incubation system. This allowed us to assess the accumulation of gas in the headspace, which can be measured with a manometer. Gas production is a measure of microbial activity and, therefore, also of the fermentation rate of potential prebiotic substrates. It can be used as an indicator of bloating and digestive health after consuming prebiotics.

[0295] As shown in the examples, active agents, preferably vitamin B2 and / or vitamin C, can be used to reduce the amount of gas produced in the intestine.

[0296] Therefore, another embodiment of the present invention is a method of treating, preventing or reducing the amount of gas produced in the intestinal tract, the method comprising delivering to the large intestine an active agent selected from vitamin B2, vitamin C, and combinations thereof.

[0297] Strengthens the intestinal barrier

[0298] Co-culture of Caco-2 and HT29-MTX-E12 cells was used to evaluate the protective effects of the supernatants before and after fermentation. Transepithelial electrical resistance (TEER) readings correlate with the tightness of confluent monolayers (Srinivasan et al. 2015) and can be used as an indicator of intestinal barrier strength. Reduced intestinal barrier strength has been observed in conditions associated with many conditions including irritable bowel disease, Crohn's disease, ulcerative colitis, leaky gut, and malnutrition.

[0299] 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. Thus, another embodiment of the present invention is a method of treating, preventing, or alleviating the symptoms of a condition selected from 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 riboflavin, vitamin C, vitamin E, omega-3 fatty acids, vitamin D, vitamin A, folic acid, vitamin K, and mixtures thereof.

[0300] Another embodiment of the present invention is the use of an active agent for improving intestinal health, wherein the improvement comprises improving intestinal barrier function. It can be used in animals, including humans, suffering from a condition selected from the group consisting of irritable bowel disease, Crohn's disease, ulcerative colitis, leaky gut, and malnutrition. Preferably, the active agent 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.

[0301] Dosage and concentration

[0302] The dosage used herein is intended to be in addition to the active ingredients taken in by mammals for general nutritional purposes. Instead, they act on the intestinal microbial environment as a whole at the level of the genus, species and strain of intestinal microorganisms. The active agents are not intended to be directly metabolized by mammals, but they are intended to be utilized by the bacterial population of the colon. Therefore, the amount reported below will also be consumed by animals in addition to the usual diet, but they cannot be directly used in animals due to their delayed release.

[0303]

[0304] *The upper limits for all vitamins are based on the recommendations of the Council for Responsible Nutrition (https: / / www.crnusa.org / resources / vitamin-mineral-safety). For DHA and EPA, the upper concentration limits are based on the UL levels in the EFSA.

[0305] 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.

[0306] In one embodiment suitable for poultry, the supraphysiological amount may be at least about 10 times or even 20 times the recommended dose, for example, if the recommended daily dose is 5 mg, the amount administered in the feed is 50 mg or 100 mg, so that riboflavin is present in the colon.

[0307] Preferably, vitamin E is administered in an amount that provides a local concentration in the colon of at least 0.01 mg / ml, preferably at least 0.05 mg / ml, and 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, and most preferably from about 0.05 mg / ml to about 3 mg / ml.

[0308] Preferably, ascorbic acid is administered in an amount such that the local concentration in the colon is at least 0.05 mg / ml, preferably at least 0.1 mg / ml, and most preferably at least 0.5 mg / ml. The preferred local concentration in the colon is from about 0.05 mg / ml to about 50 mg / ml, more preferably from about 0.1 mg / ml to about 20 mg / ml, and most preferably from about 1 mg / ml to about 10 mg / ml.

[0309] Preferably, vitamin D is administered in an amount that provides a local concentration in the colon of 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. The preferred local concentration in the colon is 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. Alternatively, from about 0.1 μg / ml to about 2 μg / ml.

[0310] Preferably, vitamin A is administered in an amount such that the local concentration in the colon is at least 0.05 μg / ml, preferably at least 0.1 μg / ml, and most preferably at least 0.2 μg / ml. The preferred local concentration in the colon is from about 0.05 μg / ml to about 20 μg / ml, more preferably from about 0.1 μg / ml to about 10 μg / ml, and more preferably from about 0.2 μg / ml to about 5 μg / ml.

[0311] Preferably, vitamin K is administered in an amount such that the local concentration in the colon is at least 0.01 mg / ml, preferably at least 0.03 mg / ml, and 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, and most preferably from about 0.05 mg / ml to about 0.5 mg / ml.

[0312] Preferably, folic acid is administered in an amount such that the 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. The preferred local concentration in the colon is 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.

[0313] Preferably, beta-carotene is administered in an amount such that the 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.

[0314] 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, and most preferably at least 20 μg / ml. The preferred local concentration in the colon is from about 1 μg / ml to about 10 mg / ml, more preferably from about 10 μg / ml to about 5 mg / ml, and most preferably from about 20 μg / ml to about 1 mg / ml.

[0315] Preferably, niacin is administered in an amount such that the 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. If niacin is administered, it is not the only active agent.

[0316] Preferably, vitamin B5 is administered in an amount such that the local concentration in the colon is at least 0.05 mg / ml, preferably at least 0.1 mg / ml, and most preferably at least 0.4 mg / ml. The preferred local concentration in the colon is from about 0.05 mg / ml to about 100 mg / ml, more preferably from about 0.1 mg / ml to about 50 mg / ml, and most preferably from about 0.4 mg / ml to about 20 mg / ml.

[0317] Preferably, vitamin B6 is administered in an amount such that the local concentration in the colon is at least 1 μg / ml, preferably at least 5 μg / ml, and most preferably at least 10 μg / ml. The preferred local concentration in the colon is from about 1 μg / ml to about 3 mg / ml, more preferably from about 5 μg / ml to about 1 mg / ml, and most preferably from about 10 μg / ml to about 500 μg / ml.

[0318] Preferably, biotin is administered in an amount such that the local concentration in the colon is at least 1 μg / ml, preferably at least 2 μg / ml, most preferably at least 4 μg / ml. The preferred local concentration in the colon is 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.

[0319] Preferably, vitamin B12 is administered in an amount such that the local concentration in the colon is at least 0.01 μg / ml, preferably at least 0.03 μg / ml, and 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, and most preferably from about 0.05 μg / ml to about 2 μg / ml.

[0320] Preferably, ω-3 fatty acids are used at least 0.02mg / ml, preferably at least 0.05mg / ml, most preferably at least 0.1mg / ml in the local concentration in the colon.Preferred local concentration ranges in the colon are from about 0.02mg / ml to about 2mg / ml, more preferably from about 0.05mg / ml to about 1.5mg / ml, most preferably from about 0.1mg / ml to about 1mg / ml.In another embodiment, ω-3 fatty acids are used at least 0.02mg / ml, preferably at least 0.05mg / ml, most preferably at least 0.1mg / ml in the local concentration in the colon.Preferred local concentration in the colon is from about 0.02mg / ml to about 100mg / ml, more preferably from about 0.05mg / ml to about 75mg / ml, most preferably from about 0.1mg / ml to about 60mg / ml. In another embodiment, the active agent is administered to a mammal at a daily dose of up to 2.9 mg riboflavin / kg body weight, up to 42.9 μg vitamin A / kg body weight, up to 3.6 μg vitamin D / kg body weight, up to 28.6 mg vitamin C / kg body weight, up to 14.3 mg vitamin E / kg body weight, up to 143 μg vitamin K / kg body weight, up to 14.3 μg folic acid / kg body weight, up to 0.4 mg beta-carotene, 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 / kg body weight, or a combination thereof, wherein preferably EPA is up to 25.7 mg / kg body weight and DHA is up to 14.3 mg / kg body weight.

[0321] Another embodiment is a delayed release formulation for delivering an active agent to the intestine, the formulation comprising an active agent and an enteric layer or coating, wherein the active agent consists 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, wherein preferably EPA is up to 1800 mg and DHA is up to 1000 mg, and wherein release of the active agent is delayed until the intestine.

[0322] Another embodiment is a delayed release formulation for delivering an active agent to the intestine, the formulation comprising an active agent and an enteric layer or coating, wherein the active agent consists 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 a combination thereof, and wherein the release of the active agent is delayed until the intestine.

[0323] Another embodiment is a delayed release formulation of an article, wherein the active agent consists 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 a combination thereof.

[0324] In one embodiment, riboflavin is administered as the only active agent.

[0325] In another embodiment, vitamin A is administered as the only active agent.

[0326] In another embodiment, vitamin C is administered as the only active agent.

[0327] In another embodiment, vitamin D is administered as the only active agent.

[0328] In another embodiment, vitamin E is administered as the only active agent.

[0329] In another embodiment, vitamin K is administered as the only active agent.

[0330] In another embodiment, one or more omega-3-fatty acids are administered as the only active agent.

[0331] In one embodiment, folic acid is administered as the only active agent.

[0332] In another embodiment, beta-carotene is administered as the only active agent.

[0333] In another embodiment, vitamin B1 is administered as the only active agent.

[0334] In another embodiment, vitamin B5 is administered as the only active agent.

[0335] In another embodiment, vitamin B6 is administered as the only active agent.

[0336] In another embodiment, biotin is administered as the only active agent.

[0337] In another embodiment, vitamin B12 is administered as the only active agent.

[0338] In another embodiment, DHA is administered as the only active agent.

[0339] In another embodiment, EPA is administered as the only active agent.

[0340] In another embodiment, EPA and DHA are administered as the only active agents.

[0341] In another embodiment, the active agent consists 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 consists 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 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, EPA and DHA.

[0342] In one embodiment, the delayed release formulation of the invention is administered once a day. In other embodiments, the formulation is administered twice a day or once every two days.

[0343] In one embodiment, the active agent is administered to a mammal. The mammal may be a horse, a cow, a pig, a dog or a cat. More preferably, the mammal is a human, such as an adult. In one embodiment, the adult has a body mass index (BMI) of at least 20, or at least 25, or even at least 30. For example, the range of BMI may be 25 to 35, or 26 to 34, or 27 to 33, or 28 to 32.

[0344] Condition to be treated or prevented

[0345] According to a first aspect of the invention, an active agent is administered to increase the concentration of one or more SCFAs. Thus, any condition associated with impaired SCFA levels in the colon may be treated or prevented. SCFA levels are impaired if they are significantly reduced by at least 10%, or at least 20%, or at least 30% relative to levels in healthy individuals. Fecal concentrations may be a measure of concentrations in the colon.

[0346] Disease to be treated or prevented according to the present invention comprises inflammatory disease, disease and atheroma formation characterized by inflammation, particularly chronic inflammation. In addition, metabolic disorders or obesity such as type 2 diabetes can be treated or prevented or improved. In addition, immunodeficiency or allergy can be treated.

[0347] According to another aspect of the invention, in order to improve or enhance the barrier function in the intestine, an active agent is administered. In one embodiment, the barrier function in the small intestine is improved or enhanced. In another embodiment, the barrier function in the large intestine is improved or enhanced. In another embodiment, the barrier function in the colon is improved or enhanced. Thus, any condition or disorder associated with increased intestinal permeability can be treated, improved or prevented. Disorders to be treated include, but are not limited to, disorders characterized by increased intestinal permeability, enteropathy (e.g., environmental enteropathy, enteropathy caused by malnutrition), inflammatory bowel disease (e.g., Crohn's disease or ulcerative colitis), and irritable bowel syndrome.

[0348] 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 fluorescein from the apical to the basolateral compartment (see Examples). In humans, intestinal permeability can be measured indirectly by determining the circulating concentration of bacterial lipopolysaccharide or antibodies to the core portion of lipopolysaccharide, or directly by a dual sugar absorption test (Dorshow et al., Scientific Reports Vol. 7, Article No.: 10888 (2017). For example, the ratio of lactose: mannitol in urine after disaccharide ingestion can be determined. Alternatively, blood alpha-1 antitrypsin, myeloperoxidase and / or REG1B protein can be determined.

[0349] Another aspect of the invention is to reduce gas production during large intestinal fermentation, which will improve intestinal health, reduce bloating and potentially lead to greater amenability to fiber and prebiotic treatments.

[0350] preparation

[0351] As used herein, "delayed release" refers to the release of an active agent at a time later than immediate release after administration. Preferably, "delayed release" refers to the delivery of an active agent to the large intestine in a delayed manner relative to an immediate release formulation when administered orally.

[0352] An "enteric layer" is a layer around the core, wherein the core contains the active agent and the layer confers resistance to gastric juice. An "enteric coating" is a shell or matrix surrounding or encapsulating the active agent, wherein the shell confers resistance to gastric juice. Alternatively, a matrix-based delivery system can be used. A matrix-based system has no discrete layers of coating material, but the active agent is more or less evenly distributed within the matrix. In addition, there are colonic release systems in which the active agent is embedded in, for example, a fiber matrix (enzymatically initiated) and an enteric coating on top.

[0353] In a preferred embodiment for use in humans, the formulation of the invention is a solid dosage form for oral administration. The formulation may be a capsule, pill, bead, sphere, pellet, tablet, mini-tablet or granule, optionally coated with a delayed release coating or shell that prevents the release of the active agent before the small intestine, preferably before the colon.

[0354] When administered orally, coatings, shells or matrix materials for delayed release of active agents, particularly for targeted release in the ileum or large intestine are known in the art. They can be subdivided into coating materials that disintegrate at a higher than a specific pH value, coating materials that disintegrate after a specific residence time in the gastrointestinal tract, and coating materials that disintegrate due to enzymatic initiation specific to the microflora in a specific area of ​​the intestine. Different categories of coating or shell materials are usually used in combination. For example, these three different categories of coating or shell materials for targeting the large intestine are reviewed in Bansal et al. (Polim. Med. 2014, 44, 2, 109-118). In one embodiment of the present invention, the delayed release coating comprises at least one component selected from a coating material for pH-dependent disintegration, a coating material for time-dependent disintegration, a coating material for disintegration due to enzymatic initiation in an intestinal environment (e.g., in the intestinal environment of the ileum and large intestine), and a combination thereof.

[0355] Coating materials that rely on pH for disintegration include polyvinyl acetate phthalate, cellulose acetate trimellitate, hydroxypropyl methylcellulose phthalate HP-50, HP-55 or HP-558, cellulose acetate phthalate, shellac, hydroxypropyl methylcellulose acetate succinate (HPMCAS), poly(methacrylic acid, ethyl acrylate) 1:1 ( L100-55, L30D-55), poly(methacrylic acid, methyl methacrylate) 1:1( L-100, L12.5), poly(methacrylic acid, methyl methacrylate) 1:2( S-100, S12,5, and FS30D).

[0356] Coating materials that disintegrate over time include RL, RS and ethyl cellulose.

