Methods to increase the number of Dialister species in the gut microbiota.

Abstract

People with various diseases have reduced numbers of Dialister spp. in their intestinal flora. The present inventors have found that administration of a vitamin / vitamin combination such as β-carotene, vitamin A, vitamin D, vitamin B5, vitamin B2, a combination of vitamin B2 and vitamin C, vitamin K, DHA, or EPA can result in an increase in Dialister spp. when administered directly to the large intestine to provide nutrients directly to the intestinal flora.

Description

Detailed Description of the Invention

[0001] The present invention relates to a method for increasing the number of bacteria of the genus Dialister in the intestinal flora of animals, preferably humans, by directly delivering vitamins or combinations of vitamins to the intestinal flora. This can be achieved, for example, by using delayed release formulations of selected vitamins or PUFAs, preferably beta-carotene, vitamin A, vitamin D, vitamin B5, vitamin B2, combinations of vitamin B2 and vitamin C, vitamin K, DHA, EPA and combinations of EPA and DHA.

[0002] [Background of the Invention] Dialister is the genus name of bacteria found in the intestinal flora, including D. pneumosintes, D. invisus, D. micraerophilus, D. hominis, D. massiliensis, D. succinatiphilus and D. propionicifaciens found in the intestinal flora. D. pneumosintes was previously known as Bacteriodes pneumosintes.

[0003] Studies have shown that sub-normal levels of Dialister spp. in the gut are associated with a variety of seemingly unrelated medical and / or adverse conditions, including: irritable bowel syndrome, autism spectrum disorder, calcium malabsorption, food allergies and sensitizations (milk, eggs, peanuts, soy, walnuts, and wheat), generalized anxiety disorder, childhood obesity, infant and child growth disorders and pathological conditions, immune-mediated inflammatory diseases, atopic diseases (including childhood asthma and allergies), Crohn's disease, rheumatoid arthritis and osteoarthritis, pre-eclampsia, type 1 diabetes, juvenile idiopathic arthritis, depression, attention deficit hyperactivity disorder (ADHD), chronic hepatitis B, major depressive disorder, airway allergies, multiple sclerosis, chronic inflammation, and Alzheimer's disease.

[0004] It would be desirable to provide a method for increasing the number of Dialister species in the gut microbiota, particularly in individuals experiencing or at risk of experiencing the aforementioned diseases / adverse conditions or symptoms associated with one of these diseases / adverse conditions.

[0005] [Detailed description of the invention] Numerous studies have shown that when animals, preferably humans, suffer from a particular disease / adverse condition, the number of Dialister spp. bacteria in the gut microbiota decreases compared to the number of bacteria present in animals that do not suffer from that particular disease / adverse condition. However, none of these studies have suggested a method to increase the number of Dialister spp. bacteria and thereby alleviate at least one of the symptoms of that disease / adverse condition. According to the present invention, it has been found that by directly delivering certain vitamins or polyunsaturated fatty acids ("PUFAs") to the large intestine of animals, preferably humans, it is possible to nourish the gut microbiota and increase the resident number of Dialister spp. bacteria. Therefore, the present invention relates to a method for preventing, reducing the risk of developing, or delaying the development of, and / or treating, a disease / adverse condition characterized by sub-normal numbers of Dialister spp. bacteria in the gut microbiota by administering at least one vitamin or PUFA delivered directly to the intestine.

[0006] In accordance with the present invention, the inventors have found that certain vitamins and PUFAs selected from the group consisting of β-carotene, vitamin A, vitamin D, vitamin B5, vitamin B2, a combination of vitamin B2 and vitamin C, vitamin K, docohexaenoic acid "DHA", eicosapentaenoic acid "EPA", and a combination of EPA and DHA are suitable for increasing the number of Dialister spp. bacteria in the intestines when provided to the intestinal microbiota.

[0007] Accordingly, one aspect of the present invention is a method for increasing the number of Dialister spp. bacteria in the intestinal microbiota, comprising directly administering to the large intestine of an animal, preferably a human, an amount effective in increasing the number of bacteria, a vitamin or PUFA selected from the group consisting of β-carotene, vitamin A, vitamin D, vitamin B5, vitamin B2, a combination of vitamin B2 and vitamin C, vitamin K, DHA, EPA, and a combination of EPA and DHA.

