Vitamins for use in improving intestinal health

Abstract

The present invention relates to vitamins for use in improving intestinal health in animals and humans. In particular, it has been found that vitamins B2, C, and D can be used to increase the abundance of the beneficial bacterium Bifidobacterium animalis ssp. lactis in the gastrointestinal tract by delivery to the large intestine.

Description

Detailed Description of the Invention

[0001] [Field of the Invention] The present invention relates to certain vitamins for use in improving the health of the intestines of animals and humans. It has been found that upon delivery to the large intestine, vitamin B2, vitamin C, and vitamin D can each be used to increase the abundance of the beneficial bacterium Bifidobacterium animalis ssp in the intestinal tract.

[0002] [Background of the Invention] Increasing evidence indicates that an imbalance in the human gut microbiota (also referred to as "intestinal endotoxemia") may be associated with Western diseases including obesity and type 2 diabetes, as well as cardiovascular diseases, autoimmune diseases, and intestinal inflammatory diseases. Therefore, targeted modulation of the human gut microbiome with the intention of restoring the imbalance is a potential therapeutic and preventive strategy that has attracted the attention of scholars and those engaged in various industries. The public's awareness and acceptance of substances that modulate the human gut microbiome continue to grow.

[0003] There is a consensus that certain live microorganisms called probiotics have a beneficial effect on human health. The Bifidobacterium animalis ssp lactis strain is a well-known commercially used health-promoting probiotic that actively modulates the composition of the gut microbiota and the function of the immune system (Jungersen M, The science behind the probiotic strain bifidobacterium animalis ssp.lactis BB-12 (registered trademark) (2014)).

[0004] Recently, it has been demonstrated that vitamins can modulate the human gut microbiota. WO 2020 / 043797 pamphlet discloses that vitamins may be useful for increasing the growth of certain beneficial bacteria in the intestine. However, the human intestine is a habitat for hundreds of different microorganisms, and it would be desirable to be able to selectively enhance certain beneficial bacteria. In particular, it would be desirable to increase the abundance of Bifidobacterium animalis ssp. in the intestine in order to improve health, improve digestive health, and support the immune system.

[0005] [Summary of the Invention] The present invention relates to the following items: 1) Vitamin B2, vitamin C, and / or vitamin D for use in increasing the abundance of Bifidobacterium animalis ssp. lactis in the intestine of an animal, preferably a human, wherein said use comprises delivering said vitamin to the large intestine, vitamin B2, vitamin C, and / or vitamin D. 2) Vitamin B2, vitamin C, and / or vitamin D for use according to item 1, wherein said vitamin is delivered to the large intestine by a delayed-release formulation. 3) The use according to item 1 or 2, wherein said use comprises administering to an animal a formulation comprising a vitamin B2 dose of up to 200 mg / day, preferably 5 - 100 mg / day, more preferably 10 - 50 mg / day; a vitamin C dose of up to 2000 mg / day, preferably 100 - 2000 mg / day; more preferably 200 - 1000 mg / day; and / or a vitamin D dose of up to 200 μg / day, preferably 100 μg / day; more preferably 20 μg / day. 4) Vitamin B2, vitamin C, and / or vitamin D for use according to any one of items 1 - 4, wherein said vitamin is co-administered with Bifidobacterium animalis ssp. lactis. 5) Use of vitamin B2, vitamin C, and / or vitamin D according to any one of items 1 to 4 for an animal including a human who is experiencing a condition selected from the group consisting of irritable bowel syndrome, functional intestinal gas symptoms, diarrhea, constipation, abdominal pain and bloating, upper airway symptoms, respiratory tract infections, colon inflammation, colon cancer, liver damage, nutrient loss, and folate deficiency. 6) Use of vitamin B2, vitamin C, and / or vitamin D according to any one of items 1 to 5 for Bifidobacterium animalis ssp. lactis, wherein the Bifidobacterium animalis ssp. lactis is Bifidobacterium animalis ssp. lactis BB - 12, preferably Bifidobacterium animalis ssp. lactis DSM 32269. 7) An oral formulation comprising vitamin B2, vitamin C, and / or vitamin D and Bifidobacterium animalis ssp. lactis. 8) The oral formulation according to item 7, which is a delayed - release formulation. 9) A method for increasing the abundance of Bifidobacterium animalis ssp. lactis in the intestine of an animal, preferably a human, the method comprising administering to the animal a formulation comprising an effective dose of vitamin B2, vitamin C, and / or vitamin D. 10) The method according to item 9, wherein the animal is a human and the vitamin is delivered to the large intestine. 11) The method according to item 9 or 10, wherein the vitamin is delivered by a delayed - release formulation. 12) The method according to any one of claims 9 to 11, wherein the vitamin is co - administered with Bifidobacterium animalis ssp. lactis. 13) A method according to any one of items 9 to 12 for treating, preventing or reducing symptoms of irritable bowel syndrome, functional intestinal gas symptoms, diarrhea, constipation, abdominal pain and bloating, upper airway symptoms, respiratory tract infections, colon inflammation, colon cancer, liver damage, nutrient loss, or folate deficiency in animals including humans in need thereof. 14) A method according to any one of items 9 to 13, wherein Bifidobacterium animalis ssp. lactis is Bifidobacterium animalis ssp. lactis BB-12, preferably Bifidobacterium animalis ssp. lactis DSM 32269.