[0357] Coating materials that disintegrate enzymatically in the large intestine environment include chondroitin sulfate, pectin, guar gum, chitosan, inulin, lactose, raffinose, stachyose, alginate, dextran, xanthan gum, locust bean gum, arabinogalactan, cyclodextrin, pullulan, carrageenan, scleroglucan, chitin, curcumin, levan, pullulan, starch, amylose, resistant starch and azo compounds degraded by azo bond cleaving bacteria.

[0358] In one embodiment, the formulation comprises an enteric-coated capsule filled with a composition comprising an active agent. Enteric-coated capsules are resistant to the acidic environment of the stomach. For example, soft gel formulations can deliver active agents in solution, but still provide the advantages of solid dosage forms. Soft gel capsules are particularly suitable for hydrophobic active agents that are not easily soluble in water. Vitamin K and omega-3 fatty acids are preferably formulated as soft capsules.

[0359] In another embodiment, the formulation is a tablet comprising (i) a core comprising the active agent, and (ii) a coating that delays release, such as an enteric coating. This may be a hard gelatin capsule.

[0360] The release of the drug may be delayed until the small intestine. In another embodiment, the release of the drug is delayed until the distal small intestine. In another embodiment, the release of the drug is delayed until the colon.

[0361] For example, a formulation may comprise an active agent and an enteric layer or coating, wherein the active agent is selected from: 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, wherein preferably EPA is up to 1800 mg and DHA is up to 1000 mg, wherein release of the active agent is delayed until the intestine.

[0362] Another aspect of the invention is the non-therapeutic use of an active agent selected from 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 a mammal not suffering from any disorder or pathological condition of the intestine, provided that if niacin is an active agent, it is not the only active agent. The mammal is preferably a healthy human.

[0363] 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 maintaining or improving intestinal function in a healthy mammal, preferably a healthy human, with the proviso that if niacin is the active agent, it is not the only active agent.

[0364] One consideration of the present invention is that the active agents used are not intended to provide nutrition to the mammalian recipient; rather, they are intended to directly benefit the large intestinal microbial population, thereby indirectly benefiting the mammal. Thus, the dosages represent dosages in addition to the normal daily consumption of vitamins, fatty acids, and other nutrients as part of the mammal's diet. Thus, by bypassing the stomach and preferring absorption in the small intestine, the microbial population can be nourished, maintaining optimal performance while keeping the intestinal barrier intact. Thus, the formulations of the present invention help maintain a healthy intestine.

[0365] We also observed a beneficial effect on overall digestion. As shown in the examples, pigs supplemented with vitamin B2 showed a higher DM digestibility of substrates (feed or stomach contents of pigs) incubated in pepsin-HCl compared to pigs not supplemented with vitamin B2. This is surprising and clearly has a positive effect on gastrointestinal function. In addition, we observed a beneficial effect on large intestine fermentation when using cecal contents from pigs, namely reducing the production of methane. Although not wishing to be bound by theory, we believe that this can be explained by the fact that vitamin B2 can reduce the activity of methanogenic bacteria or archaeal populations, thereby having a positive impact on the environment.

[0366] In order to further illustrate certain aspects of the present invention, the present invention also specifically provides the following non-limiting embodiments:

[0367] Embodiment 1. An active agent for use in improving intestinal health in animals including humans, the active agent being selected from 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 comprises or consists of:

[0368] i. increasing the concentration of at least one short-chain fatty acid or a salt thereof in the intestine,

[0369] ii. increasing the diversity of the microbiome in the intestine,

[0370] iii. increasing the abundance of beneficial bacteria in the intestine,

[0371] iv. promoting or increasing the butyrate synthesis pathway in the intestine,

[0372] v. improving the intestinal barrier function,

[0373] vi. Reduce the redox potential of the intestine,

[0374] vii. reducing the amount of gas produced in the intestine, and / or

[0375] viii. Stimulate intestinal immune response;

[0376] The use comprises administering to an animal

[0377] a) a formulation comprising an effective dose of an active agent, wherein the active agent is delivered to the large intestine; or

[0378] b) a supraphysiological dose of the active ingredient, such that at least an effective amount of the active ingredient is present in the intestine of the animal;

[0379] The premise is that if niacin is present, it is not the only active ingredient and that if riboflavin is present, it has an ameliorative effect other than increasing the number of Faecalibacterium prausnitzii.

[0380] Embodiment 2. The active agent according to embodiment 1, wherein the animal is a human and the effective dose of the active agent is delivered by a delayed release formulation.

[0381] Embodiment 3. The active agent according to embodiment 1 or 2, wherein the improvement comprises increasing the concentration of at least one short-chain fatty acid or a salt thereof in the intestine, or promoting or increasing the butyrate synthesis pathway in the intestine;

[0382] And wherein the short-chain fatty acid is selected from acetic acid, propionic acid and butyric acid or their salts.

[0383] Embodiment 4. An active agent according to embodiment 3, wherein the active agent is selected from: vitamin A, β-carotene, vitamin C, riboflavin, vitamin D, vitamin E, vitamin K, folic acid, ω3 fatty acids and combinations thereof.

[0384] Embodiment 5. The active agent according to embodiment 3 or 4, wherein the animal, including humans, suffers from a condition selected from metabolic disorders, type 2 diabetes, obesity, chronic inflammation and atherogenesis.

[0385] Embodiment 6. An agent according to embodiment 1 or 2, wherein the improvement comprises increasing the diversity of the microbiome in the intestine.

[0386] Embodiment 7. An active agent according to embodiment 6, wherein the diversity of the microbiome in the colon is increased and / or the abundance of beneficial bacteria is increased.

[0387] Embodiment 8. An active agent according to embodiment 6 or 7, wherein the increased beneficial bacteria are selected from the group consisting of Acidaminococcus, Akkermansia species Bacteroides ovatus, Bifidobacterium, Brucella, Clostridium perfringens, Collinsella perfringens, Dorea longicatena, Escherichia coli, Eubacterium, Faecalibacterium prausnitzii, Lachnospira, Lactobacillus, Parabacteroides distichous, Raoultella, Roseburia, Ruminococcus, Streptococcus and combinations thereof.

[0388] Embodiment 9. An active agent according to any one of embodiments 6 to 8, wherein the increased bacteria are selected from Bifidobacterium, Akkermansia, Faecalibacterium and Bacteroides

[0389] Embodiment 10. The active agent according to any one of embodiments 6-9, wherein the animal, including humans, suffers from a condition selected from irritable bowel disease, metabolic diseases, obesity, and inflammatory conditions.

[0390] Embodiment 11. An active agent according to any one of embodiments 6-10, wherein the active agent is selected from: vitamin B1, vitamin B2, vitamin B5, vitamin B6, vitamin B12, β-carotene, biotin, vitamin C, vitamin E, vitamin D, folic acid, vitamin K, ω3 fatty acids and combinations thereof.

[0391] Embodiment 112. An agent according to embodiment 1 or 2, wherein the improvement comprises improving intestinal barrier function.

[0392] Embodiment 13. The active agent according to embodiment 12, wherein the animal, including humans, suffers from a condition selected from irritable bowel disease, Crohn's disease, ulcerative colitis, leaky gut, and malnutrition.

[0393] Embodiment 14. An active agent according to embodiment 12 or 13, wherein the active agent is selected from: vitamin B2, vitamin C, vitamin E, omega-3 fatty acids, vitamin D, vitamin A, folic acid, vitamin K1 and mixtures thereof.

[0394] Embodiment 15. An agent according to embodiment 1 or 2, wherein the improvement comprises lowering the redox potential of the intestine.

[0395] Embodiment 16. An active agent according to embodiment 15, wherein the reduced redox potential produces a benefit selected from the following: promoting the growth of strictly anaerobic beneficial bacteria in the colon; reducing aerobic pathogenic bacteria in the intestine, reducing plasma free thiols, reducing inflammation in the intestine and reducing cell damage caused by inflammation in the intestine.

[0396] Embodiment 17. The active agent according to embodiment 15 or 16, wherein the active agent is selected from vitamin C, vitamin E and mixtures thereof.

[0397] Embodiment 18. An agent according to embodiment 1 or 2, wherein the improvement is a reduction in the amount of gas produced in the intestine.

[0398] Embodiment 19. The active agent according to embodiment 18, wherein the active agent is vitamin B2, vitamin C, vitamin E and combinations thereof.

[0399] Embodiment 20. An agent according to embodiment 1 or 2, wherein the improvement is stimulation of the intestinal immune response.

[0400] Embodiment 21. The active agent according to embodiment 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.

[0401] Embodiment 22. An active agent according to embodiment 20 or 21, wherein the active agent is selected from: vitamin B2, vitamin E, vitamin D, vitamin A and vitamin K and mixtures thereof.

[0402] Embodiment 23. A method for improving intestinal health in an animal, including a human, wherein the improvement comprises or consists of:

[0403] i. increasing the concentration of at least one short-chain fatty acid or a salt thereof in the intestine,

[0404] ii. increasing the diversity of the microbiome in the intestine,

[0405] iii. increasing the abundance of beneficial bacteria in the intestine,

[0406] iv. promoting or increasing the butyrate synthesis pathway in the intestine,

[0407] v. Improve intestinal barrier function,

[0408] vi. reducing the redox potential of the intestine,

[0409] vii. reducing the amount of gas produced in the intestine, and / or

[0410] viii. Stimulate intestinal immune response;

[0411] It comprises administering to the animal an active agent selected from 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 present in

[0412] a) a formulation comprising an effective dose of said active agent, wherein said active agent is delivered to the large intestine; or

[0413] b) supraphysiological doses of active ingredients, so that at least an effective amount of the active ingredient is present in the intestine of the animal;

[0414] The premise is that if niacin is present, it is not the only active ingredient and that if riboflavin is present, it has an ameliorative effect other than increasing the number of Faecalibacterium prausnitzii.

[0415] Embodiment 24. The method of embodiment 23, wherein the animal is a human and the active agent is delivered to the intestine.

[0416] Embodiment 25. The method of embodiment 23, wherein the active agent is administered in a supraphysiological dose.

[0417] Embodiment 26. A method according to embodiment 23, wherein the improvement is increasing at least one short chain fatty acid and / or increasing the activity of a butyrate synthesis pathway in the animal.

[0418] Embodiment 27. The method of embodiment 26, wherein the animal has a condition selected from the group consisting of metabolic disorders, type 2 diabetes, obesity, chronic inflammation, and arteriogenesis.

[0419] Embodiment 28. The method according to embodiment 26 comprises administering at least one active agent selected from vitamin A, vitamin C, riboflavin, vitamin E, folic acid, beta-carotene, omega 3 fatty acids, and combinations thereof.

[0420] Embodiment 29. The method according to embodiment 23, wherein the improvement is increasing the diversity of the microbiome in the intestine, or increasing the abundance of beneficial bacteria in the intestine.

[0421] Embodiment 30. A method according to embodiment 29, wherein the active agent is selected from vitamin B1, riboflavin, vitamin B5, vitamin B12, β-carotene, biotin, vitamin C, vitamin E, vitamin D, folic acid, vitamin K, ω3 fatty acids and combinations thereof; provided that if riboflavin is the active agent, it is present for a purpose other than increasing the abundance of Faecalibacterium prausnitzii.

[0422] Embodiment 31. A method according to embodiment 29, wherein the method is a method for treating, preventing or alleviating the symptoms of irritable bowel syndrome, metabolic diseases, obesity or inflammatory conditions in animals including humans in need thereof, the method comprising: administering to the colon of the animal an amount of beneficial bacteria that enhances an active agent; the active agent is selected from vitamin B1, riboflavin, vitamin B5, vitamin B12, β-carotene, biotin, vitamin C, vitamin E, vitamin D, folic acid, vitamin K, ω3 fatty acids and combinations thereof; provided that if riboflavin is the active agent, it is present for purposes other than increasing the abundance of Faecalibacterium prausnitzii.

[0423] Embodiment 32. The method of embodiment 23, wherein the improvement is stimulation of the intestinal immune response.

[0424] Embodiment 33. A method according to embodiment 32, wherein the animal, including humans, suffers from acute or chronic inflammation.

[0425] Embodiment 34. The method of embodiment 33, wherein the active agent is selected from riboflavin, vitamin E, vitamin A, vitamin K, and combinations thereof.

[0426] Embodiment 35. The method of embodiment 32, wherein the improvement is a reduction in the redox potential of the intestinal tract, which results in a benefit selected from the group consisting of:

[0427] Promotes the growth of strictly anaerobic beneficial bacteria in the colon;

[0428] reducing aerobic pathogenic bacteria in the intestinal tract;

[0429] reducing inflammation in the intestine; and

[0430] Reducing intestinal cell damage caused by inflammation in the intestine.

[0431] Embodiment 36. A method according to embodiment 35, wherein the agent is selected from riboflavin, vitamin C, vitamin E and mixtures thereof.

[0432] Embodiment 37. The method of embodiment 23, wherein the improvement is the treatment, prevention or reduction of the amount of gas produced in the intestine.

[0433] Embodiment 38. The method of embodiment 23 or 37, wherein the active agent is selected from vitamin B2, vitamin C, and combinations thereof.

[0434] Embodiment 39. The method according to embodiment 23, wherein the improvement is improvement of intestinal barrier function.

[0435] Embodiment 40. The method of embodiment 39, wherein improving barrier function is a method of treating, preventing or alleviating symptoms of a condition selected from irritable bowel disease, Crohn's disease, ulcerative colitis, leaky gut, and malnutrition.

[0436] Embodiment 41. A method according to embodiment 40, wherein the agent is selected from riboflavin, vitamin C, vitamin E, omega-3 fatty acids, vitamin D, vitamin A, folic acid, vitamin K and mixtures thereof.

[0437] Example

[0438] Example 1

[0439] Materials and methods

[0440] Donor and sample preparation

[0441] In the first set of in vitro experiments at the beginning of enteric batch fermentation, all test components were added from stock solutions to a modified nutrient medium containing (g / l): 2.5K 2 HPO 4 , 10.9KH 2 PO 4 , 2NaHCO 3 , 2 yeast extract, 2 peptone, 1 mucin, 0.5 cysteine, 2 Tween 80, 2 glucose, 2 starch, 2 cellobiose, 0.1 NaCl, 0.01 MgSO 4 .7H 2 O, 0.01CaCl 2 .6H 2 O, 0.05 heme, 0.5 bile salts.

[0442] To the stock solution prepared in water add the following compounds:

[0443] Riboflavin grade (abbreviated as RTG),

[0444] Riboflavin-5-phosphate (R5P),

[0445] Ascorbic acid granules powder (AA),

[0446] · Dry Vitamin E 50CWS-S (E 50CWS-S),

[0447] · Dry vitamin D3100CWS (D3100CWS),

[0448] ·Dry Vitamin A Acetate 325CWS / A,

[0449] · Riboflavin grade + ascorbic acid,

[0450] Folic acid,

[0451] Other compounds were prepared in ethanol:

[0452] Vitamin K1 (VK1),

[0453] ·MEG-30080EE oil (MEG-30080EE),

[0454] ·MEG-34535EE oil (MEG-34535EE),

[0455] ·MEG-38000EE oil (MEG-38000EE).