[0008] Another embodiment of the present invention is a treatment and / or prevention of a disease or adverse condition associated with a reduced number of Dialister spp. in the intestinal microbiota, comprising directly administering a vitamin or PUFA selected from the group consisting of β-carotene, vitamin A, vitamin D, vitamin B5, vitamin B2, a combination of vitamin B2 and vitamin C, vitamin K, DHA, EPA, and a combination of EPA and DHA, to the large intestine of an animal in need, preferably a human. Conditions or diseases characterized by a reduced number of Dialister spp. include irritable bowel syndrome, autism spectrum disorder, calcium malabsorption, food allergies and sensitizations (milk, eggs, peanuts, soy, walnuts, and wheat), generalized anxiety disorder, childhood obesity, infant and child growth disorders and pathological conditions, immune-mediated inflammatory diseases, atopic diseases (including childhood asthma and allergies), Crohn's disease, rheumatoid arthritis and osteoarthritis, pre-eclampsia, type 1 diabetes mellitus, juvenile idiopathic arthritis, depression, attention deficit hyperactivity disorder (ADHD), chronic hepatitis B, major depressive disorder, airway allergies, multiple sclerosis, chronic inflammation, and Alzheimer's disease.

[0009] Another embodiment of the present invention is an orally delivered formulation comprising an amount effective in increasing the number of Dialister spp. bacteria, a vitamin or PUFA selected from the group consisting of β-carotene, vitamin A, vitamin D, vitamin B5, vitamin B2, a combination of vitamin B2 and vitamin C, vitamin K, DHA, EPA, and a combination of EPA and DHA, and an excipient, characterized in that the vitamin or PUFA is delivered to the intestinal microbiota of the large intestine.

[0010] Other embodiments of the present invention include irritable bowel syndrome, autism spectrum disorder, calcium malabsorption, food allergies and sensitizations (milk, eggs, peanuts, soy, walnuts, and wheat), generalized anxiety disorder, childhood obesity, infant growth disorders and pathological conditions, immune-mediated inflammatory diseases; atopic diseases (including childhood asthma and allergies), Crohn's disease, rheumatoid arthritis and osteoarthritis, pre-eclampsia, type 1 diabetes mellitus, juvenile idiopathic arthritis, depression, attention deficit hyperactivity disorder (ADHD), chronic hepatitis B, major depressive disorder, airway allergies, and multiple sclerosis. A medical food that addresses the nutritional needs of patients experiencing at least one symptom of a disease / adverse condition selected from the group consisting of chronic inflammation and Alzheimer's disease, and which contains nutrients selected from the group consisting of beta-carotene, vitamin A, vitamin D, vitamin B5, vitamin B2, a combination of vitamin B2 and vitamin C, vitamin K, DHA, EPA, and a combination of EPA and DHA, wherein these nutrients are formulated to be delivered directly to the intestinal microbiota.

[0011] [Definition] As used throughout this specification and the claims, the following definitions apply:

[0012] "Dialister spp." refers to at least one species of the genus Dialister, and may include D. pneumosintes, D. invisus, D. micraerophilus, D. hominis, D. massiliensis, D. succinatiphilus, and D. propionicifaciens.

[0013] "Decreased bacterial count" means that the amount of Dialister spp. present in an individual is lower compared to the amount found in a population of healthy individuals.

[0014] As used herein, "healthy" means that an animal, including humans, is not experiencing any disease or adverse condition known to be associated with a reduced number of Dialister spp. bacteria in the gut microbiota.

[0015] The terms "vitamin B2" and "riboflavin" are used interchangeably and include its esters, particularly riboflavin-5'-phosphate.

[0016] The term "vitamin K" includes either or both vitamin K1 and vitamin K2.

[0017] "DHA" includes its free acid form, as well as salts and esters such as ethyl esters. This also includes various concentrations, such as those with a purity of at least 90%. This also includes fish oil and algae oil that contain DHA, either in naturally occurring amounts or in concentrated amounts, and may also contain EPA. This further includes DHA triglycerides.

[0018] "EPA" includes its free acid form, as well as its salts and esters. This includes various concentrations, such as those with a purity of at least 90%. This also includes fish oil and algae oil, which may contain some amount of EPA, and may also contain DHA. Furthermore, this also includes EPA triglycerides.

[0019] As used herein, the term "vitamin D" refers to vitamin D3. 25-hydroxyvitamin D3 may be used in place of or in addition to vitamin D3, preferably in non-human species. The relative strength of 25-hydroxyvitamin D3 to vitamin D3 is approximately 40:1, and therefore, when administering 25-hydroxyvitamin D3, adjustments must be made accordingly.