Brief Description of the Drawings

[0006]

Figure 1A

Figure 1B

Figure 2

[0007] [Detailed Description of the Invention] Bifidobacterium animalis ssp. lactis is a bacterial strain known for its beneficial effects on human health, particularly intestinal health. The inventors have found that vitamin B2, vitamin C, and vitamin D can promote the growth of Bifidobacterium animalis ssp. lactis and result in an increase in the levels of Bifidobacterium animalis ssp. lactis in the intestine.

[0008] Accordingly, in a first aspect, the present invention relates to vitamin B2, vitamin C, and / or vitamin D for use in improving intestinal health in an animal. Said improvement comprises or consists of increasing the abundance of Bifidobacterium animalis ssp. lactis in the intestine of said animal. Specifically, vitamin B2, vitamin C, and / or vitamin D are for use in increasing the abundance of Bifidobacterium animalis ssp. lactis in the large intestine (colon) of an animal, and preferably, said use comprises delivering vitamin B2, vitamin C, and / or vitamin D to the large intestine. Preferably, the animal is a human. Preferably, the vitamin is vitamin B2.

[0009] Vitamin B2 (also known as riboflavin) is one of the water-soluble B vitamins that is an essential component of two major coenzymes, flavin mononucleotide (FMN; also known as riboflavin-5'-phosphate) and flavin adenine dinucleotide (FAD). These coenzymes play a major role in energy production; cell function, growth, and development; and the metabolism of fats, drugs, and steroids. Riboflavin can be purchased from DSM GmbH. Alternative suppliers are TER Chemicals Distribution Group, BIOCHEM Bernburg GmbH, DVA International GmbH, Falken Trade GmbH, and Neupert Ingredients GmbH.

[0010] Vitamin C, also known as L-ascorbic acid, is a water-soluble vitamin necessary for the integrity of collagen, L-carnitine, and certain neurotransmitters. Vitamin C is also involved in protein metabolism. Furthermore, vitamin C is an important physiological antioxidant. Vitamin C plays an important role in immune function and improves nutrient absorption. Vitamin C can be purchased from DSM GmbH. Alternative suppliers are TER Chemicals Distribution Group, BIOCHEM Bernburg GmbH, DVA International GmbH, Falken Trade GmbH, and Neupert Ingredients GmbH.