[0456] Each compound was tested at three different concentrations; an overview is given in Table 1. Since ethanol is known to have antimicrobial effects, two blanks were included for each stock type. As a source of microbial communities, freshly prepared fecal suspensions from selected donors were added to the reactors. The volume of each reactor was 70 mL. All tests, except the blanks, were repeated once, and 40 independent incubations were performed as shown in Table 1. The incubation conditions for the pre-screening experiment and the final experiment were 48 hours at 37°C under shaking (90 rpm) and anaerobic conditions. DNA was extracted from effluent samples collected from each fermentation bottle before fermentation (0h fermentation) and after fermentation (24h fermentation), and microbiome composition analysis was performed by Shotgun sequencing. Effluent samples were collected from each fermentation bottle before fermentation (0h fermentation) and after fermentation (48h fermentation), sterilized by filtration through a 0.22μμm filter and used for quantification of short-chain fatty acids (SCFA), immunological biomarkers and in vitro analysis of cell culture systems.

[0457] Table 1: Experimental setup for the final experiment

[0458]

[0459]

[0460] 1: Retinol equivalent

[0461] 2 : α -Tocopherol equivalent

[0462] In a second set of in vitro experiments, the test ingredients were added to a nutrient medium containing basal nutrients present in the colon (e.g., host-derived glycans such as mucin) at the beginning of a short-term colonic incubation. Three doses were tested for each product. The nutrient medium used for this particular project was again supplemented with NaCl, MgSO 4 , CaCl 2 , heme and bile salts, and the carbon sources glucose, starch and cellobiose. Each donor included a blank containing the same nutrient medium as that used for the product incubation, but without the addition of product. This allowed the assessment of the background activity of the bacterial community, which is due to the fermentation of compounds in the nutrient medium. As a source of colonic microbiota, fecal inocula from two healthy adults were added. Incubation was carried out at 37°C for 48h under shaking (90rpm) and anaerobic conditions, and protected from light. The following compounds were tested:

[0463] Thiamine hydrochloride

[0464] β-Carotene 10% CWS / S

[0465] Nicotinamide PC

[0466] ·Calcium D-pantothenate

[0467] Pyridoxine hydrochloride

[0468] D-Biotin

[0469] Vitamin B12 crystals.

[0470] Each compound was tested at three different concentrations; a summary is given in Table 2 below:

[0471] Table 2 Second experimental results

[0472]

[0473] Short-chain fatty acids

[0474] Short-chain fatty acids (SCFA) are an assessment of microbial carbohydrate metabolism (acetate, propionate and butyrate) or protein metabolism (branched SCFAs) and can be compared with typical fermentation patterns of normal GI microbiota. Samples for SCFA analysis were analyzed for the above two sets of in vitro experiments after 0, 6, 24 and 48 h of incubation.

[0475] SCFA was extracted from the sample with ether after adding 2-methylhexanoic acid as an internal standard. The extract was analyzed using a GC-2014 gas chromatograph (Shimadzu, 's-Hertogenbosch, the Netherlands), which was equipped with a capillary-free fatty acid EC-1000Econo-Cap column (size: 25mm x 0.53mm, film thickness 1.2mM; Alltech, Laarne, Belgium), a flame ionization detector and a split injector. The injection volume was 1ml and the temperature curve was set to 110 to 160°C, with a temperature increase of 6°C min-1. The carrier gas was nitrogen and the temperatures of the injector and detector were 100 and 220°C, respectively. The production of non-branched and branched SCFA was calculated by adding the molar concentrations of acetate, propionate, butyrate, valerate and caproate, and adding the molar concentrations of isobutyrate, isovalerate and isocaproate. The total yield of SCFA is defined as the sum of non-branched and branched SCFA.

[0476] Cell culture

[0477] Co-culture of Caco-2 and HT29-MTX-E12 cells was used to evaluate the protective effects of the supernatants from the above two sets of in vitro experiments before (0 h) and after (48 h) fermentation.

[0478] CaCo-2ECACC 86010202 and HT29-MTX-E12ECACC12040401 cells (both from the European Cell Culture Center, Salisbury, UK) were cultured in DMEM medium at 37°C in an atmosphere of 5% CO2, and the DMEM medium was supplemented with 4.5 g / L D-glucose, 4 mM L-glutamine, 1 mM sodium pyruvate, 1% MEM non-essential amino acids, 50 μ μ g / mL gentamicin (Life Technologies Europe BV, Zug, Switzerland) and 10% heat-inactivated FBS (Sigma-Aldrich, Buchs, Switzerland). The sub-confluent cells were trypsinized using 0.25% trypsin / EDTA (Life Technologies Europe BV, Zug, Switzerland) and passaged twice a week at a ratio of 1:5-1:10. Cells between the 55th and 85th generations were used for this study.

[0479] 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 with a pore size of 0.4 μM and a cell growth area of ​​0.33 cm 2 / well (Corning BV, Amsterdam, The Netherlands) and cultured as described above. The culture medium was changed every second to third day.

[0480] Measurement of barrier integrity in a healthy intestinal model

[0481] For both sets of in vitro experiments, initial transepithelial electrical resistance (TEER) readings were obtained after culturing the cells for 13 days using an EVOM2 voltmeter (EVOM, World Precision Instruments, Berlin) equipped with STX100 electrodes. TEER values ​​are correlated with the tightness of the confluent monolayer (Srinivasan et al., 2015).

[0482] Background TEER (insert) was subtracted from the total TEER (cell monolayer plus insert) to generate the monolayer resistance and then normalized to the surface area by multiplying by the area of ​​the insert (Srinivasan et al. 2015). The integrity of the monolayer was determined at a basal level by measuring the TEER. A TEER of 300 Ω cm2 was considered a tight monolayer (see Results section). Afterwards, cells were 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 of each insert compared to the initial TEER was calculated to represent a healthy intestinal model. All TEER measurements were performed by placing the plate containing the insert on a heating plate at 37 °C and were performed for the same amount of time.

[0483] Barrier integrity measurements in the leaky gut model

[0484] For both sets of in vitro experiments, the integrity of the cell monolayer after the stressor test was assessed by TEER values ​​and by measuring the transport of lucifer yellow from the apical to the basolateral compartment (leaky gut model). Preheated HBSS transport buffer, consisting of Hank's pH 7.4 balanced salt solution with Ca2+ and Mg2+, containing 5.5mM D-(+)-glucose, sodium bicarbonate, and supplemented with 4mM L-glutamine, 20mM HEPES (LifeTechnologies Europe BV, Zug, Switzerland) was used for all permeability experiments. The insert was rinsed twice with HBSS using a reservoir tray, and then 100 μl of HBSS was added to the top of the insert, and then moved to a new well filled with fresh HBSS and equilibrated in an incubator at 37°C for 60min, followed by TEER measurement to obtain a baseline resistance reading.

[0485] Lucifer Yellow CH dilithium salt (Sigma-Aldrich, Buchs, Switzerland) was dissolved in HBSS at a final concentration of 100 μg / ml. The basolateral stressor EtOH (Merck, KgaA, Darmstadt, Germany) was diluted to 5% in HBSS. The apical stressor 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.

[0486] First aspirate the HBSS transport buffer from the basolateral compartment, then aspirate the HBSS from the apical compartment and replace it with the Lucifer Yellow solution. For the apical stress test, the Lucifer Yellow solution containing 350 μg / ml rhamnolipid or the Lucifer Yellow solution without stressor for the control wells was loaded into the apical compartment, followed by the addition of blank HBSS to the basolateral compartment. For the basolateral stress test, the Lucifer Yellow solution was added to the apical side of all wells, and 5% EtOH in HBSS or blank HBSS in the control wells was placed into the basolateral compartment.

[0487] After incubation for 3 h 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.

[0488] After TEER measurement, 100 μl aliquots were taken in duplicate from the basolateral sites 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).

[0489] Microbiome composition analysis by shotgun sequencing

[0490] For the first set of in vitro experiments, total DNA was extracted from all samples collected throughout the study using the QiAmp DNA Stool Mini Kit (Qiagen, Crawley, UK) according to the manufacturer’s instructions, except for the addition of a bead beating step and increasing the lysis temperature to 95 °C as previously described (McCormack et al., 2018).

[0491] After DNA separation, DNA was quantified using Qubit high-sensitivity DNA assay (Thermo Fisher). Complete metagenomic libraries were then prepared using Illumina Nextera XT kits (Illumina) according to the manufacturer's instructions, which had the following modifications: First, the labeling time was extended to 7 min, and secondly, after the index and Amp purification of the product were incorporated, the samples were individually analyzed for size by running on Agilent high-sensitivity chips (Agilent) and quantified using Qubit high-sensitivity DNA assays (Thermo Fisher) according to the Teagasc sequencing platform SOP. The samples were then merged equimolarly and sequenced (300 cycles) on IlluminaNextSeq500 with NextSeq 500 / 550v2 high-output kits. All sequencing was performed in Teagasc sequencing facilities according to standard Illumina sequencing protocols.

[0492] The delivered raw FASTQ sequence files were subjected to the following quality checks: poor quality and duplicate reads were removed, and trimmed using a combination of SAM and Picard tools. Taxonomies were assigned to reads using Kraken software and functional analysis was performed using Superfocus.

[0493] For the second set of in vitro experiments, two techniques were combined according to Vandeputte et al. (2017) to characterize in detail the community shifts caused by the different treatments: 1) 16S targeted Illumina sequencing, a PCR-based method by which microbial sequences can be amplified to saturation, thus providing proportional abundances of different taxa at different phylogenetic levels (microbial phyla, family, and OTU levels). 2) Accurate quantification of total bacterial cells in the samples by flow cytometry. Combining the high-resolution phylogenetic information of 16S targeted Illumina with accurate quantification of cell counts by flow cytometry resulted in quantitative counts of different taxonomic entities within the reactor.

[0494] HT29 cell culture and quantification of immune biomarkers

[0495] For both 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 into 12-well plates at 8x105 cells per well and used after 2 days of pre-culture. Before treatment, they were starved for 18 h in DMEM containing 0.25% FBS. Cells were treated for 24 h with 100 ng / mL of TNF-α or fermentation supernatant, which was sterile filtered and diluted 1:20 in DMEM containing 0.25% FBS. All treatments were repeated three times. Therefore, cytokines, chemokines and interleukins in HT29 supernatants can be quantified using Luminex technology (LiquiChip Workstation IS 200, Qiagen, Hilden, Germany) with the Bio-Plex Pro Human Cytokine Panel Kit (Bio-Rad, Hercules, CA) or with the Luminex Screening Assay Kit (R&D Systems, Inc., Minneapolis, MN) according to the manufacturer's instructions. Data were acquired with Luminex IS 2.3 software and evaluated with the LiquiChip Analyzer software (Qiagen, Hilden, Germany).

[0496] Gas generation (generation)

[0497] For both sets of in vitro experiments, colonic incubations were performed in a closed incubation system as described above. This allowed the assessment of gas accumulation in the headspace, which can be measured using a manometer. Gas production is a measure of microbial activity and, therefore, also of the fermentation rate of potential prebiotic substrates.

[0498] result

[0499] The first set of experiments Figure 1-15 shown.

[0500] Regarding acetate production after 48 h of fermentation after administration of vitamins and ω-3 fatty acids, the following observations were made: compared to the corresponding controls

[0501] - Supplementation with 1x and 5x doses of vitamin E to improve acetate production

[0502] - Supplementation with 1x dose of vitamin A to improve acetate production

[0503] - Supplement with 1x dose of vitamin B2 + C to increase acetate production

[0504] - Supplementation with 0.2x, 1x, and 5x doses of folic acid to increase acetate production

[0505] - Supplementation with 0.2x, 1x, and 5x doses of vitamin K1 increased acetate production

[0506] - Supplementation with 0.2x, 1x and 5x doses of DHA increased acetate production

[0507] - Supplementation with 0.2x and 1x doses of DHA+EPA improves acetate production

[0508] - Supplementation with 0.2x and 1x doses of EPA increased acetate production.

[0509] Regarding propionate production after 48 h of fermentation after administration of vitamins and ω-3 fatty acids, the following observations were made: compared to the corresponding controls

[0510] - Supplement with 1x dose of vitamin E to increase propionate production

[0511] - Supplementation with 1x and 5x doses of vitamin D increased propionate production

[0512] - Supplementation with 0.2x, 1x, and 5x doses of vitamin A increased propionate production

[0513] - Supplement with 0.2x dose of vitamin B2 + C to increase propionate production

[0514] - Supplementation with 0.2x, 1x, and 5x doses of folic acid to increase propionate production

[0515] - Supplementation with 0.2x, 1x, and 5x doses of vitamin K1 increased propionate production

[0516] - Supplementation with 0.2x, 1x and 5x doses of DHA increased propionate production

[0517] - Supplementation with 0.2x, 1x and 5x doses of DHA+EPA to increase propionate production

[0518] - Supplementation with 0.2x, 1x and 5x doses of EPA increased propionate production.

[0519] Regarding butyrate production after 48 h of fermentation following administration of vitamins and ω-3 fatty acids, the following observations were made:

[0520] -Supplementation with 5x dose of vitamin B2 to increase butyrate production

[0521] - Supplementation with 5x dose of vitamin B2-5-phosphate to increase butyrate production

[0522] - Supplementation with 0.2x and 1x doses of vitamin D improves butyrate production

[0523] - Supplementation with 0.2x, 1x, and 5x doses of vitamin A increased butyrate production

[0524] - Supplementation with 0.2x, 1x, and 5x doses of folic acid to improve butyrate production

[0525] - Supplementation with 0.2x, 1x, and 5x doses of vitamin K1 increased butyrate production

[0526] - Supplementation with 0.2x, 1x and 5x doses of DHA increased butyrate production

[0527] - Supplementation with 0.2x and 1x doses of DHA+EPA improves butyrate production

[0528] - Supplementation with 0.2x, 1x and 5x doses of EPA increased butyrate production.

[0529] Regarding total SCFA production after 48 h of fermentation following administration of vitamins and ω-3 fatty acids, the following observations were made:

[0530] -Supplementation with 1x dose of vitamin E increases total SCFA production

[0531] -Supplementation with 1x and 5x doses of vitamin D increased total SCFA production

[0532] -Supplementation with 0.2x and 5x doses of vitamin A increased total SCFA production

[0533] - Supplementation with 0.2x, 1x, and 5x doses of folic acid increased total SCFA production

[0534] -Supplementation with 0.2x, 1x, and 5x doses of vitamin K1 increased total SCFA production

[0535] - Supplementation with 0.2x, 1x and 5x doses of DHA increased total SCFA production

[0536] - Supplementation with 0.2x and 1x doses of DHA+EPA improves total SCFA production

[0537] - Supplementation with 0.2x and 1x doses of EPA increased total SCFA production.