[0020] Animals, preferably humans, that "need to increase the number of Dialister spp." are at risk of or currently experiencing at least one disease / adverse condition selected from the group consisting of irritable bowel syndrome, autism spectrum disorder, calcium malabsorption, food allergies and sensitizations (milk, eggs, peanuts, soy, walnuts and wheat), generalized anxiety disorder, childhood obesity, infant growth disorders and pathological conditions, immune-mediated inflammatory diseases, atopic diseases (including childhood asthma and allergies), Crohn's disease, rheumatoid arthritis and osteoarthritis, pre-eclampsia, type 1 diabetes mellitus, juvenile idiopathic arthritis, depression, attention deficit hyperactivity disorder (ADHD), chronic hepatitis B, major depressive disorder, airway allergies, multiple sclerosis, chronic inflammation and Alzheimer's disease.

[0021] "Prevention" is not limited to situations in which a disease / harmful condition has never occurred. Instead, as used throughout this specification and the claims, it may include reducing the severity of a disease / harmful condition or its symptoms; delaying the onset of a disease / harmful condition or its symptoms; intervening early in a disease / harmful condition or its symptoms; and reducing the risk of a disease / harmful condition or its symptoms developing.

[0022] "Direct delivery" means administering vitamins or PUFAs so that they are not absorbed in the stomach and / or small intestine, but rather the vitamins and / or combinations are present in the distal intestinal tract, preferably the large intestine, where the microbiome can utilize them. These vitamins or PUFAs are administered in amounts exceeding, but not part of, the normal daily nutritional requirements of humans (generally obtained through diet and conventional vitamin or PUFA supplementation). For use in humans, the preferred method is via a form that delays delivery until it reaches the intestinal tract. For non-human animals, preferred delivery includes administering a sufficiently large dose such that only a portion of the delivered vitamins or PUFAs are absorbed in the stomach, and the remainder, which constitutes an effective dose, is available in the intestinal tract; however, this preferred method of delivery may also be used in humans. [Brief explanation of the drawing]

[0023] [Figure 1A] This shows the relative abundance of Dialister species after vitamin administration in in vitro experiment 1. It indicates the log10 ratio change in the abundance of Dialister species in the fermentation supernatant after 24 hours compared to the control. [Figure 1B] This shows the relative abundance of Dialister species after vitamin administration in in vitro experiment 2. It shows the log10 ratio change in the abundance of Dialister species in the fermentation supernatant after 24 hours compared to the control. [Figure 1C] This shows the relative abundance of Dialister species after vitamin administration in human trials. It indicates the relative abundance of Dialister species in feces after administration of colon-targeting vitamins in human trials.

[0024] [Disease / Adverse Condition] Another embodiment of the present invention involves the use of PUFAs or vitamins formulated for direct delivery to the intestinal microbiota of animals, preferably humans, characterized in that, upon delivery, the PUFAs or vitamins increase the number of Dialister spp. in the intestinal microbiota. Preferably, the vitamins and / or combinations of vitamins are selected from the group consisting of β-carotene, vitamin A, vitamin D, vitamin B5, vitamin B2, a combination of vitamin B2 and vitamin C, vitamin K, DHA, EPA, and a combination of EPA and DHA.

[0025] In some embodiments, animals including humans in whom it is necessary to increase the number of Dialister spp. bacteria are at risk of or have experienced a disease or condition selected from the group consisting of irritable bowel syndrome, autism spectrum disorder, calcium malabsorption, food allergies and sensitization (milk, eggs, peanuts, soybeans, walnuts and wheat), generalized anxiety disorder, childhood obesity, growth disorders and morbid states in infants, immune-mediated inflammatory diseases, atopic diseases (including childhood asthma and allergies), Crohn's disease, rheumatoid arthritis and osteoarthritis, early-onset preeclampsia, type 1 diabetes, juvenile idiopathic arthritis, depression, attention deficit hyperactivity disorder (ADHD), chronic hepatitis B, major depressive disorder, airway allergies, multiple sclerosis, chronic inflammation, and Alzheimer's disease, and the number of Dialister spp. bacteria is increased by administering a vitamin or PUFA selected from the group consisting of beta-carotene, vitamin A, vitamin D, vitamin B5, vitamin B2, a combination of vitamin B2 and vitamin C, vitamin K, DHA, EPA, and a combination of EPA and DHA.

[0026] Another embodiment of the present invention is the non-therapeutic use of a PUFA and / or vitamin formulated for direct delivery to the gut microbiota, wherein, upon delivery, the PUFA and / or vitamin increases the number of Dialister spp. bacteria in the gut microbiota of an animal, preferably a human. Preferably, the PUFA or vitamin is selected from the group consisting of beta-carotene, vitamin A, vitamin D, vitamin B5, vitamin B2, a combination of vitamin B2 and vitamin C, vitamin K, DHA, EPA, and a combination of EPA and DHA.