[0011] Vitamin D is a fat-soluble vitamin that occurs naturally in some foods, is added to others, and is available as a dietary supplement. Vitamin D promotes calcium absorption in the intestine and maintains sufficient serum calcium and phosphate concentrations to enable normal bone mineralization. This is also necessary for bone growth and bone remodeling by osteoblasts and osteoclasts. Vitamin D has other roles in the body, including reduction of inflammation and regulation of processes such as cell proliferation, neuromuscular and immune function, and glucose metabolism. Many genes encoding proteins that control cell proliferation, differentiation, and apoptosis are partially regulated by vitamin D. Vitamin D3, also known as cholecalciferol, is a type of vitamin D produced by the skin when exposed to sunlight; it is also found in some foods and can be taken as a dietary supplement. Vitamin D can be purchased from DSM GmbH. Alternative suppliers are, for example, F Hoffmann-La Roche, Shandong Octagon Chemicals Limited, Fermenta Biotech,.Emkay Pharma Pvt Ltd, Dishman, Polymed Therapeutics, Zhejiang Garden Bio.

[0012] The most common Bifidobacterium animalis ssp. lactis strain is Bifidobacterium animalis ssp. lactis BB-12. This can be purchased, for example, from Chr. Hansen as BB-12®. Bifidobacterium animalis ssp. lactis DSM 32269 (Biocare Copenhagen) has a genomic sequence that is 99.99% identical to the genomic sequence of BB-12®. Therefore, for practical purposes, B. animalis ssp. lactis DSM 32269 can be considered identical or equivalent to B. animalis ssp. lactis BB-12®. Thus, Bifidobacterium animalis ssp. lactis DSM 32269 (Biocare Copenhagen) is referred to herein as the Bifidobacterium animalis ssp. lactis BB-12 strain.

[0013] Alternative Bifidobacterium animalis ssp. lactis strains are, for example, Bifidobacterium lactis Bi-07® (Howaru; Danisco / IFF / DuPont), Bifidobacterium lactis Bl-04® (Howaru; Danisco / IFF / DuPont), and Bifidobacterium lactis HN019 (Howaru; IFF / DuPont).

[0014] Bifidobacterium animalis ssp. lactis DSM 32269 (Biocare Copenhagen) is a preferred strain for the purposes of the present invention. Bifidobacterium animalis ssp. lactis DSM 32269 has been deposited in accordance with the Budapest Treaty on 26 February 2016 at Leibniz Institute DSMZ - German Collection of Microorganisms and Cell Cultures, Inhoffenstr. 7B, D - 38124 Braunschweig, Germany. The accession number assigned by the International Depository Authority is DSM 32269.

[0015] To achieve an increase in the abundance of Bifidobacterium animalis ssp. lactis in the large intestine, vitamins B2, C and / or D are preferably delivered directly to the large intestine. That is, the vitamins are delivered / administered in such a way that the vitamins are not absorbed in the stomach and / or small intestine; the vitamins are delivered / administered to the distal gastrointestinal tract, preferably the large intestine (colon). This is preferably done by delivering / administering vitamins B2, C and / or D in a delayed - release formulation. Oral administration is preferred.

[0016] Preferably, the dose of vitamin B2 administered to an animal is up to 200 mg / day, preferably 5 - 100 mg / day, more preferably 10 - 50 mg / day. In one embodiment, vitamin B2 is administered in an amount such that its local concentration in the colon is at least 0.001 g / L, preferably at least 0.01 g / L, more preferably 0.02 g / L. The preferred local concentration in the colon ranges from about 0.001 g / L to about 0.5 g / L, or from about 0.005 g / L to about 0.2 g / L, preferably from about 0.01 to about 0.02 g / L.

[0017] Preferably, the dosage of vitamin C (ascorbic acid) administered to an animal is up to 2000 mg / day, preferably 100 - 2000 mg / day; more preferably 200 - 1000 mg / day. In one embodiment, vitamin C 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, most preferably at least 0.33 g / L. The preferred local concentration in the colon ranges from about 0.05 g / L to about 1.5 g / L, more preferably from about 0.1 g / L to about 1 g / L, most preferably from about 0.2 g / L to about 0.5 g / L.