[0538] About the healthy intestinal cell model ( Figure 6 ) were treated with 48 h fermentation samples after subtracting the TEER of cells treated with 0 h fermentation samples, and the following observations were made: compared with the corresponding controls,

[0539] - Treating cells with samples from fermentation of 0.2x dose of vitamin B2-5-phosphate increases TEER

[0540] - Treating cells with samples from fermentation with 1x dose of vitamin C increases TEER

[0541] - Treating cells with samples from fermentation with 1x and 5x doses of vitamin E increased TEER

[0542] - Treating cells with fermentation samples from 0.2x, 1x and 5x doses of DHA+EPA increased TEER

[0543] - Treating cells with samples from fermentations at 0.2x, 1x and 5x doses of EPA increased TEER.

[0544] Regarding the leaky gut cell model ( Figure 7 ) in the basolateral induction of the cells treated with the 48h fermentation sample after subtracting the TEER of the cells treated with the 0h fermentation sample, the following observations were made: compared with the corresponding control,

[0545] - Treating cells from fermentation samples with 0.2x dose of vitamin B2 increases TEER

[0546] - Treating cells with fermentation samples from 0.2x dose of vitamin B2 + vitamin C to increase TEER

[0547] - Treating cells with samples from fermentation with 0.2x dose of vitamin E to increase TEER

[0548] - Treating cells with samples from fermentation with 0.2x and 1x doses of DHA+EPA increased TEER

[0549] - Treatment of cells with samples from fermentation with 0.2x and 1x doses of EPA increased TEER.

[0550] Regarding the leaky gut cell model ( Figure 7 ) in the basolateral induction of the cells treated with the 48h fermentation samples. The following observations were made by measuring the fluorescein yellow after TEER of the cells treated with the 48h fermentation samples: compared with the corresponding control,

[0551] - Treatment of cells with 0.2x dose of vitamin B2 from fermentation samples reduced fluorescent yellow

[0552] - Reduced fluorescent yellow from fermentation samples treated with 0.2x dose of vitamin B2 + vitamin C

[0553] - Reduced fluorescent yellow from fermentation samples treated with 0.2x dose of vitamin E

[0554] - Treatment of cells with samples from fermentation with 0.2x dose of DHA+EPA reduced lucifer yellow.

[0555] Regarding the leaky gut cell model ( Figure 8 ) in apical induction, the TEER of cells treated with 48 h fermentation samples was measured after subtracting the TEER of cells treated with 0 h fermentation samples, and the following observations were made: compared with the corresponding controls,

[0556] - Treating cells from fermentation samples with 0.2x dose of vitamin B2 increases TEER

[0557] - Treating cells with samples from fermentation of 0.2x dose of vitamin B2-5-phosphate increases TEER

[0558] - Treating cells with fermentation samples from 0.2x and 1x doses of vitamin C increased TEER

[0559] - Treating cells with fermentation samples from 0.2x dose of vitamin B2 + vitamin C to increase TEER

[0560] - Treating cells with samples from fermentation with 0.2x, 1x and 5x doses of vitamin E increased TEER

[0561] - Treating cells with samples from fermentation with 5x dose of vitamin D increases TEER

[0562] - Treating cells with samples from fermentation with 0.2x, 1x and 5x doses of vitamin A increased TEER

[0563] - Treating cells with samples from fermentation with 0.2x and 1x doses of folic acid increased TEER

[0564] - Treating cells with samples from fermentation with 0.2x dose of DHA+EPA to increase TEER

[0565] - Treating cells with samples from fermentations at 0.2x, 1x and 5x doses of EPA increased TEER.

[0566] Regarding the leaky gut cell model ( Figure 8 ) in the apical induction of cells treated with 48h fermentation samples. The following observations were made by measuring the fluorescein yellow after TEER of cells treated with 48h fermentation samples: compared with the corresponding control,

[0567] - Treatment of cells with 0.2x dose of vitamin B2 from fermentation samples reduced fluorescent yellow

[0568] - Treatment of cells with samples from fermentation with 0.2x dose of vitamin B2-5-phosphate reduces fluorescent yellow

[0569] - Reduced fluorescent yellow in cells treated with fermentation samples from 0.2x and 1x doses of vitamin C

[0570] - Reduced fluorescent yellow from fermentation samples treated with 0.2x dose of vitamin B2 + vitamin C

[0571] - Treatment of cells with samples from fermentation with 1x and 5x doses of vitamin E reduced fluorescent yellow

[0572] - Treatment of cells with samples from fermentation with 1x and 5x doses of vitamin A reduced fluorescent yellow

[0573] - Treatment of cells with fermentation samples from 0.2x dose of vitamin K1 reduced fluorescent yellow

[0574] - Treatment of cells with samples from fermentation with 0.2x dose of DHA+EPA reduced lucifer yellow.

[0575] Regarding the alpha diversity of the microbiome after 24 h of fermentation after administration of vitamins and ω-3 fatty acids, the following observations were made: compared to the corresponding controls,

[0576] - Supplementation with 1x and 5x doses of vitamin B2 improves microbiome diversity

[0577] - Supplementation with 0.2x and 1x doses of vitamin B2-5-phosphate improves microbiome diversity

[0578] -Supplementation with 5x the dose of vitamin C improves microbiome diversity

[0579] -Supplementation with 0.2x, 1x, and 5x doses of vitamin E improved microbiome diversity

[0580] -Supplementation with 0.2x and 5x doses of vitamin D improved microbiome diversity

[0581] - Supplementation with 1x and 5x doses of vitamin A improved microbiome diversity

[0582] - Supplementation with 1x and 5x doses of vitamin B2 + vitamin C improves microbiome diversity

[0583] - Supplementation with 0.2x and 1x doses of folic acid improves microbiome diversity

[0584] - Supplementation with 0.2x, 1x, and 5x doses of vitamin K1 improved microbiome diversity

[0585] - Supplementation with 0.2x, 1x and 5x doses of DHA improves microbiome diversity

[0586] - Supplementation with 1x and 5x doses of DHA+EPA improves microbiome diversity

[0587] -Supplementation with 0.2x, 1x, and 5x doses of EPA improved microbiome diversity.

[0588] With regard to the relative abundance of Bifidobacteria after 24 h of fermentation following administration of vitamins and ω-3 fatty acids, the following observations were made: compared to the corresponding controls,

[0589] - Supplementation with 1x and 5x doses of vitamin B2 increased the relative abundance of Bifidobacterium

[0590] - Supplementation with 0.2x and 1x doses of vitamin B2-5-phosphate increased the relative abundance of Bifidobacterium

[0591] -Supplementation with 1x dose of vitamin C increased the relative abundance of Bifidobacterium

[0592] -Supplementation with 0.2x, 1x and 5x doses of vitamin E increased the relative abundance of Bifidobacterium

[0593] -Supplementation with 0.2x and 5x doses of vitamin D increased the relative abundance of Bifidobacterium

[0594] - Supplementation with 1x dose of vitamin A increased the relative abundance of Bifidobacterium

[0595] - Supplementation with 1x and 5x doses of vitamin B2 + vitamin C increased the relative abundance of Bifidobacterium

[0596] - Supplementation with 0.2x and 1x doses of folic acid increased the relative abundance of Bifidobacterium

[0597] -Supplementation with 0.2x dose of vitamin K1 increased the relative abundance of Bifidobacterium

[0598] - Supplementation with 0.2x and 5x doses of DHA increased the relative abundance of Bifidobacterium

[0599] - Supplementation with 5x dose of DHA+EPA increases the relative abundance of Bifidobacterium

[0600] - Supplementation with 0.2x, 1x and 5x doses of EPA increased the relative abundance of Bifidobacteria.

[0601] Similar observations were made for other beneficial bacteria.

[0602] The following observations were made regarding the relative abundance of genes involved in the acetyl-CoA fermentation to butyrate pathway after 24 h of fermentation following administration of vitamins and ω-3 fatty acids:

[0603] -Supplementation with 1x and 5x doses of vitamin B2 increased the relative abundance of genes involved in the acetyl-CoA fermentation to butyrate pathway

[0604] -Supplementation with 0.2x and 1x doses of vitamin B2-5-phosphate increased the relative abundance of genes involved in the acetyl-CoA fermentation to butyrate pathway

[0605] -Supplementation with 5x dose of vitamin C increased the relative abundance of genes involved in the acetyl-CoA fermentation to butyrate pathway

[0606] -Supplementation with 0.2x dose of vitamin E increased the relative abundance of genes involved in the acetyl-CoA fermentation to butyrate pathway

[0607] -Supplementation with 0.2x dose of vitamin D increased the relative abundance of genes involved in the acetyl-CoA fermentation to butyrate pathway

[0608] -Supplementation with 1x and 5x doses of vitamin A increased the relative abundance of genes involved in the acetyl-CoA fermentation to butyrate pathway

[0609] -Supplementation with 5x dose of vitamin B2 + vitamin C increased the relative abundance of genes involved in the acetyl-CoA fermentation to butyrate pathway

[0610] - Supplementation with 0.2x dose of folic acid increased the relative abundance of genes involved in the acetyl-CoA fermentation to butyrate pathway

[0611] -Supplementation with 0.2x, 1x, and 5x doses of vitamin K1 increased the relative abundance of genes involved in the acetyl-CoA fermentation to butyrate pathway

[0612] - Supplementation with 0.2x, 1x, and 5x doses of DHA increased the relative abundance of genes involved in the acetyl-CoA fermentation to butyrate pathway

[0613] - Supplementation with 0.2x, 1x, and 5x doses of DHA+EPA increased the relative abundance of genes involved in the acetyl-CoA fermentation to butyrate pathway

[0614] - Supplementation with 0.2x, 1x, and 5x doses of EPA increased the relative abundance of genes involved in the acetyl-CoA fermentation to butyrate pathway.

[0615] With regard to the production of GROa-CXCL1 by HT29 cells after treatment with effluent from 24 h fermentation after administration of vitamins and ω-3 fatty acids, the following observations were made: compared to the corresponding controls,

[0616] - Supplementation with 1x and 5x doses of vitamin B2 increased GROa-CXCL1 production

[0617] -Supplementation with 5x dose of vitamin E increases GROa-CXCL1 production

[0618] - Supplementation with 1x dose of vitamin B2 + vitamin C increases GROa-CXCL1 production

[0619] - Supplementation with 0.2x, 1x and 5x doses of DHA increased GROa-CXCL1 production

[0620] - Supplementation with 0.2x, 1x and 5x doses of DHA+EPA increased GROa-CXCL1 production

[0621] -Supplementation with 1x and 5x doses of EPA increased GROa-CXCL1 production.

[0622] With regard to the production of IL-8 by HT29 cells after treatment with effluent from 24 h fermentation after administration of vitamins and ω-3 fatty acids, the following observations were made: compared to the corresponding controls,

[0623] - Supplementation with 1x and 5x doses of vitamin B2 increased IL-8 production

[0624] -Supplementation with 5x dose of vitamin B2-5-phosphate increases IL-8 production

[0625] -Supplementation with 0.2x, 1x, and 5x doses of vitamin E increased IL-8 production

[0626] -Supplementation with 0.2x and 5x doses of vitamin A increased IL-8 production

[0627] - Supplement 1x dose of Vitamin B2 + Vitamin C IL-8 production

[0628] - Supplementation with 0.2x and 5x doses of folic acid increased IL-8 production

[0629] -Supplementation with 0.2x dose of vitamin K1 increases IL-8 production

[0630] - Supplementation with 0.2x, 1x and 5x doses of DHA increased IL-8 production

[0631] - Supplementation with 0.2x, 1x and 5x doses of DHA+EPA increased IL-8 production

[0632] - Supplementation with 0.2x, 1x and 5x doses of EPA increased IL-8 production.

[0633] With regard to the production of MIP3A-CCL20 by HT29 cells after treatment with effluent from 24 h fermentation after administration of vitamins and ω-3 fatty acids, the following observations were made: compared to the corresponding controls,

[0634] -Supplementation with 0.2x, 1x and 5x doses of vitamin E increased MIP3A-CCL20 production

[0635] - Vitamin D supplementation at 0.2x, 1x, and 5x doses increased MIP3A-CCL20 production

[0636] - Supplementation with 0.2x, 1x, and 5x doses of vitamin A increased MIP3A-CCL20 production

[0637] -Supplementation with 0.2x, 1x and 5x doses of vitamin K1 increased MIP3A-CCL20 production

[0638] - Supplementation with 0.2x, 1x and 5x doses of DHA increased MIP3A-CCL20 production

[0639] - Supplementation with 0.2x, 1x and 5x doses of DHA+EPA increased MIP3A-CCL20 production

[0640] - Supplementation with 0.2x, 1x and 5x doses of EPA enhanced MIP3A-CCL20 production.

[0641] With regard to gas production after 48 h of fermentation after administration of vitamins and ω-3 fatty acids, the following observations were made: compared to the corresponding controls,

[0642] Supplementation with 1x and 5x doses of vitamin C reduces gas production

[0643] Supplementation with 1x and 5x doses of vitamin E reduces gas production

[0644] Supplementation with 1x and 5x doses of vitamin C and B2 reduced gas production.

[0645] The second in vitro experiment Figure 16-19 As shown:

[0646] With regard to acetate production after 48 h of fermentation following administration of vitamins and β-carotene, the following observations were made:

[0647] - Supplementation with 1x and 5x doses of β-carotene increased acetate production in both donors compared to the corresponding controls.

[0648] Regarding propionate production after 48 h of fermentation after vitamin administration, the following observations were made:

[0649] - Supplementation with 1x and 5x doses of β-carotene increased propionate production in one of the two donors (donor C)

[0650] - Supplementation with 0.2x vitamin B5 increased propionate production in one of the two donors (donor C).

[0651] With regard to butyrate production after 48 h of fermentation after vitamin administration, the following observations were made:

[0652] - Supplementation with 1x dose of β-carotene increased butyrate production in both donors

[0653] - Supplementation with a 0.2x dose of β-carotene increased butyrate production in one of the two donors (donor C).

[0654] With regard to total SCFA production after 48 h of fermentation following administration of vitamins and β-carotene, the following observations were made:

[0655] - Supplementation with 1x and 5x doses of β-carotene increased total SCFA production in both donors compared to corresponding controls.

[0656] - Supplementation with a 5x dose of vitamin B5 reduced branched SCFA production in both donors compared to the corresponding controls.