[0027] Another embodiment of the present invention is the use of a combination of vitamins or PUFAs in the manufacture of a medicament formulated for direct delivery, wherein, upon delivery, the combination of vitamins or PUFAs increases the number of Dialister spp. in the intestinal microbiota of an animal, preferably a human. Preferably, the PUFA or vitamin is selected from the group consisting of β-carotene, vitamin A, vitamin D, vitamin B5, vitamin B2, a combination of vitamin B2 and vitamin C, vitamin K, DHA, EPA, and a combination of EPA and DHA.

[0028] In some embodiments, the PUFA or vitamin is β-carotene, vitamin A, vitamin D, vitamin B5, vitamin B2, a combination of vitamin B2 and vitamin C, vitamin K, DHA, EPA, or a combination of EPA and DHA, to increase the number of Dialister spp. in an animal or human at risk of or experiencing a disease or condition selected from the group consisting of irritable bowel syndrome, autism spectrum disorder, calcium malabsorption, food allergies and sensitizations (milk, egg, peanut, soy, walnut, and wheat), generalized anxiety disorder, childhood obesity, growth disorders and morbid states in infants and young children, immune-mediated inflammatory diseases, atopic diseases (asthma including childhood asthma and allergies), Crohn's disease, rheumatoid arthritis and osteoarthritis, early-onset preeclampsia, type 1 diabetes, juvenile idiopathic arthritis, depression, attention deficit hyperactivity disorder (ADHD), chronic hepatitis B, major depressive disorder, airway allergies, multiple sclerosis, chronic inflammation, and Alzheimer's disease, by administering the selected vitamin or vitamins.

[0029] The vitamins and vitamin combinations mentioned above may be administered as single activators, or in combination with conventionally used prebiotics, probiotics, other components that can modulate the gut microbiota, and conventional pharmaceuticals or nutritional supplements. Therefore, DHA can be selected as a single activator; EPA can be selected as a single activator; vitamin K can be selected as a single activator; β-carotene can be selected as a single activator; vitamin A can be selected as a single activator; vitamin D can be selected as a single activator; vitamin B5 can be selected as a single activator; vitamin B2 can be selected as a single activator; a combination of vitamin B2 and vitamin C can be selected as a single activator; or a combination of EPA and DHA can be selected as a single activator.

[0030] [animal] "Animals" include mammals, poultry, and preferably humans. Preferred non-human animals are companion animals, including dogs, cats, and horses. Among agriculturally important animals, preferred animals include poultry, pigs, cattle, sheep, goats, and horses.

[0031] [Dosage] The dosages used herein are intended to be added to active ingredients taken for general nutritional purposes. Instead, they act at the genus, species, and strain levels on the entire gut microbiota environment of intestinal bacteria. These activators are not intended to be directly metabolized by animals, including humans. Rather, they are intended to be utilized by the bacterial population of the colon. Therefore, the amounts reported below will be ingested by animals in addition to their normal diet, but will not be directly utilized by the animals due to delayed release.

[0032] The preferred daily dose is as follows: Riboflavin: up to 200 mg per day; preferably 1 to 85 mg per day; more preferably 70 to 80 mg per day. In one embodiment, about 75 mg per day is used. Preferably, riboflavin is administered in amounts such that its local concentration in the colon is at least 0.05 g / L, preferably at least 0.1 g / L, and more preferably 0.125 g / L. Preferred local concentrations in the colon are in the range of about 0.1 g / L to about 0.5 g / L or about 0.1 g / L to about 0.2 g / L, and preferably about 0.125 g / L. One preferred daily dose may be up to 200 mg.

[0033] Beta-carotene: up to 150 mg per day (additional range). Preferably, beta-carotene is administered in an amount such that its local concentration in the colon is at least 0.1 g / L, preferably at least 0.15 g / L, and most preferably at least 0.2 g / L. Preferred local concentrations in the colon are in the range of about 0.05 g / L to about 0.4 g / L, more preferably about 0.15 g / L to about 0.25 g / L. One preferred daily dose is up to 150 mg.

[0034] Vitamin B5: up to 1500 mg per day (additional range). Preferably, vitamin B5 is administered in an amount such that its local concentration in the colon is at least 1 g / L, preferably at least 1.5 g / L, and most preferably at least 2 g / L. Preferred local concentrations in the colon are in the range of about 0.5 g / L to about 4 g / L, more preferably about 1.5 g / L to about 2.5 g / L. One preferred daily dose is up to 1500 mg.