[0018] Preferably, the dosage of vitamin D administered to an animal is up to 200 μg / day, preferably 100 μg / day; more preferably 20 μg / day. In one embodiment, vitamin D is administered in an amount such that its local concentration in the colon is at least 0.00025 mg / L, preferably at least 0.0025 mg / L, most preferably at least 0.025 mg / L. The preferred local concentration in the colon ranges from about 0.0001 mg / L to about 5 mg / L, more preferably from about 0.001 mg / L to about 0.5 mg / L, most preferably from about 0.01 mg / L to about 0.05 mg / L.

[0019] The probiotic Bifidobacterium animalis ssp. lactis can be co-administered with vitamin B2, C, or D. Accordingly, the present invention also relates to vitamin B2, C, and / or D for use in increasing the abundance of Bifidobacterium animalis ssp. lactis in the intestine of an animal, preferably a human, said use comprising delivering both vitamin B2, C, and / or D and Bifidobacterium animalis ssp. lactis to the large intestine. This can be done, for example, when Bifidobacterium animalis ssp. lactis has not yet colonized the intestine or only a small number are present. Bifidobacterium animalis ssp. lactis BB-12, particularly Bifidobacterium animalis ssp. lactis DSM 32269 (Biocare Copenhagen), is preferred for co-administration.

[0020] For co-administration, which includes simultaneous administration / delivery / consumption, the vitamins and probiotics can be present in the same compartment or in separate compartments. For example, the vitamins and bacteria can be present in the same tablet / capsule or in the same sachet. Alternatively, the vitamins and bacteria can be present in separate tablets / capsules or in different sachets. In the latter case, administration / delivery / consumption within 24 hours is still considered co-administration.

[0021] Bifidobacterium animalis ssp. lactis is known to improve digestive health and actively regulate the immune system. Many studies have shown that the population of Bifidobacterium animalis ssp. lactis in the gut microbiota decreases in animals, preferably humans, when they are suffering from a particular disease / poor condition, compared to the population present in animals not suffering from that particular disease / poor condition. However, none of these studies have suggested how to increase the population of Bifidobacterium animalis ssp. lactis and thereby reduce at least one of the symptoms of the disease / poor condition. According to the present invention, it has been found that direct delivery of vitamin B2, C, or D to the large intestine of an animal, preferably a human, can increase the population of Bifidobacterium animalis ssp. lactis in the large intestine.

[0022] In a preferred embodiment, the animal (including humans) is experiencing at least one condition selected from the group consisting of irritable bowel syndrome, functional intestinal gas symptoms, diarrhea, constipation, abdominal pain and bloating, upper airway symptoms, respiratory infections, colon inflammation, colon cancer, liver damage, nutrient loss, and folate deficiency.

[0023] Preferably, the Bifidobacterium animalis ssp. lactis to be enhanced is Bifidobacterium animalis ssp. lactis BB-12, for example Bifidobacterium animalis ssp. lactis DSM 32269 (Biocare Copenhagen). Thus, in one embodiment, vitamins B2, C, and / or D are for use in increasing the abundance of Bifidobacterium animalis ssp. lactis BB-12 (preferably Bifidobacterium animalis ssp. lactis DSM 32269) in the large intestine of an animal.

[0024] In another aspect, the present invention relates to an oral formulation comprising vitamins B2, C, and / or D, and Bifidobacterium animalis ssp. lactis. Preferably, the oral formulation is a delayed release formulation. Preferably, the vitamin is vitamin B2.

[0025] In yet another aspect, the present invention relates to a method for increasing the abundance of Bifidobacterium animalis ssp. lactis in the intestine of an animal, preferably in the large intestine, the method comprising administering to the animal an effective dose of vitamin B2, C, and / or D. This method is for improving the health of the animal's intestine, and said improvement comprises increasing the abundance of Bifidobacterium animalis ssp. lactis, preferably Bifidobacterium animalis ssp. lactis BB-12 (in particular, Bifidobacterium animalis ssp. lactis DSM 32269) in the large intestine of said animal. Preferably, the animal is a human. Preferably, the vitamin is vitamin B2.