[0657] With regard to the relative abundance of Bifidobacterium after 24 h of fermentation after vitamin administration, the following observations were made:

[0658] - Supplementation with 1x and 5x doses of β-carotene increased the relative abundance of Bifidobacterium in both donors

[0659] -Supplementation with 5x dose of vitamin B5 increased the relative abundance of Bifidobacterium in both donors

[0660] - Supplementation with 0.2x, 1x, and 5x doses of biotin increased the relative abundance of Bifidobacteria in one of the two donors (donor D)

[0661] - Supplementation with 1x and 5x doses of pyridoxine increased the relative abundance of Bifidobacteria in one of the two donors (donor D).

[0662] With regard to the relative abundance of Eggerthellaceae after 24 h of fermentation after vitamin administration, the following observations were made:

[0663] - Supplementation with 5x dose of β-carotene increased the relative abundance of Eagleziaceae in both donors compared to the corresponding controls.

[0664] The following observations were made regarding the relative abundance of Desulfovibrioideae after 24 h of fermentation after vitamin administration:

[0665] - Supplementation with 5x dose of β-carotene reduced the relative abundance of Desulfovibrioideae in both donors

[0666] - Supplementation with a 5x dose of vitamin B5 reduced the relative abundance of Desulfovibrioides in one of the two donors (donor C).

[0667] With regard to the relative abundance of Akkermansia after 24 h of fermentation after vitamin administration, the following observations were made:

[0668] - Supplementation with 5x dose of β-carotene increased the relative abundance of Akkermansia in both donors

[0669] - Supplementation with 1x dose of thiamine increased the relative abundance of Akkermansia in one of the two donors (donor C)

[0670] - Supplementation with 0.2x dose of niacinamide increased the relative abundance of Akkermansia in one of the two donors (donor C)

[0671] - Supplementation with 0.2x the vitamin B5 dose increased the relative abundance of Akkermansia in one of the two donors (donor D)

[0672] - Supplementation with 0.2x, 1x, and 5x doses of pyridoxine increased the relative abundance of Akkermansia in one of the two donors (donor D)

[0673] - Supplementation with a 0.2x dose of biotin increased the relative abundance of Akkermansia in one of the two donors (donor D)

[0674] - Supplementation with 0.2x and 1x doses of vitamin B12 increased the relative abundance of Akkermansia in one of the two donors (donor D).

[0675] Regarding the relative abundance of Prevotellaceae after 24 h of fermentation after vitamin administration, the following observations were made:

[0676] - Supplementation with 1x dose of β-carotene increased the relative abundance of Prevotellaceae in one of the two donors (donor C)

[0677] - Supplementation with a 5x dose of pyridoxine increased the relative abundance of Prevotellaceae in one of the two donors (donor C).

[0678] Regarding the relative abundance of Clostridiaceae after 24 h of fermentation after vitamin administration, the following observations were made:

[0679] - Supplementation with a 1x dose of β-carotene increased the relative abundance of Clostridiaceae in one of the two donors (donor C).

[0680] With regard to the relative abundance of Erysipelotrichaceae after 24 h of fermentation after vitamin administration, the following observations were made:

[0681] - Supplementation with 5x dose of thiamine increased the relative abundance of Erysipelothrix in one of the two donors (donor C)

[0682] - Supplementation with 1x and 5x doses of niacinamide increased the relative abundance of Erysipelothrix in one of the two donors (donor C)

[0683] - Supplementation with 1x dose of pyridoxine increased the relative abundance of Erysipelothrix in one of the two donors (donor C)

[0684] - Supplementation with 0.2x and 1x doses of biotin increased the relative abundance of Erysipelothrix in one of the two donors (donor C)

[0685] - Supplementation with 1x and 5x doses of vitamin B12 increased the relative abundance of Erysipelothrix in one of the two donors (donor C)

[0686] - Supplementation with a 1x dose of vitamin B5 increased the relative abundance of Erysipelothrix in one of the two donors (donor D).

[0687] With regard to the relative abundance of the family of Psoraleaceae after 24 h of fermentation after vitamin application, the following observations were made:

[0688] - Supplementation with 1x and 5x doses of β-carotene increased the relative abundance of Pseudomonasaceae in one of the two donors (donor D).

[0689] With regard to the relative abundance of Barnesiellaceae after 24 h of fermentation after vitamin administration, the following observations were made:

[0690] - Supplementation with 5x dose of β-carotene increased the relative abundance of Barnesellaceae in one of the two donors (donor D)

[0691] - Supplementation with a 1x dose of biotin increased the relative abundance of Barnesellaceae in one of the two donors (donor D).

[0692] Regarding the relative abundance of F. prausnitzii after 24 h of fermentation after vitamin administration, the following observations were made:

[0693] - Supplementation with 1x and 5x doses of β-carotene increased the relative abundance of F. prausnitzii in one of the two donors (donor D)

[0694] -Supplementation with 0.2x, 1x, and 5x doses of vitamin B5 increased the relative abundance of F. prausnitzii in one of the two donors (donor D)

[0695] - Supplementation with 0.2x, 1x, and 5x doses of pyridoxine increased the relative abundance of F. prausnitzii in one of the two donors (donor D)

[0696] - Supplementation with 0.2x, 1x, and 5x doses of biotin increased the relative abundance of F. prausnitzii in one of the two donors (donor D)

[0697] -Supplementation with 0.2x, 1x and 5x doses of vitamin B12 increased the relative abundance of F. prausnitzii in one of the two donors (donor D).

[0698] Regarding the relative abundance of Veillonellaceae after 24 h of fermentation after vitamin administration, the following observations were made:

[0699] - Supplementation with 1x and 5x doses of β-carotene increased the relative abundance of Veillonellaceae in one of the two donors (donor D)

[0700] - Supplementation with 0.2x the vitamin B5 dose increased the relative abundance of Veillonellaceae in one of the two donors (donor D)

[0701] - Supplementation with a 5x dose of pyridoxine increased the relative abundance of Veillonellaceae in one of the two donors (donor D).

[0702] Regarding microbiome diversity, the following observations were made:

[0703] Supplementation with 1x and 5x doses of β-carotene increased β-diversity in both donors. Supplementation with 0.2x dose of β-carotene increased β-diversity in one of the two donors (donor D).

[0704] Supplementation with 5x dose of vitamin B5 increased β-diversity in both donors. Supplementation with 0.2x and 1x doses of β-carotene increased β-diversity in one of the two donors (donor D).

[0705] Supplementation with 0.2x, 1x, and 5x doses of vitamin B6 increased β-diversity in one of the two donors (donor D).

[0706] Supplementation with 0.2x, 1x, and 5x doses of vitamin B12 increased β-diversity in one of the two donors (donor D).

[0707] Supplementation with 1x dose of vitamin B7 increased β-diversity in both donors.

[0708] Supplementation with a 5x dose of vitamin B7 increased β-diversity in one of the two donors (donor D).

[0709] Supplementation with a 1x dose of vitamin B1 increased β-diversity in one of the two donors (donor C). Supplementation with a 5x dose of vitamin B1 increased β-diversity in one of the two donors (donor D).

[0710] Supplementation with 0.2x and 5x doses of vitamin B3 increased the β-diversity of one of the two donors (donor D).

[0711] Supplementation with a 1x dose of vitamin B1 increased the β-diversity of one of the two donors (donor C) compared to the corresponding control.

[0712] Supplementation with 0.2x and 5x doses of vitamin B5 increased the α -Diversity.

[0713] Supplementation with 5x the dose of vitamin B6 increased the α -Diversity.

[0714] Supplementation with 0.2x and 1x doses of vitamin B12 increased the α -Diversity.

[0715] Supplementation with 0.2x dose of vitamin B7 increased α -Diversity. Supplementation with 5x the dose of vitamin B7 increased the α -Diversity.

[0716] Supplementation with 0.2x and 1x doses of vitamin B1 increased the α -Diversity.

[0717] Supplementation with a 1x dose of vitamin B3 increased α-diversity in one of the two donors (donor C).

[0718] Example 2

[0719] Preparations for use in humans

[0720] The following formulations each containing a single active ingredient were prepared and loaded into a S 100 polymer in enteric hard gelatin capsules (Capsugel).

[0721] Table 3. Preparations for human use

[0722]

[0723] 1 : Retinol equivalent

[0724] 2 : Tocopherol equivalent

[0725] The formulation is suitable for once-daily administration, preferably for at least 28 consecutive days.

[0726] Example 3

[0727] Poultry Model

[0728] Three groups of 120 chickens (all female) with the same genetic characteristics were housed in three separate rooms with 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.

[0729] Each group was sampled three times on specific days (ie S1 = 14 days, S2 = 28 days, S3 = 38 / 42 days) corresponding to the times when feeds were changed in the feeding program.

[0730] The 42-day broiler (end of the trial) weighed 2.6 kg and ate about 205 g per head per day. Therefore, the three test doses corresponded to riboflavin intakes of 0.38, 3.82 and 7.65 mg / kg body weight at 5, 50 and 100 mg / kg feed, respectively, and the intake per kg body weight was much higher at lower body weights. Table 4 below shows the theoretical growth of broilers and the amount of riboflavin added.

[0731] Table 4. Theoretical growth

[0732]

[0733] During each sampling period (ie, S1, S2, and S3), a total of 40 birds / group were humanely euthanized and 9 litters were collected (ie, 3 samples / group).

[0734] Cecal sample microbiota analysis

[0735] A total of 741 cecal samples were analyzed by the Microbial Ecology Unit of the Ministry of Health, including 120 samples from the first time point, 119 from the second time point (1 chicken was lost in group C), and 118 from the third time point (1 chicken was lost in group B and 1 chicken was lost in group C).

[0736] Materials and methods.

[0737] DNA extraction from cecal samples: The commercial kit DNeasy PowerSoil kit (Qiagen, Hilden, Germany) has been selected and applied to all available cecal samples following the manufacturer's instructions. DNA yields were measured using a Nanodrop instrument.

[0738] 16S rRNA gene PCR amplification and sequencing.Use 341F and 785R primers with Illumina extension adapter sequence to 16S rRNA gene V3-V4 hypervariable region PCR amplification.By using AMPure XP Beads magnetic beads (BeckmanCoulter, Brea, CA) to carry out amplicon purification.For the library of indexing, use NexteraXT DNA library preparation kit (Illumina, San Diego, CA).Carry out further magnetic bead purification step and use Qubit 3.0 fluorometer (Invitrogen) to quantify the library, then merge at 4nM.The library pool is denatured with 0.2N NaOH and diluted to 6pM.According to the manufacturer's instructions (Illumina), 2x 250bp paired end scheme is used for sequencing on the Illumina MiSeq platform.

[0739] 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 removing chimeric sequences, high-quality reads were filtered and divided into high-resolution operational taxa (OTUs) according to a 99% classification threshold by an open reference strategy with dada2 (Callahan et al., 2016). Taxonomy was assigned using the vsearch classifier (Rognes et al., 2016) and the SILVA database as a reference (Quast et al., 2013). Alpha diversity was measured using the Faith PD index and the number of observed OTUs. Statistics were performed using RStudio software version 1.0.136 (implemented with vegan, made4, PMCMR libraries) running on R software 3.1.3 (https: / / www.r-project.org / ). Beta diversity was assessed by calculating weighted UniFrac distances and visualized by principal coordinate analysis (PCoAs). The significance of separation between sample groups was tested by multivariate analysis of variance using the Adonis function of the vegan package. Bacterial phylogenetic groups (genus, family, phylum) that showed a minimum relative abundance of 0.5% in at least 1% of the samples were retained for further analysis and graphical display. Compositional differences between sample groups were tested using the Kruskal-Wallis test. P values ​​were corrected for multiple comparisons using the Benjamini-Hochberg method.

[0740] result

[0741] 16S rRNA amplicons obtained from 741 DNA samples were sequenced, yielding 4,986,865 high-quality reads ranging between 1,099 and 15,182, with an average of 6,490 ± 2715 reads per sample. Reads were grouped into 20,950 operational taxonomic units (OTUs) based on 99% similarity.

[0742] First, beta diversity analysis of all available samples of group B chickens ( Fig. 20 ) showed that the microbiota structure in the cecum had different longitudinal trajectories relative to groups A and C.

[0743] This trend is particularly evident when the weighted UniFrac distance is used to plot the entire sample set ( Fig. 20 A), while the PCoA obtained using the unweighted UniFrac distance showed a higher overlap between the different groups ( Fig. 20 B). This suggests that the differences in the microbiota of broiler chickens in group B are likely to be found in the most abundant bacterial species rather than in the minor bacterial species.

[0744] The same analysis was performed using samples from time points T1, T2, and T3, respectively ( Fig.21 At T1, samples from groups A, B, and C largely overlapped ( Fig.21 A and 21D), while group B samples began to cluster with each other at T2 and T3, especially when using the weighted UniFrac metric ( Fig.21 B and 21C).

[0745] On the contrary, in terms of biodiversity ( α Diversity, Fig.18 ), the nutritional regimen administered to chickens in Group C resulted in a significant increase in species richness (measured by “observed OTUs”) at time points T2 and T3 (P<0.0001) (Figures 22E and 22F).

[0746] Visualization of the mean microbiota distribution emphasizes that the compositional differences between the three experimental groups are appreciable at the phylum level ( Fig.23 In group B, the relative abundance of Bacteroidetes ( Fig.23 The blue in the figure) increased significantly at T2, while in group A, the relative abundance of Bacteroidetes only occurred at T3. The diet of group C seemed to partially inhibit this increase. From the composition analysis at the family level ( Fig.24 ) It can be seen that the diet administered only to group B promoted a gradual increase in Bacteroidetes ( Fig.24 ), while in groups A and C, the Bacteroidetes phylum was mainly composed of bacteria belonging to the family Rikenellaceae ( Fig.24 The light blue in the figure is composed of Fig.25 ) and genus level ( Fig.25 ). The Bacteroidetes family and the Bacteroides genus in group B showed higher abundance at all available time points. In contrast, the Rikenellaceae family and its Arisbezia genus showed higher abundance in groups A and C, but only at T3.

[0747] Differently, the average family-level distribution obtained for broilers in group C showed a gradual increase in the abundance of Bifidobacteriaceae from T1 to T3 ( Fig.24 ), which is not visible in panels A and B ( Fig.24 The importance of this difference is underlined by the differences between T2 and T3 ( Fig.25 ) and genus level (Bifidobacterium, Fig.26 ) confirmed. At the family level, it was also emphasized that the increase in Bifidobacteriaceae was harmful to Corynebacteraceae, another family belonging to the Actinobacteria phylum ( Fig.25 ).