[0035] Vitamin C: up to 2000 mg per day; preferably 400-600 mg per day; more preferably 450-550 mg per day. Preferably, ascorbic acid is administered in an amount such that its local concentration in the colon is at least 0.05 g / L, preferably at least 0.1 g / L, and most preferably at least 0.8 g / L. Preferred local concentrations in the colon are in the range of about 0.05 g / L to about 1.5 g / L, more preferably about 0.5 g / L to about 1 g / L, and most preferably about 0.8 g / L to about 0.9 g / L. One of the preferred daily doses is up to 2000 mg.

[0036] Vitamin D3: up to 250 micrograms per day; preferably 5 to 80 micrograms per day; more preferably 15 to 25 micrograms per day.

[0037] Vitamin K: up to 10,000 micrograms per day, preferably 80 to 140 micrograms per day, more preferably 100 to 120 micrograms per day. In one embodiment, 110 micrograms per day is administered.

[0038] DHA and EPA combination: up to 5,000 mg per day. The DHA to EPA ratio is not important. In one embodiment, the EPA to DHA ratio may be the same as the ratio found in natural fish oil or algal oil. Other non-limiting available ratios include DHA to EPA ratios of 10 to 1:1 to 10.

[0039] DHA: up to 1000 mg per day; preferably 80-120 mg per day; more preferably 90-110 mg. In one embodiment, about 100 mg is used.

[0040] EPA: up to 1800 mg per day, preferably up to 80-120 mg per day; more preferably up to 90-110 mg per day. In one embodiment, about 100 mg per day is used.

[0041] Regarding the combination of vitamins B2 and C, the ratio of vitamin B2 to C is not important. Generally, the amount of vitamin C is much higher than the amount of B2; for example, vitamin C is 500mg to 1000mg, while vitamin B2 is 1mg.

[0042] To enable direct delivery using a delayed-release formulation, the dosage is preferably taken once daily, but may be taken multiple times at lower doses as needed (i.e., half the dose twice daily or one-third the dose three times daily).

[0043] When administered in high doses to animals, rather than directly to the intestines as is commonly done when they are not humans, the dose may be at least about 10 times or even 20 times the recommended dose. For example, if the recommended daily dose is 5 mg, then 50 mg or 100 mg is preferably administered in food, dosage form, or feed to ensure the vitamin and / or PUFA is present in the colon. In humans, it may be necessary to adjust to a higher dose.

[0044] It is preferable to continue taking this dose for a period of time, for example, at least one week, preferably at least two weeks, and more preferably at least one month. This dose may be taken daily for a longer period of time, if necessary.

[0045] [formulation] A preferred formulation may contain a sufficiently high dose in which some of the vitamins / PUFAs are normally absorbed, while the remainder is available in an effective amount to the intestinal microbiota. Other formulations include those administered via parenteral routes, such as suppositories or injections. A preferred formulation is an oral delayed-release formulation.

[0046] As used herein, "delayed release" refers to the release of the activator at a later date than immediately after administration. Preferably, "delayed release" means that, after oral administration, the activator is delivered to the large intestine, preferably the colon, at a later date compared to an immediate-release formulation.

[0047] The "enteric coating layer" is a layer surrounding the core substance, which contains the activator, and the layer provides resistance to gastric juice. The "enteric coating shell" is a shell or matrix that surrounds or encapsulates the activator, and the shell provides resistance to gastric juice. Alternatively, a matrix-based delivery system can be used. In a matrix-based system, there is no separate layer of coating material, and the activator is distributed somewhat homogeneously within the matrix. Furthermore, there is a colon-release system, for example, in which the activator is embedded in a fibrous matrix (which responds to enzymes), and the outermost layer is an enteric coating.

[0048] In preferred embodiments for humans, the formulation of the present invention is a solid dosage form for oral administration. The formulation may be in the form of capsules, pellets, beads, spheres, spheres, tablets, small tablets, or granules, optionally coated with a delayed-release coating or shell to prevent the release of the activator before the small intestine, preferably before the colon.

[0049] Coatings, shells, or matrix materials for delayed release of activators via oral administration, particularly for targeted release in the ileum or large intestine, are known in the art. These can be subdivided into coatings that disintegrate above a certain pH, coatings that disintegrate after a certain residence time in the gastrointestinal tract, and coatings that disintegrate in response to enzymes specific to the microbiome of a particular region of the intestine. It is common to use combinations of coatings or shells classified into different types. These three different types of coatings or shells for targeting the large intestine are outlined, for example, 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 pH-dependently disintegrating coating material, a time-dependently disintegrating coating material, a coating material that disintegrates in response to trigger enzymes in the intestinal environment (e.g., the intestinal environment of the ileum and large intestine), and combinations thereof.