[0026] Preferably, vitamin B2, C or D is delivered directly to the large intestine. Delivery to the large intestine can be achieved by administering vitamin B2, C, and / or D as a sustained-release formulation.

[0027] In one embodiment, Bifidobacterium animalis ssp. lactis is co-administered with vitamin B2, C, and / or D. Preferably, an effective dose of Bifidobacterium animalis ssp. lactis BB-12 (such as Bifidobacterium animalis ssp. lactis DSM 32269) is co-administered.

[0028] The method of the present invention can be used to treat, prevent, and / or reduce the symptoms of irritable bowel syndrome, functional intestinal gas symptoms, diarrhea, constipation, abdominal pain and bloating, upper airway symptoms, airway infections, colon inflammation, colon cancer, liver damage, nutrient loss, or folate deficiency in animals, including humans, in need thereof.

[0029] In a further aspect, the present invention relates to the use of vitamins B2, C, and / or D to increase the abundance of Bifidobacterium animalis ssp. lactis in the intestine of an animal, preferably a human, said use comprising delivering the vitamins to the large intestine. Preferably, the use comprises delivering / administering the vitamins to the large intestine by means of a delayed-release formulation. The vitamins can be co-administered with Bifidobacterium animalis ssp. lactis. Preferably, the animal, including a human, is experiencing a condition selected from the group consisting of irritable bowel syndrome, functional intestinal gas symptoms, diarrhea, constipation, abdominal pain and bloating, upper airway symptoms, airway infections, colon inflammation, colon cancer, liver damage, nutrient loss, and folate deficiency. Preferably, Bifidobacterium animalis ssp. lactis is Bifidobacterium animalis ssp. lactis BB-12. Bifidobacterium animalis ssp. lactis DSM 32269 is particularly preferred. Preferably, the vitamin is vitamin B2.

[0030] The preferred method of (co)-administration is oral administration.

[0031] [Definitions] When used throughout, the following definitions apply.

[0032] The term "vitamin B2", which is used interchangeably with "riboflavin", includes riboflavin and its esters, particularly riboflavin-5'-phosphate and other pharmaceutically acceptable forms.

[0033] The term "vitamin C", which may be used interchangeably with "ascorbic acid", also includes its pharmaceutically acceptable salts (e.g., sodium ascorbate and calcium ascorbate) and its pharmaceutically acceptable esters (particularly ascorbyl palmitate) and other pharmaceutically acceptable forms.

[0034] As used herein, the term "vitamin D" means vitamin D3. 25-Hydroxyvitamin D3 can preferably be used in non-human species in place of or in addition to vitamin D3. The relative potency of 25-hydroxyvitamin D3 to vitamin D3 is about 40:1, and thus the dose of 25-hydroxyvitamin D3 needs to be adjusted accordingly.

[0035] "Increasing the abundance of" Bifidobacterium animalis ssp. lactis means increasing the level (or amount, or number, or population size) of Bifidobacterium animalis ssp. lactis compared to each control (i.e., the level / amount / number / population size of Bifidobacterium animalis ssp. lactis without the addition of vitamin B2, C, or D).

[0036] As used herein, the term "intestine" (or "gut") refers to a part of the gastrointestinal tract consisting of the small intestine and the large intestine. The "large intestine" (intestinum crassum) is the lower part of the gastrointestinal tract and is also referred to herein as the "colon".

[0037] "Direct delivery" or "delivered directly" means that the vitamin is not absorbed in the stomach and / or small intestine; the vitamin is formulated to be available in the distal gastrointestinal tract, preferably the large intestine (colon), where it is available to the microbiota. The vitamin is administered in excess, rather than as part of a person's normal daily nutritional requirements (typically obtained via diet and conventional vitamin supplementation). For human use, the preferred method according to the invention is via a form that delays release until reaching the large intestine (colon). Alternatively, a high enough dose can be administered such that only a portion of the vitamin administered is absorbed in the proximal small intestine and the remaining, effective dose is available in the large intestine; although less preferred, the latter delivery method can also be used in humans.