[0748] 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, with the relative abundance of Faecalibacterium being significantly higher in groups B and C relative to that in group A at T1 ( Fig.26 ). The relative abundance of Faecalibacterium in the three groups did not differ significantly at the following time points. This longitudinal trend reflects the observation that the Ruminococcus family, to which Faecalibacterium belongs, Fig.25 At time point T2, another genus belonging to the Ruminococcus family, the coprosterol-producing [eubacteria] group, was more abundant in groups B and C relative to control group A ( Fig.26 ).

[0749] Description of the ileal microbiota in three groups of broiler chickens (Groups A, B, C) along the production cycle (time points T1, T2 and T3).

[0750] At the time of the initial observation, beta diversity analysis of all available ileum samples ( Fig. 27 ) showed that samples collected from three different groups of chickens (A, B and C) did not follow different longitudinal trajectories in terms of microbiota structure, unlike what was observed in the case of the cecal microbiota. In fact, in the case of both the weighted and unweighted UniFrac metrics used for sample distance calculation, the three groups of samples overlapped ( Fig. 27 A and B).

[0751] 16S rRNA amplicons obtained from 741 DNA samples were sequenced, yielding 4,986,865 high-quality reads ranging between 1,099 and 15,182, with an average of 6,490 ± 2715 reads per sample. Reads were grouped into 20,950 operational taxonomic units (OTUs) based on 99% similarity.

[0752] This was confirmed by plotting samples collected at different time points on separate PCoAs ( Fig.28 ).

[0753] In terms of biodiversity (α diversity, Fig.29 ), the nutritional regimens administered to the chickens in groups B and C were significantly lower than those in groups B at the first time point (T1, P < 0.001, Fig.29 A) resulted in a significant decrease in the diversity of the ileal ecosystem (calculated by Faith's PD index) relative to group A. In contrast, the same diet (groups B and C) resulted in an increase in species richness (calculated by the "observed OTU" metric) at time points T2 and T3 (significant only for time point T2, P < 0.0001) (29E and 29F).

[0754] Visualization of the mean microbiota distribution highlights that at the phylum level, the ileal microbiota reflects that reported for the cecum, with the majority of bacteria belonging to the phylum Firmicutes ( Fig.30 However, at lower phylogenetic levels (family), the specificity of the ileal microbiota is evident, with the Lactobacillus family (a family belonging to the phylum Firmicutes) dominating at all available time points ( Fig.31 of lavender).

[0755] Longitudinal analysis highlighted the Enterobacteriaceae ( Fig.31 The red color in the figure shows a gradual decrease from T1 to T3, independent of the nutritional regimen (groups A, B, and C). In addition, it is possible to notice a gradual decrease in the Clostridiaceae family from T1 to T3 ( Fig.31 dark green in the middle), which is beneficial to digesting Streptococcus ( Fig.31 This was particularly evident in T3 samples collected from birds in groups A and C (P < 0.0001, Fig.32 ). Peptostreptococci are a bacterial taxon with unknown function in the gut of broiler chickens. Studies focusing on the cecal microbiome of chickens have highlighted that the abundance of this taxon varies with diet, supplementation and antibiotic treatment (Munyaka et al., 2016; Costa et al., 2017; Zeits et al., 2019), whereas studies of the ileal microbiome remain too sparse to speculate on possible functional roles of changes in Peptostreptococci.

[0756] Description of the litter microbiota of three groups of broiler chickens (Groups A, B, C) along the production cycle (time points T1, T2 and T3).

[0757] At the time of initial observation, beta diversity analysis of all available litter samples ( Fig.33) showed that, in terms of microbiota structure, samples obtained from the boxes of chickens in group C were clustered separately from the other samples when the distances between samples were calculated using the unweighted Unifrac metric (Adonis test), and visualization of the average microbiota distribution emphasized that, at the phylum level, the ileal microbiota reflected that reported for the cecum, with the vast majority of bacteria belonging to the phylum Firmicutes ( Fig.30 However, at lower phylogenetic levels (family), the specificity of the ileal microbiota is evident, with the Lactobacillus family (a family belonging to the phylum Firmicutes) dominating at all available time points ( Fig.31 of lavender).

[0758] Longitudinal analysis highlighted the Enterobacteriaceae ( Fig.31 The red color in the figure shows a gradual decrease from T1 to T3, independent of the nutritional regimen (groups A, B, and C). In addition, it is possible to notice a gradual decrease in the Clostridiaceae family from T1 to T3 ( Fig.31 dark green in the middle), which is beneficial to the Peptostreptococcus family ( Fig.31 This was particularly evident in T3 samples collected from birds in groups A and C (P < 0.0001, Fig.32 ). Peptostreptococci is a bacterial taxon of unknown function in the gut of broiler chickens. Studies focusing on the cecal microbiome of chickens have highlighted that the abundance of this taxon varies with diet, supplementation and antibiotic treatment (Munyaka et al., 2016; Costa et al., 2017; Zeits et al., 2019), whereas studies of the ileal microbiome remain too sparse to speculate on possible functional roles of changes in Peptostreptococci.

[0759] Description of the litter biota of three groups of broiler chickens (Groups A, B, C) along the production cycle (time points T1, T2 and T3).

[0760] At the time of initial observation, beta diversity analysis of all available litter samples ( Fig.33 ) showed that, in terms of microbiota structure, samples obtained from the boxes of chickens from group C clustered separately from the other samples when the distances between samples were calculated using the unweighted Unifrac metric (Adonis test), and visualization of the mean microbiota distribution emphasized that, at the phylum level, the ileal microbiota reflected that reported for the cecum, with the vast majority of bacteria belonging to the phylum Firmicutes ( Fig.30 However, at lower phylogenetic levels (family), the specificity of the ileal microbiota is evident, with the Lactobacillus family (a family belonging to the phylum Firmicutes) dominating at all available time points ( Fig.31 Light purple in the middle; P = 0.006, Fig.33 B).

[0761] This was not confirmed by analysis based on weighted UniFrac distances ( Fig.33 A), indicating that the differences between the C group bedding microbiota and other bedding microbiota may exist in minor bacterial groups. Similarly, the separation between the C group bedding and other bedding samples in the PCoA based on the unweighted UniFrac distance ( Fig.33 B) Positioning along the second ordination axis (MDS2), which only helps explain 9.4% of the total variation in the dataset.

[0762] Visualization of average microbiota distribution ( Fig.34 and 35 ) highlighted that there was no single dominant taxon at either the phylum or family level. However, the bacterial composition (at the family or genus level) and species abundance / diversity (data not shown) of the litter microbiome did not differ significantly among the analyzed datasets.

[0763] Metabolomics analysis

[0764] Sample analysis

[0765] A total of 63 samples have been provided to the Bio-NMR team of the Department of Agricultural and Food Sciences and the Interdepartmental Center for Research on Industrial Agro-products (CIRI) at the University of Bologna, Italy, including 21 cecal samples collected at the first time point, 21 at the second time point, and 21 at the third time point.

[0766] Sample preparation

[0767] Initial analysis was performed on 63 samples divided into the following: control sample (A) fed 10 mg / kg vitamin B2, treated sample (B) fed 50 mg / kg vitamin B2, and treated sample (C) fed 100 mg / kg vitamin B2. Samples for nuclear magnetic resonance (NMR) analysis were prepared by vortexing the feces with 1 ml of deionized water for 5 minutes and then centrifuging at 14,000 rpm and 4°C for 10 minutes. Approximately 540 mL of supernatant was added to 100 μL of D 2 O1.5M phosphate buffer solution containing 0.1% TSP (3-(trimethylsilyl) propionic acid-d4), NaN 3 2 mM, set to pH 7.40. Prior to analysis, the samples were centrifuged again for 10 minutes and then 590 uL were transferred to NMR tubes.

[0768] NMR spectral acquisition

[0769] Proton NMR ( 1H-NMR) spectra. The deuterium hydroxide (HOD) residual signal was suppressed by presaturation, while the broad signal from the slowly tumbling molecules was removed by including a Carr-Purcell-Meiboom-Gill filter 27 in the free induction decay sequence. The filter consisted of a series of 400 echoes (800 μμs apart) with a total time of 328 ms. Each spectrum was acquired by summing 256 transients using 32K data points on a 7211.54 Hz spectrum (acquisition time of 2.27 seconds). The cycle delay was set to 8 seconds taking into account the longitudinal relaxation time of the protons studied. Each spectrum was processed with Top Spin 3.0 (Bruker) using the automatic command apkO.noe and by applying a line width factor of 1 Hz, which performed baseline and phase correction at once. The peaks were assigned by comparing their chemical shifts and multiplicities with the literature and by using the Chenomx NMR suit 8.1 software.

[0770] 1 H-NMR spectrum pretreatment

[0771] After Fourier transformation, phase and baseline correction, the spectra were calibrated with reference to the chemical shift of 0.00ppm assigned to the internal standard TSP; the peripheral areas of the spectra as well as the water signal were removed. Thereafter, the spectra were normalized (regional scaling) using the probability quotient algorithm (PQN) on two different regions, respectively, as this is best suited for such samples. After normalization and before any possible statistical analysis, the spectra were binned into 100 data points, each at an interval of 0.0183ppm. As a result, the new spectra consisted of 410 binned data, which were saved as matrices in text files and imported into R and Python spaces for multivariate statistical analysis (MvSA).

[0772] Statistical analysis

[0773] MvSA has been applied to hierarchical dataset matrices. Specifically, principal component analysis (PCA) and partial least squares discriminant analysis (PLS-DA) have been employed. PCA is used to explore a single type of omics data and identify the largest sources of variation, whereas the multilevel approach aims to emphasize within-subject treatment effects (in this case, samples belonging to groups B and C were treated with 50 and 100 mg / kg vitamin B2, respectively) separately from biological variation between biological samples.

[0774] result

[0775] NMR Spectra

[0776] Fig.36 The sample designated C281 at pH 7.13 was reported 1 H-NMR spectrum.

[0777] Several metabolites were assigned by comparing their chemical shifts and multiplicities with those in the literature and using Chenomx NMR Suit8.1 software (abbreviations for multiplicity are: s = singlet, dd = doublet of a doublet, d = doublet, t = triplet, 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, d: 1.030, m: 2.261 ppm, d: 3.583 ppm); 4) propionate (t:

[0778] 1.060ppm, q: 2.188ppm); 5) ethanol (t: 1.189ppm, q: 3.664ppm); 6) lactate (d: 1.335ppm, q: 4.124ppm); 7) alanine (d: 1.485ppm, q: 3.792ppm); 8) acetate (s: 1.925ppm); 9) glutamate (m: 2.047ppm, m: 2.1 34ppm, 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:

[0779] 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).

[0780] Multivariate statistical analysis.

[0781] First, different unsupervised dimension reduction methods (PCA, Factor Analysis, Independent Component Analysis, Kernel-driven PCA) and classifiers (Random Forest, ADABoosted Decision Tree, Support Vector Machine) were combined to find the best dimension reduction model that allowed separation between the groups and identification of any outlier samples. Fig.37 Display UnscaledResults of PCA performed on the bin and region datasets. Fig.37 The PCA in A shows the presence of two outliers (samples 44 and 46 are T3-A-C250 and T3-A-C261, respectively). After removing these outliers, the results of the new PCA are Fig.37 This is shown in B. The first two principal components account for 84% of the total variance but do not show any visible trend or separation, nor do they reveal any additional outliers.

[0782] Since the difference from the unmanaged approach is not emphasized, it is necessary to apply a managed approach ( Fig.38 ). After outlier filtering, 61 subjects with 437 spectral features were each used for the final PLS-DA (Szymahska, E. et al., 2012). A 5-component PLS-DA model was trained on the standard scaled data, where the combination of the first 3-PLS directions was the most informative in distinguishing the time group intervals (especially the first direction) ( Fig.38 A). Therefore, the best prediction accuracy was obtained by feeding the first 3-PLS directions into a linear kernel support vector classifier, with an average prediction accuracy of about 89%. The ADABoosting tree classifier was also evaluated for comparison, yielding slightly worse average performance (Valdes, G. et al., 2016). This result is consistent with the 2d-PLS-DA space projection, where the 3 groups appear to be linearly separable considering time as a variable; whereas no clear separation appears considering the control samples vs. the treated samples ( Fig.38 B). All steps of the pipeline used were cross-validated internally, with particular attention to the classification and prediction steps, which were subjected to stratified 10-fold cross-validation to avoid overfitting. The PLS-DA average weights were associated with the most informative signals in the spectra, where spectra were extracted after smoothing and baseline correction to enhance the signal-to-noise ratio and filter non-informative spectral signals.

[0783] The major metabolites involved in the separation are: uracil ( Fig.39 ), glutamate ( Fig.40 ), alanine( Fig.41 ), Creatine( Fig.42 ), butyrate ( Fig.43 ), propionate ( Fig.44 ) and acetate ( Fig.45 ). In these figures, the integrated area of ​​each metabolite of samples at different times (T0-T1-T2, corresponding to 14 days, 28 days and 38 / 42 days, respectively) has been compared.

[0784] In all treatments, uracil ( Fig.39 ) seem to increase in concentration until 42 days (T2), especially for sample C whose concentration is always higher than other concentrations. Fig.40 ) showed a trend of increasing concentration after 28 days (T1), especially in samples A and B, and remained constant at T3. Similarly, the integral of alanine ( Fig.41 ) Overall, it increased at T1 for sample C. In contrast, creatine showed an increasing trend only at T2 for samples A and B, but then its concentration decreased at T3 ( Fig.42 ).

[0785] Fig.43 and 44 related to the organic acids identified in the spectra. As before, the integrals of the interval peaks were compared between the three groups according to the time point. Fig.43 and 44 Results are shown for butyrate and propionate.

[0786] Butyrate ( Fig.43 ) does not seem to be affected by treatment and time. In fact, the average integrated area at T0, T1 and T2 in all samples is about the same amount. For acetate, the trend results are the same ( Fig.45 ). For propionate, there is an increasing trend ( Fig.44 ).

[0787] It is important to highlight that the results were affected by the shortage of data and therefore no clear trends could be generated from them. Therefore, further analysis has been performed and trends have been fitted for the 4 main metabolites: Fig.46 Shows how the integrated area of ​​a specified organic acid in each group changes over time.

[0788] Metagenomic analysis

[0789] Sample analysis

[0790] A total of 63 samples were provided to the Food Safety Laboratory of the Department of Agricultural and Food Sciences of the University of Bologna (UNIBO), including 21 cecal samples collected from the first time point, 21 cecal samples collected from the second time point, and 21 samples collected from the third time point.