[0050] Examples of coating materials that degrade in a pH-dependent manner include polyvinyl acetate phthalate, trimellitic cellulose acetate, hydroxypropyl methylcellulose phthalate HP-50, HP-55, or HP-55S, cellulose phthalate acetate, shellac, hydroxypropyl methylcellulose acetate succinate (HPMCAS), poly(methacrylate-ethyl acrylate) 1:1 (Eudragit® L100-55, Eudragit® L30D-55), poly(methacrylate-methyl methacrylate) 1:1 (Eudragit® L-100, Eudragit® L12.5), and poly(methacrylate-methyl methacrylate) 1:2 (Eudragit® S-100, Eudragit® S12.5, and Eudragit® FS30D).

[0051] Examples of coating materials that degrade in a time-dependent manner include Eudragit®RL, Eudragit®RS, and ethylcellulose.

[0052] Coating materials that degrade in response to enzyme triggers in the colonic environment include chondroitin sulfate, pectin, guar gum, chitosan, inulin, lactulose, raffinose, stachyose, alginate, dextran, xanthan gum, locust bean gum, arabinogalactan, cyclodextrin, pullulan, carrageenan, scleroglucan, chitin, carduran, levan, amylopectin, starch, amylose, indigestible starch, and azo compounds that are degraded by bacteria that cleave azo bonds.

[0053] In one embodiment, the formulation comprises an enteric-coated capsule filled with a composition containing an activator. The enteric-coated capsule provides resistance to the acidic environment of the stomach. For example, a softgel formulation can deliver the activator in solution while still offering the advantages of a solid dosage form. Softgel capsules are particularly suitable for hydrophobic activators that are poorly soluble in water. Vitamin K and omega-3 fatty acids are preferred for formulation as softgel capsules.

[0054] In other embodiments, the formulation is a tablet comprising (i) a core material containing an activator, and (ii) a delayed-release coating such as an enteric coating. This may be a hard gel capsule.

[0055] The release of the activator can be delayed until the small intestine. In another embodiment, the release of the activator is delayed until the distal small intestine. In yet another embodiment, the release of the activator is delayed until the colon.

[0056] The present invention will be better illustrated by the following non-limiting examples.

[0057] [Examples] [Example 1] [In vitro fermentation test 1] [Donor and sample preparation] At the start of this batch intestinal fermentation culture, all test components were added from stock to a modified nutrient medium containing the following (g / l): K2HPO4 (2.5), KH2PO4 (10.9), NaHCO3 (2), yeast extract (2), peptone (2), mucin (1), cysteine ​​(0.5), Tween 80 (2), glucose (2), starch (2), cellobiose (2), NaCl (0.1), MgSO4.7H2O (0.01), CaCl2.6H2O (0.01), hemin (0.05), and bile salts (0.5).

[0058] The following compounds were added to stock solutions of vitamin B2 and B9 prepared in water, and stock solutions of vitamin K1, DHA, and EPA prepared in ethanol.

[0059] [Table 1]

[0060] Each compound was tested at three different concentrations. As a source of microbial communities, a fecal suspension prepared immediately after collection from the donor was added to the reactor. Each reactor had a capacity of 70 ml. All tests except the blank test were performed in a single series. The culture conditions were anaerobic conditions at 37°C with shaking (90 rpm) for 48 hours.

[0061] [measurement] Microbial composition: From all fermentation samples collected throughout this study, total DNA was extracted using the QIAamp DNA stool minikit (Qiagen, Crawley, United Kingdom) according to the manufacturer's instructions, except that the bead crushing step was added as previously described and the dissolution temperature was raised to 95°C. After DNA isolation, the DNA was quantified using the Qubit High Sensitivity DNA assay (Thermo Fisher). Subsequently, a total metagenomic library was prepared using the Illumina Nextera XT kit (Illumina) according to the manufacturer's instructions, with the following modifications: firstly, the tagmentation time was extended to 7 minutes; and secondly, after indexing and Ampure purification of the product, each sample was individually sized by electrophoresis on an Agilent High Sensitivity Chip (Agilent) and quantified using the Qubit High Sensitivity DNA assay (Thermo Fisher) according to the Teagasc Sequencing Platform SOP. Next, the samples were pooled in equimolar quantities and sequenced using an Illumina NextSeq 500 with the NextSeq 500 / 550 v2 high-power reagent kit (300 cycles). All sequencing was performed at the Teagasc sequencing facility according to the standard Illumina sequencing protocol. The delivered FASTQ format raw sequence files were quality-checked as follows: Low-quality and duplicate reads were removed and trimmed using a combination of SAM and Picard tools. Reads were assigned to taxa using Metaphlan2 software.