[0038] As used herein, "delayed release" refers to the release of the vitamin and / or probiotics being slower than immediately after administration. Preferably, "delayed release" means that the delivery of the vitamin (and / or probiotics) to the large intestine (colon) after oral administration is delayed compared to an immediate release formulation.

[0039] "Enteric layer" or "enteric coating" is a layer surrounding the core, where the core contains the active agent and the layer confers resistance to gastric juice.

[0040] "Prevent" can include reducing the risk of occurrence of an adverse condition, reducing the symptoms of an adverse condition, reducing the severity of an adverse condition, and extending the time of occurrence of an adverse condition.

[0041] "Oral formulation" means that the vitamin and / or probiotics are formulated for oral administration / consumption.

[0042] "Co-administer" or "co-administration" means that the vitamin and / or probiotics are delivered / administered / consumed simultaneously (i.e., together), or separately but within a 24-hour time frame. The vitamin can be delivered / administered / consumed first. Similarly, the probiotics can be delivered / administered / consumed first.

[0043] [Dosage] Vitamin B2 can be administered in an amount such that its local concentration in the colon is at least 0.001 g / L, preferably at least 0.01 g / L, more preferably 0.02 g / L. The preferred local concentration in the colon ranges from about 0.001 g / L to about 0.5 g / L or from about 0.005 g / L to about 0.2 g / L, preferably from about 0.01 to about 0.02 g / L. The specific daily dosage can range up to 200 mg / day, preferably 5 - 100 mg / day, more preferably 10 - 50 mg / day.

[0044] Preferably, vitamin C / 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, most preferably at least 0.33 g / L. The preferred local concentration in the colon ranges from about 0.05 g / L to about 1.5 g / L, more preferably from about 0.1 g / L to about 1 g / L, most preferably from about 0.2 g / L to about 0.5 g / L. The specific daily dosage can range up to 2000 mg / day, preferably 100 - 2000 mg / day; more preferably 200 - 1000 mg / day.

[0045] Preferably, vitamin D3 is administered in an amount such that its local concentration in the colon is at least 0.00025 mg / L, preferably at least 0.0025 mg / L, most preferably at least 0.025 mg / L. The preferred local concentration in the colon ranges from about 0.0001 mg / L to about 5 mg / L, more preferably from about 0.001 mg / L to about 0.5 mg / L, most preferably from about 0.01 mg / L to about 0.05 mg / L. The specific daily dosage can range up to 200 μg / day, preferably 100 μg / day; more preferably 20 μg / day.

[0046] The dosage of probiotics can be up to 5E+10 cfu / day. Preferably, the dosage range of probiotics is 1E+08 - 1E+10 cfu / day, more preferably 1E+09 - 5E+10 cfu / day.

[0047] [Formulation] Vitamins (when the vitamin is co-administered with a probiotic, preferably the probiotic as well) are preferably present in a formulation that preferentially makes the vitamins (and / or probiotics) available in the large intestine.

[0048] Oral formulations are preferred. Other formulations include parenteral routes such as suppositories or injections.

[0049] For human use, the preferred method is via a delayed-release form that delays delivery until it reaches the gastrointestinal tract. For non-human animals, the preferred delivery includes administering a sufficiently high dose such that only a portion of the delivered vitamin is absorbed in the stomach and the remaining, effective dose portion is available in the gastrointestinal tract; although not preferred, this delivery method can also be used in humans.

[0050] Delayed-release formulations are known in the art. Preferably, the delayed-release formulation has an enteric coating (also referred to as an enteric layer).

[0051] In one embodiment of the present invention, vitamins (when the vitamin is co-administered with a probiotic, preferably the probiotic as well) are present in a formulation comprising enteric-coated capsules filled with a composition containing the vitamin. The enteric-coated capsules confer resistance to the acidic environment of the stomach. For example, soft gel formulations deliver the active drug in solution but can still offer the advantages of a solid dosage form.