[0791] Methodology of metagenomic analysis

[0792] DNA extraction for metagenomic sequencing

[0793] DNA was extracted from each sample of cecal contents using a bead-beating procedure. Briefly, 0.25 g of cecal contents were suspended in 1 ml of lysis buffer (500 mM NaCl, 50 mM Tris-C1, pH 8.0, 50 mM EDTA, 4% SDS) with MagNA Lyser Green Beads (Roche, Milan, Italy) and homogenized on a MagNA Lyser (Roche) at 6500 rpm for 25 seconds. The samples were then heated at 70°C for 15 min and then centrifuged to separate DNA from bacterial cell debris. This process was repeated with a second 300 pi aliquot of lysis buffer. The samples were then subjected to 10 M v / v ammonium acetate precipitation (Sigma, Milan, Italy), followed by isopropanol (Sigma) precipitation and 70% ethanol (Carlo Erba, Milan, Italy) washes and resuspended in 100 ul 1X Tris-EDTA (Sigma). The samples were then treated with DNase-free RNase (Roche) and incubated overnight at 4 °C and then purified by DNA StoolMini Kit (Qiagen, Milan, Italy). (Eppendorf, Milan, Italy) to assess the quantity and quality of DNA. All samples that meet the DNA quality score (i.e., A260 / 280nm ratio between 1.8 and 2.0) will be used for library preparation using the Nextera XT DNA library preparation kit (Illumina, San Diego, CA). Then, all libraries with an average size between 300 and 500 bp and a minimum concentration of 4 pM will be sequenced in paired-end mode using the NextSeq500 (Illumina) at 150 bp.

[0794] Sequencing strategies and platforms

[0795] All samples were sequenced in paired-end format. The average read length was 300 bp. Sequencing runs were performed using Illumina's NextSeq500. The submitted genomes were characterized by outputs between 20 Mbp and 12 Gbp.

[0796] Bioinformatics analysis

[0797] The raw reads were filtered and curated using the MGRast (MR) (https: / / www.mg-rast.org) (Keegan et al., 2016) bioinformatics pipeline. Additionally, functional annotation was performed on the MG-RAST v3.6 metagenome 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). For functional classification, genes were mapped to SEED and KEGG (Kyoto Encyclopedia of Genes and Genomes) classifications using SEED and KEGG identifiers, respectively.

[0798] Statistical analysis

[0799] Comparative functional analysis was performed using STAMP v2.0.8 software (Parks and Beiko, 2010; Parks et al., 2014) for statistical analysis. Gene counts were normalized by dividing the number of gene hits in an individual function by the number of gene hits in each metagenome dataset to eliminate biases due to differences in sequencing efforts. To identify SEED or KEGG functions with differential abundances in the metagenome, statistical tests on relative gene abundances were performed by applying a two-sided Welch's exact test with Benjamini-Hochberg false discovery rate (FDR) multiple testing correction method and a p-value < 0.05.

[0800] Results

[0801] First, principal component analysis (PCA) based on the "functional trait data" aimed to find the best dimensionality reduction model that could separate between groups and identify any outlier samples. In Fig.47 it, the PCA results for 63 subjects with 6361 functional traits were reported. The first two principal components accounted for 81% of the total variance; representing a clearly visible separation between the treatment groups (groups B and C) and the control group (group A). Regarding time points, no separation could be identified.

[0802] Description of the main KEGG metabolic pathways of three groups of broiler chickens (groups A, B, C) along the production cycle (time points T1, T2, and T3).

[0803] For each available time point (T1, T2, T3), visualization of the average functional gene distribution at level 1 based on the KEGG database of the ceca of broiler chickens in three different groups A, B, and C emphasized that the compositional differences between the three experimental groups were evident at level 1 ( Fig.49 ). In particular, in group A, the relative abundance of genetic information processing compared to the treatment groups ( Fig.49In contrast, the relative abundance of the environmental information treatment ( Fig.50 A) had a statistically significant increase in both groups A and B at each time point compared to group A (Table 5). In addition, the relative abundance of the metabolites ( Fig.50 B) was statistically increased in Groups B and C compared to Group A, and statistically increased in Group A compared to Groups B and C at 28 days of age (Table 5).

[0804] Table 5 Relative abundance of major KEGG metabolic pathways in broiler cecum ≥ 0.1%, showing statistical differences between treatment groups (groups B and C) and control group (group A) at each time point (T1, T2, T3)

[0805]

[0806] * = When statistical significance was reached (P < 0.05), Bejamini Hocherg corrected P values ​​are reported.

[0807] Description of secondary KEGG metabolic pathways in three groups of broiler chickens (groups A, B, and C) along the production cycle (time points T1, T2, and T3).

[0808] Visualization of secondary KEGG metabolic pathways with relative abundance ≥ 0.5% emphasizes that compositional differences between the three experimental groups were evident for each time point ( Fig.51 ) (i.e. translation and transcription at 14 days; membrane transport, translation, replication and repair at 28 days; membrane transport, translation, replication and repair, nucleotide metabolism at 42 days).

[0809] In particular, in groups B and C, the relative abundance of cofactors and vitamins at each time point day was statistically significantly increased compared to group A (light grey in Tables 6, 7, and 8 below). In contrast, the relative abundance of translation, transcription, and environmental adaptation at each time point was statistically significantly increased in group A compared to groups B and C (white in Table 5).

[0810] Considering other pathways, the relative abundance of membrane transport, replication and repair, biosynthesis of other secondary metabolites, metabolism of terpenoids and polyketides, and metabolism of other amino acids in groups B and C were statistically increased at 28 and 42 days of age compared with group A (orange in Tables 7 and 8). In contrast, the relative abundance of translation, energy metabolism, signal transduction, transcription, infectious diseases, and environmental adaptation in group A were statistically increased at 28 and 42 days of age compared with groups B and C (white in Tables 7 and 8). Finally, at 28 days of age, the relative abundance of lipid metabolism in groups B and C was significantly increased compared with group A (light gray in Tables 6 and 7), while folding, sorting and degradation, cell communication and signaling molecules, and interactions in group A were statistically increased at 28 days of age compared with groups B and C (white in Table 7).

[0811] Table 6 Secondary KEGG metabolic pathways with relative abundance ≥ 0.5% in cecum of broiler chickens and showing statistically significant differences between (Groups B and C) and the control group (Group A) at T1 (14 days of age)

[0812]

[0813] * = When statistical significance was reached (P < 0.05), Bejamini Hocherg corrected P values ​​are reported.

[0814] Table 7 Secondary KEGG metabolic pathways with relative abundance ≥ 0.5% in cecum of broiler chickens and showing statistically significant differences between the treatment groups (Groups B and C) and the control group (Group A) at T2 (28 days of age)

[0815]

[0816]

[0817] * = When statistical significance was reached (P < 0.05), Bejamini Hocherg corrected P values ​​are reported.

[0818] Table 8 Secondary KEGG metabolic pathways with relative abundance ≥ 0.5% in cecum of broiler chickens and showing statistically significant differences between the treatment groups (Groups B and C) and the control group (Group A) at T3 (42 days of age)

[0819]

[0820]

[0821] * = When statistical significance was reached (P < 0.05), Bejamini Hocherg corrected P values ​​are reported.

[0822] Description of the three-level KEGG metabolic pathways of three groups of broiler chickens (groups A, B, and C) along the production cycle (time points T1, T2, and T3).

[0823] When analyzing tertiary KEGG metabolic pathways, no differences were seen between experimental groups at 14 days of age (data not reported), while a number of pathways showed significant differences at 28 and 42 days of age (Tables 9 and 10 below).

[0824] In particular, at the ages of 28 and 42 days, 19 pathways with relative abundance ≥ 0.1% (ABC transporters, DNA replication, pyrimidine metabolism, homologous recombination, pentose phosphate pathway, phenylalanine, tyrosine and tryptophan biosynthesis, nicotinate and niacinamide metabolism, phenylpropanoid biosynthesis, sulfur relay system, glycerophospholipid metabolism, methane metabolism, pantothenate and CoA biosynthesis, selenocompound metabolism, folate biosynthesis, phosphoinositide metabolism, lysosome, butyrate metabolism, biotin metabolism, phosphonate and phosphinate metabolism) were statistically significantly increased in groups B and C compared with group A (light gray in Tables 9 and 10). In contrast, nine pathways with relative abundance ≥ 0.1% (alanine, aspartate and glutamate metabolism, purine metabolism, RNA polymerase, RNA degradation, two-component system, 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 (self-expressed in Tables 9 and 10).

[0825] In addition, 27 pathways (aminoacyl-tRNA biosynthesis, glycolysis / gluconeogenesis, oxidative phosphorylation, starch and sucrose metabolism, arginine and proline metabolism, galactose metabolism, amino sugar and nucleotide sugar metabolism, peptidoglycan biosynthesis, histidine metabolism, phosphotransferase system (PTS), terpenoid backbone biosynthesis, fructose and mannose metabolism, fatty acid biosynthesis, citrate cycle (TCA cycle), base excision repair, mismatch repair, tyrosine metabolism, glyoxylate and dicarboxylate metabolism, bacterial chemotaxis, peroxisome, other glycan degradation, glutathione metabolism, vitamin B6 metabolism, zeatin biosynthesis, lipopolysaccharide biosynthesis, tryptophan metabolism, calcium signaling pathway) were significantly increased in Groups B and C compared to Group A (light grey in Table 9) at 28 days of age.

[0826] Of particular interest were metabolic pathways such as fatty acid biosynthesis, phosphotransferase system (PTS), and butyrate metabolism, which were increased in the treatment groups and are associated with SCFA production. In addition, statistically significant increases in the treatment groups (Groups B and C) were reported for vitamin B pathways such as nicotinate and niacinamide metabolism, pantothenate biosynthesis, folate biosynthesis, biotin metabolism, and vitamin B6 metabolism.

[0827] Table 9 Tertiary KEGG metabolic pathways in the cecum of broiler chickens and showing statistically significant differences between the treatment groups (Groups B and C) and the control group (Group A) at 28 days of age

[0828]

[0829]

[0830]

[0831]

[0832] * = When statistical significance was reached (P < 0.05), Bejamini Hocherg corrected P values ​​are reported.

[0833] Table 10 Level 3 KEGG metabolic pathways with relative abundance ≥ 0.1% in cecum of broiler chickens and showing statistically significant differences between the treatment groups (Groups B and C) and the control group (Group A) at 42 days of age

[0834]

[0835]

[0836] * = When statistical significance was reached (P < 0.05), Bejamini Hocherg corrected P values ​​are reported.

[0837] Evaluation of gene abundance related to SCFA and glucosidase

[0838] Since short-chain fatty acids (SCFA) play an important role in intestinal physiology, we specifically analyzed the metabolic pathways leading to the synthesis of butyrate and propionate. As described in the literature, there are 4 potential pathways leading to butyrate production. The first and main one is based on acetyl-CoA acetyltransferase ( Fig.39 ).

[0839] Considering the relative abundance of genes related to butyrate production in our study, we identified that at least 7 metabolic functions at 28 days of age and 5 metabolic functions at 42 days of age were statistically significantly increased in the treatment groups (Groups B and C) compared to Group A (Table 11 below). This evidence may be related to the cross-feeding activity of different microbial genera such as Faecalibacterium and Eubacterium (Polansky et al., 2016, Rowland et al., 2018) and Bifidobacterium (as reported by Reviere et al., 2016). All of these genera have been identified in our study by microbiota analysis and these results can predict the potential production of butyrate in animals treated with vitamin B2.

[0840] Table 11 Percentage of relative abundance of functional genes related to butyrate production in broiler cecum (KEGG and SEED databases), showing statistically significant differences between the treatment groups (Groups B and C) and the control group (Group A) at 28 and 42 days of age

[0841]

[0842] * = When statistical significance was reached (P < 0.05), Bejamini Hocherg corrected P values ​​are reported.

[0843] Very interestingly, there was evidence of a statistically significant increase in the relative abundance of functional genes related to β-galactosidase in the cecum of broiler chickens in the treated groups (groups B and C) compared to group A (Table 12 below). This result could be explained by the increase in Bifidobacterium in the treated groups as well as Lactobacillus species as reported in the microbiota analysis (Shaima et al., 2017). In humans, this enzyme is found in high abundance in the colon, where it promotes lactose fermentation, and its activity measured indicates the ability of the colonic microbiota to ferment intestinal lactose.

[0844] Table 12 Relative abundance percentage of functional genes related to β-galactosidase (KEGG and SEED databases) in broiler cecum, showing statistically significant differences between the treatment groups (Groups B and C) and the control group (Group A)

[0845]

[0846] * = When statistical significance was reached (P < 0.05), Bejamini Hocherg corrected P values ​​are reported.

[0847] in conclusion

[0848] Both dietary regimens administered to groups B and C induced changes in the cecal microbiota, including an increase in the well-known anti-inflammatory and health-promoting bacteria belonging to the genus Faecalibacterium during the first period of the production cycle of broiler chickens (T1) relative to the control diet. In the second and third phases of the production cycle (T2 and T3), the relative abundance of Faecalibacterium did not differ significantly between the three groups. In summary, our results appear to emphasize that Faecalibacterium increases naturally during the production cycle of broiler chickens (control diet, group A), while the dietary regimens administered to broiler chickens in groups B and C appear to accelerate this increase.

[0849] The diet administered to group C also induced a gradual increase in the abundance of another health-promoting group of Bifidobacteria, as well as an increase in species richness, relative to the control diet (administered to group A). ​​In contrast, the diet administered to group B favored an increase in the abundance of Bacteroidetes throughout the production period.

[0850] Increases in Bacteroidetes would harm another member of the Bacteroidetes phylum, Rikenellaceae (Alicebeckia).

[0851] • Both diets administered to broiler chickens in groups B and C had a slight effect on ileal microbiota composition and diversity, and no significant effect on the microbiota was detected in the litter samples.

[0852] As mentioned before, the shortage of samples selected for metabolic studies affects the quality of the data and results. Therefore, it is important to have a more robust data set that includes the analysis of all samples. However, it seems clear that metabolites such as acetate, glutamate, propionate and butyrate are all affected by the treatment.

[0853] • PCA performed on 63 subjects with 6361 functional characteristics showed a clear vitamin B2 effect, which resulted in separation between the treatment groups (Groups B and C) and the control group (Group A). ​​The results were not highlighted as a function of analysis time.

[0854] Considering the major KEGG metabolic pathways, the relative abundance of genetic information processing and biological systems increased in group A (5 ppm vitamin B2), while environmental information processing and metabolism increased in the treatment groups (50 and 100 ppm vitamin B2).

[0855] · Observing the secondary KEGG metabolic pathways with a relative abundance ≥ 0.5%, the results highlighted clear compositional differences between the three experimental groups for each time point. In particular, the relative abundance of metabolism of cofactors and vitamins, membrane transport, replication and repair, biosynthesis of other secondary metabolites, metabolism of terpenes and polyketides, and metabolism of other amino acids increased in the treatment groups at 50 and 100 ppm of vitamin B2. In contrast, translation, energy metabolism, signal transduction, transcription, infectious diseases, and environmental adaptation were statistically increased in the control group at 5 ppm of vitamin B2.