[0062] [Example 2] [In vitro fermentation test 2] [Donor and sample preparation] At the start of this batch intestinal fermentation culture, all test components were added from stock to a modified nutrient medium containing the following (g / l): K2HPO4 (2.5), KH2PO4 (10.9), NaHCO3 (2), yeast extract (2), peptone (2), mucin (1), cysteine ​​(0.5), Tween 80 (2), glucose (2), starch (2), cellobiose (2), NaCl (0.1), MgSO4.7H2O (0.01), CaCl2.6H2O (0.01), hemin (0.05), and bile salts (0.5).

[0063] The following compounds were added from stock solutions prepared in water: β-carotene, Vitamin B3, Vitamin B5, Vitamin B7, Fructooligosaccharides (positive control).

[0064] Each compound was tested at three different concentrations, and the results are summarized in Table 2 below. Fecal suspension, prepared immediately after collection from the donor, was added to the reactor as the source of the microbial community. Each reactor had a capacity of 70 ml. All tests except the blank test were performed in a single series. The culture conditions were anaerobic conditions at 37°C with shaking (90 rpm) for 48 hours.

[0065] [Table 2]

[0066] [measurement] Microbial composition: Illumina sequencing was performed at the start of culture and after 24 hours of culture. This technique targets 16S rRNA genes, which have a structure in which variable and conserved regions are distributed throughout the gene. Because these regions play a crucial role in protein expression, the conserved regions are characterized by a very slow evolutionary rate.

[0067] The methodology applied involves primers extending to two hypervariable regions (V3-V4) of the 16S rRNA gene. A 424 bp amplicon is generated by sequencing 2 × 250 bp using paired-end sequencing. Such fragments contain more taxonomic information than shorter fragments. Samples analyzed by Illumina sequencing were also analyzed by flow cytometry to determine the total bacterial cell count, thereby converting the relative values ​​obtained from Illumina to absolute values. Samples were analyzed using BDFacs verse. Samples were flowed at high flow rates. Bacterial cells were isolated from culture medium residue and signal noise by setting the threshold to 200 in a SYTO channel. Appropriate gates were set for parental and daughter cells to measure the entire population.

[0068] [Example 3] [Clinical Trials] [Human subjects] Twelve participants were assigned to each of the six vitamin groups, and 24 participants were assigned to the placebo group. All 96 participants completed the intervention. Participants were eligible to enroll in this study if they met the following criteria: able to submit an informed consent form; between 20 and 50 years of age; BMI between 18.5 and 30 kg / m2; stable weight (change rate <5%) over the past three months; generally good health as judged by the principal investigator; not having taken any dietary supplements, prebiotics, probiotics, or nutrient or fiber-rich supplements within four weeks prior to baseline visit and willing to refrain from taking such supplements until the end of the study; willing to refrain from liver intake throughout the study period; willing to maintain their current level of physical activity throughout the study period; and willing to take IP daily throughout the study period. Participants who met any of the following criteria were excluded: their usual fiber intake is >30g / day; they are pregnant or planning to become pregnant; they have received any prohibited medication treatment; their diet has changed significantly in the past three months or they are planning any significant changes to their lifestyle; they have participated in the study within the past 60 days; or they have any ongoing or pre-existing medical conditions that the principal investigator believes may affect the purpose of the study.

[0069] This study procedure was approved by the Clinical Research Ethics Committee of Cork Teaching Hospitals (protocol number: AFCRO-087) and conducted in accordance with the Declaration of Helsinki. Each participant submitted an informed consent form before being enrolled in the study. This study was registered on clinicaltrials.gov with ID: NCT03668964.

[0070] [Test Design] This study was a randomized, double-blind, placebo-controlled, parallel trial in which participants received either a vitamin supplement or a placebo for four weeks. There were three visits: 1) screening; 2) baseline (one week after screening); and 3) follow-up (four weeks after baseline). At the screening visit (visit 1), informed consent was given, and eligibility was assessed, including a medical history and physical examination. Eligible participants entered a one-week introductory period and were instructed to avoid extreme dieting. Participants recorded their daily activities in an eDiary, and stool samples were collected 48 hours prior to the randomization visit. Prior to the randomization visit, food frequency questionnaires were analyzed to confirm that participants' usual fiber intake was <30g fiber / day. Participants who did not meet this criterion or other eligibility criteria were excluded.

[0071] At baseline visit (visit 2), participants were asked to return a stool sample collected within 48 hours prior to the visit to assess their eligibility. Eligible participants were enrolled and assigned a randomization number, receiving a 4-week supply of investigational drug (IP) from one of seven groups. Both participants and research staff were blinded during the randomization process. Participants completed the GSRS and SF-36 questionnaires. Blood samples were collected and stored at -80°C within the facility. Participants were instructed to avoid extreme diets, keep a daily eDiary, and take one capsule daily for the following 4 weeks.

[0072] At the final visit (visit 3), participants were asked to return a stool sample collected within 48 hours prior to the visit. Participants completed the GSRS and SF-36 questionnaires. Blood samples were collected and stored at -80°C within the facility. Participants returned their IPs, and compliance was evaluated.

[0073] [Investigational drug] The investigational drugs were as follows: 1) Vitamin A (retinol equivalent (RE) 250 μg / day), 2) Vitamin C (ascorbic acid 500 mg / day), 3) Vitamin B2 (75 mg / day) + Vitamin C (500 mg / day) 4) Vitamin D3 (cholecalciferol 60 μg / day), or 5) Microcrystalline cellulose 200 mg / day (placebo).

[0074] All vitamins were supplied by DSM Nutritional Products Ltd (Kaiseraugst, Switzerland); the placebo was obtained from Fagrone (Waregem, Belgium). The investigational drug was formulated in a colon-release form as hard gelatin capsules coated with Eudragit S100 (Evonik Nutrition & Care GmbH, Darmstadt, Germany), a pH-dependent polymer that has been shown to be effective for targeting the colon (Cole et al., 2002) (Lonza, Bornem, Belgium). The selected doses were based on high-dose oral delivery of vitamins in previous studies (de Vries et al., 2006; Lakoff et al., 2014; Cantarel et al., 2015; Steinert et al., 2016; Tang et al., 2016), minus the estimated intestinal absorption of each vitamin (Graf, 1980; Basu and Donaldson, 2003; Gropper et al., 2004; Reboul, 2013). Except for vitamin B2, for which no upper limit has been established, all doses were kept below the upper limits published by EFSA (https: / / www.efsa.europa.eu / sites / default / files / assets / UL_Summary_tables.pdf).

[0075] [measurement] Microbial composition of feces: DNA extraction and sequencing were performed using the same method as that used for the fermentation sample in in vitro fermentation test 1.

[0076] [result] • In vitro experiment 1 (Figure 1A): No significant increase in the relative abundance of Dialister was observed with vitamin B9. In contrast, administration of vitamin B2, and especially vitamin K1, as well as the polyunsaturated fatty acids EPA and DHA, resulted in a substantial increase in the relative abundance of Dialister at 24 hours compared to the control group at all tested concentrations.

[0077] • In vitro experiment 2 (Figure 1B): No significant increase in the relative abundance of Dialister was observed with vitamins B3 and B7. In contrast, administration of all tested concentrations of β-carotene, as well as 0.2-fold and 5-fold vitamin B5, increased the relative abundance of Dialister in donor C. These increases were comparable to those observed with the prebiotic FOS.

[0078] • Human trials (Figure 1C): Administration of a combination of vitamins A, D3, and B2 and C over four weeks increased the relative abundance of Dialister compared to placebo. This was in contrast to the observation made with vitamin C alone.

Claims

1. A preparation for increasing the number of Dialister species in the intestinal microbiota, comprising an effective amount for increasing the number of bacteria, a vitamin selected from the group consisting of β-carotene, vitamin A, vitamin D, vitamin B5, vitamin B2, a combination of vitamin B2 and vitamin C, vitamin K, DHA, EPA, and a combination of EPA and DHA, or a PUFA, and administered directly to the large intestine of an animal.

2. A formulation for treating and / or preventing diseases or adverse conditions associated with a reduced number of Dialister species in the intestinal microbiota, the formulation according to claim 1, for direct administration to the large intestine of an animal in need thereof.

3. The preparation according to claim 2, wherein the animal requiring the preparation is experiencing a disease or adverse condition selected from the group consisting of irritable bowel syndrome, autism spectrum disorder, calcium malabsorption, food allergies and sensitization (to milk, eggs, peanuts, soy, walnuts and wheat), generalized anxiety disorder, childhood obesity, infant growth disorders and pathological conditions, immune-mediated inflammatory diseases, atopic diseases (including childhood asthma and allergies), Crohn's disease, rheumatoid arthritis and osteoarthritis, pre-eclampsia, type 1 diabetes mellitus, juvenile idiopathic arthritis, depression, attention deficit hyperactivity disorder (ADHD), chronic hepatitis B, major depressive disorder, airway allergies, multiple sclerosis, chronic inflammation and Alzheimer's disease.

4. The preparation according to any one of claims 1 to 3, wherein the animal is a human.

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