[0856] Looking at the tertiary KEGG metabolic pathways, no differences were reported between the experimental groups at 14 days of age, while many pathways showed significant differences at 28 and 42 days of age. Particularly interesting were metabolic pathways such as fatty acid biosynthesis, phosphotransferase system (PTS), and butyrate metabolism, which were increased in the treatment groups (50 and 100 ppm of vitamin B2) and are related to the production of SCFAs. In addition, statistically significant increases in the treatment groups were reported for vitamin B pathways such as nicotinate and niacinamide metabolism, pantothenate biosynthesis, folate biosynthesis, biotin metabolism, and vitamin B6 metabolism.

[0857] At least 7 metabolic functions at 28 days of age and 5 at 42 days of age related to butyrate production were statistically increased in the treated groups (50 and 100 ppm vitamin B2) compared to the control group (5 ppm vitamin B2). This evidence may be related to the cross-feeding activity of different microbial genera, such as Faecalibacterium and Eubacterium (Zam et al., 2016; Rowland et al., 2018), as well as Bifidobacterium (as reported by Reviere et al., 2016). All these genera have been identified in our study by microbiota analysis, and these results may predict the potential production of butyrate in animals treated with vitamin B2.

[0858] Example 4

[0859] Pig Model

[0860] Materials and methods

[0861] Three pigs were provided with a commercial diet for 4 weeks. The diet included: barley, wheat and soybean meal as the main ingredients, and its main chemical composition was (g / kg): 151 crude protein, 34 crude fat, 40 crude fiber, 55 and ash. The animals were weighed at the beginning of the experimental period (average; 23.9±1.01Kg) and at the end (average; 43.3±3.56Kg). After 30 days of adaptation, the animals were slaughtered. The pH of the stomach, small intestine and hindgut contents was directly measured with a glass electrode pH meter (CRISON 507, CRISON, Barcelona, ​​Spain). The stomach and small intestine contents were immediately filtered, sampled and stored at -20°C and used as digestion inoculum to evaluate in vitro digestibility. Regarding the cecal contents, one portion was immediately sampled, distributed in 20 ml test tubes, frozen in liquid nitrogen, and stored at -80°C to be used as frozen inoculum for in vitro gas production studies. The other portion was directly used as fresh fermentation inoculum.

[0862] In vitro enzymatic digestion

[0863] To determine the enzymatic digestibility in the stomach and small intestine of pigs, two experiments were performed:

[0864] A-Using exogenous enzymes:

[0865] For each treatment, Erlenmeyer flasks (total volume 100 mL) containing 0.5 g of substrate alone or supplemented with the three eubiotics were incubated in duplicate to determine their in vitro enzymatic digestibility according to the three-step method of Boisen and Fernandez (1997). The digestibility was determined by:

[0866] [1] Pepsin

[0867] [2] Pepsin + Pancreatin

[0868] [3] Pepsin + pancreatin + mucin

[0869] B. Using stomach contents and small intestine contents

[0870] A similar enzymatic digestion procedure was followed, but in this case the pepsin and trypsin solutions were replaced by the stomach and small intestinal contents of each animal, eliminating the third step of Boisen and Fernandez (1997). Digestibility was determined by:

[0871] Gastric contents (9 mL)

[0872] Gastric contents + small intestine contents (9 mL)

[0873] In vitro gas production

[0874] The microbial fermentation pattern of cecal contents was studied in vitro by the gas production technique (Theodorou et al., 1994. Anim Feed Sci Technol 48: 185-1-971994. Anim Feed Sci Technol 48: 185-1-97). A 0.8 g amount of predigested feed (incubated in pepsin-HCl and pancreatin incubations in succession according to the procedure of Boisen and Fernández, 1997 Anim Feed Sci Technol 51: 29-43) was used as substrate alone or supplemented with vitamin B2, simulating the digestive processes that occur before the cecum. First, triplicate glass bottles (total volume 110 mL) were filled with 56 ml of incubation solution for each treatment. The cecal contents of each of the three pigs in each treatment were mixed with the incubation solution under CO. 2 The mixture was mixed at a ratio of 2:1 under CO atmosphere and homogenized with a Waring blender for several minutes (the cecal contents were 0.20 of the total incubation solution). Then, 24 mL of the mixture was added to each bottle to complete the 80 mL total liquid volume. In addition, triplicate bottles without substrate were also included as blanks. The bottles were placed under CO atmosphere for 2-3 hr. 2The tubes were sealed under flow and incubated at 39°C for 12 h. During the entire incubation time, the pressure was recorded every two hours by a HD2124.02 manometer (Delta Ohm, Caselledi Selvazzano, Italy) equipped with a TP804 pressure gauge. The readings were converted to volumes by a pre-established linear regression equation between the pressure recorded in the same bottle under the same conditions and the known volume of air (n = 103; R2 = 0.996). The gas volume recorded for each incubation time was expressed per unit of organic matter (OM) incubated. The methane (CH4) concentration was measured every 6 h. At 6 and 12 h, incubation samples were collected and acidified with 0.1 N HCl in 1:1 or added to 0.5 ml of a deproteinized mixture of 0.5 M PO4H3 and 2 mg / ml 4-methylvaleric acid and stored at -20°C for the determination of ammonia and volatile fatty acid (VFA) concentrations, respectively. Note that the in vitro gas production procedure was identical for both fresh and frozen inocula, the only difference being that frozen samples of cecal contents were thawed beforehand by keeping them at 38°C for 5 min.

[0875] For gas production, the results for each incubation time were analyzed as a split plot design, with the donor pig as a block, the contents (fresh vs. frozen) as the main plot, and the treatments as subplots. The same design was used to analyze the results obtained from the digestive contents (stomach and small intestine) as the main plot. Statistical analysis was not performed on the in vitro enzymatic results as there were no sources of experimental variability. Differences were considered significant when P < 0.05 and were considered significant trends when 0.05￡P < 0.10; and when significant, differences were compared by Tukey t test at P < 0.05.

[0876] result

[0877] In vitro and enzymatic digestion

[0878] It was observed that for gastric contents, the substrate digestibility of vitamin B2 was the highest compared to the control (P<0.05). After digestion of small intestinal contents, this difference was minimized and no difference was found between treatments (P>0.05).

[0879] Although the extent of digestion was slightly lower with pepsin-HCl than with gastric contents, the substrate digestibility results observed for the exogenous enzymes were consistent with those recorded for the digesta contents. Thus, when incubated in pepsin-HCl, the digestibility recorded for the substrate supplemented with vitamin B2 was higher than that of the other treatments. However, no differences between the treatments were observed when the treatments were incubated in (P+pancreatin) and / or (P+P+mycozyme). The results indicate that both methods are effective for evaluating enzymatic digestion in pigs.

[0880] In vitro gas production

[0881] On average, the gas production patterns from treatments incubated with fresh cecal contents did not differ from those recorded with frozen cecal contents (P>0.05); however, at 4 h of incubation, the gas volumes tended to be higher with fresh contents (P=0.081). On average, the gas volumes recorded with vitamin B2 were higher than those in the control group at 4, 10, and 12 h of incubation (P<0.05).

[0882] The interaction between storage type and treatment did not reach significance (P < 0.05) throughout the incubation time, indicating that treatments performed similarly despite coagulation of cecal contents.

[0883] Methane production

[0884] Regarding CH4 production, no differences were observed between fresh and frozen inocula after 12 h of incubation (P>0.05). In incubations up to 12 h, there were no differences between treatments in the proportion of CH4 in the total gas (P>0.05), although numerically the lowest CH4 was recorded with vitamin B2. Similarly, when CH4 production was expressed as volume per unit of incubation substrate, no significant differences were recorded between treatments at 6 h and 12 h incubation, although ranking between treatments indicated that vitamin B2 recorded the lowest CH4 accumulation at 6 h incubation and at 12 h incubation.

[0885] The results observed with vitamin B2 suggest that this vitamin may have a positive effect on the activity of hindgut bacterial species, since it is known that vitamin B2 plays an important role in energy production and contributes to carbohydrate degradation. In addition, the reduction in methane production may be explained by the fact that vitamin B2 can reduce the activity of methanogens or archaeal populations.

[0886] Total volatile fatty acid concentration (TVFA), VFA molar ratio and ammonia production

[0887] No differences were observed in total VFA concentrations, molar ratios of VFAs, or ammonia concentrations between treatments and storage of cecal inoculum, indicating that cecal fermentation characteristics were not significantly affected by the experimental treatments tested.

[0888] In conclusion, vitamin B2 showed a clear positive response both at the gastric level (possibly stimulating pepsin activity) and at the hindgut level (which could enhance microbial activity).

Claims

1. An active agent for use in improving intestinal health in animals including humans, the active agent being selected from 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 comprises or consists of: i. increasing the concentration of at least one short-chain fatty acid or a salt thereof in the intestine, ii. increasing the diversity of the microbiome in the intestine, iii. increasing the abundance of beneficial bacteria in the intestine, iv. promoting or increasing the butyrate synthesis pathway in the intestine, v. improving the intestinal barrier function, vi. Reduce the redox potential of the intestine, vii. reducing the amount of gas produced in the intestine, and / or viii. Stimulate intestinal immune response; The use comprises administering to an animal a) a formulation comprising an effective dose of an 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 ingredient is present in the intestine of the animal; The premise is that if niacin is present, it is not the only active ingredient and that if riboflavin is present, it has an ameliorative effect other than increasing the number of Faecalibacterium prausnitzii.

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

3. The active agent according to claim 1 or 2, wherein the improvement comprises increasing the concentration of at least one short-chain fatty acid or a salt thereof in the intestine, or promoting or increasing the butyrate synthesis pathway in the intestine; And wherein the short-chain fatty acid is selected from acetic acid, propionic acid and butyric acid or their salts.

4. The active agent of claim 3, wherein the active agent 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. The active agent according to claim 3 or 4, wherein the animal, including humans, suffers from a condition selected from metabolic disorders, type 2 diabetes, obesity, chronic inflammation and atherogenesis.

6. An active agent according to claim 1 or 2, wherein the improvement comprises increasing the diversity of the microbiome in the intestine.

7. An active agent according to claim 6, wherein the diversity of the microbiome in the colon is increased and / or the abundance of beneficial bacteria is increased.

8. The active agent according to claim 6 or 7, wherein the increased beneficial bacteria are selected from the group consisting of Acidaminococcus, Akkermansia species, Bacteroides ovatus, Bifidobacterium, Brucella, Clostridium perfringens, Collinsella perfringens, Dorealongicatena, Escherichia coli, Eubacterium, Faecalibacterium prausnitzii, Lachnospira, Lactobacillus, Parabacteroides distichous, Raoultella, Roseburia, Ruminococcus, Streptococcus and combinations thereof.

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

10. The active agent according to any one of claims 6-9, wherein the animal, including humans, suffers from a condition selected from irritable bowel disease, metabolic diseases, obesity and inflammatory conditions.

11. An active agent according to any one of claims 6-10, wherein the active agent is selected from: vitamin B1, vitamin B2, vitamin B5, vitamin B6, vitamin B12, β-carotene, biotin, vitamin C, vitamin E, vitamin D, folic acid, vitamin K, ω3 fatty acids and combinations thereof.

12. The active agent according to claim 1 or 2, wherein the improvement comprises improving intestinal barrier function.

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

14. The active agent according to claim 12 or 13, wherein the active agent 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.

15. The active agent according to claim 1 or 2, wherein the improvement comprises reducing the redox potential of the intestinal tract.

16. An active agent according to claim 15, wherein the reduced redox potential produces a benefit selected from the group consisting of: promoting the growth of strictly anaerobic beneficial bacteria in the colon; reducing aerobic pathogenic bacteria in the intestine, reducing plasma free thiols, reducing inflammation in the intestine and reducing cell damage caused by inflammation in the intestine.

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

18. An active agent according to claim 1 or 2, wherein the improvement is a reduction in the amount of gas produced in the intestine.

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

20. An agent according to claim 1 or 2, wherein the improvement is stimulation of intestinal immune response.

21. The active agent according to 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. The active agent according to claim 20 or 21, wherein the active agent is selected from: vitamin B2, vitamin E, vitamin D, vitamin A and vitamin K and mixtures thereof.

23. A method for improving intestinal health in an animal, including a human, wherein the improvement comprises or consists of: i. increasing the concentration of at least one short-chain fatty acid or a salt thereof in the intestine, ii. increasing the diversity of the microbiome in the intestine, iii. increasing the abundance of beneficial bacteria in the intestine, iv. promoting or increasing the butyrate synthesis pathway in the intestine, v. Improve intestinal barrier function, vi. reducing the redox potential of the intestine, vii. reducing the amount of gas produced in the intestine, and / or viii. Stimulate intestinal immune response; It comprises administering to the animal an active agent selected from 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 present in a) a formulation comprising an effective dose of said active agent, wherein said active agent is delivered to the large intestine; or b) supraphysiological doses of active ingredients, so that at least an effective amount of the active ingredient is present in the intestine of the animal; The premise is that if niacin is present, it is not the only active ingredient and that if riboflavin is present, it has an ameliorative effect other than increasing the number of Faecalibacterium prausnitzii.

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

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

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

27. The method of claim 26, wherein the animal has a condition selected from the group consisting of metabolic disorders, type 2 diabetes, obesity, chronic inflammation, and arteriogenesis.

28. 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. The method of claim 23, wherein the improvement is increasing the diversity of the microbiome in the intestine, or increasing the abundance of beneficial bacteria in the intestine.

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; provided that if riboflavin is the active agent, it is present for a purpose other than increasing the abundance of Faecalibacterium prausnitzii.

31. The method of claim 29, wherein the method is a method of treating, preventing or alleviating symptoms of irritable bowel syndrome, metabolic disease, obesity or inflammatory disorders in an animal, including a human, in need thereof, the method comprising: The beneficial bacteria are administered to the colon of an animal in an amount that enhances an active agent; the active agent being 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; provided that if riboflavin is the active agent, it is present for a purpose other than increasing the abundance of Faecalibacterium prausnitzii.

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

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

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 method of claim 32, wherein the improvement is a reduction in the redox potential of the intestinal tract resulting in a benefit selected from: Promotes the growth of strictly anaerobic beneficial bacteria in the colon; reducing aerobic pathogenic bacteria in the intestinal tract; reducing inflammation in the intestine; and Reducing intestinal cell damage caused by inflammation in the intestine.

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

37. The method of claim 23, wherein the improvement is treating, preventing or reducing the amount of gas produced in the intestine.

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

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

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

41. The method of claim 40, wherein the agent 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.

Images