[0052] In another embodiment, the formulation is a tablet comprising (i) a core containing vitamins (and / or probiotics) and (ii) a delayed-release coating such as an enteric coating. This can be a hard gel capsule.

[0053] Alternatively, a matrix-based delivery system can be used for direct colon delivery. The matrix-based system does not have individual layers of coating material, but the active agent is more or less homogeneously distributed in the matrix. Further, there is a colon release system in which the active agent is embedded in a fiber matrix (enzymatically triggered) and has an enteric coating on top.

[0054] The release of the drug can be delayed until the small intestine. In another embodiment, the release of the drug is delayed until the distal small intestine. In yet another preferred embodiment, the release of the drug is delayed until the colon (large intestine).

[0055] In a preferred embodiment for humans, the vitamins are formulated into solid dosage forms for oral administration. The formulation can optionally be in the form of capsules, pellets, beads, spheres, minispheres, tablets, mini-tablets, or granules, coated with a delayed release coating that prevents the release of the active agent before the small intestine, preferably before the colon.

[0056] Coating or matrix materials for the delayed release of vitamins and / or probiotics, particularly for targeted release in the ileum or large intestine after oral administration, are known in the art. They can be subdivided into coating materials that disintegrate above a specific pH, coating materials that disintegrate after a specific residence time in the gastrointestinal tract, and coating materials that disintegrate by enzyme triggers specific to the microflora of specific regions of the intestine. Different categories of coating materials are generally used in combination. Different categories of coating materials 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 coating materials that disintegrate pH-dependently, coating materials that disintegrate time-dependently, coating materials that disintegrate by enzyme triggers in the intestinal environment (e.g., in the intestinal environment of the ileum and large intestine), and combinations thereof.

[0057] Examples of pH-dependent disintegrating coating materials include polyvinyl acetate phthalate, cellulose acetate trimellitate, hydroxypropyl methylcellulose phthalate HP-50, HP-55 or HP-55S, cellulose acetate phthalate, shellac, hydroxypropyl methylcellulose acetate succinate (HPMCAS), poly(methacrylic acid, ethyl acrylate) 1:1 (Eudragit® L100-55, Eudragit® L30D-55), poly(methacrylic acid, methyl methacrylate) 1:1 (Eudragit® L-100, Eudragit® L12.5), poly(methacrylic acid, methyl methacrylate) 1:2 (Eudragit® S-100, Eudragit® S12,5, and Eudragit® FS30D). Examples of time-dependent disintegrating coating materials include Eudragit® RL, Eudragit® RS, and ethyl cellulose. Examples of coating materials that disintegrate by enzyme triggers in the large intestine 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, curdulan, levan, amylopectin, starch, amylose, resistant starch, and azo compounds decomposed by azo bond-decomposing bacteria.

[0058] The following non-limiting examples are presented to explain the present invention in more detail.

[0059] [Examples] [Example 1] The purpose of this study was to examine the effects of vitamin B2, vitamin C, and vitamin D on the bacteria of the species Bifidobacterium animalis ssp. lactis in the human gut microbiota.

[0060] [Materials and Methods] [Design of Batch Fermentation Experiment (Colon Model)] Short-term batch - Fermentation experiments were performed by ProDigest consisting of colonic incubations of a representative dose of selected vitamins with a representative bacterial inoculum under conditions simulating the proximal colon. Under the current experiments, the bacterial inoculum was derived from fresh fecal samples of six different healthy adult donors. Incubations were performed as previously described (Van den Abbeele, P.; Taminiau, B.; Pinheiro, I.; Duysburgh, C.; Jacobs, H.; Pijls, L.; Marzorati, M. Arabinoxylo-Oligosaccharides and Inulin Impact Inter-Individual Variation on Microbial Metabolism and Composition, Which Immunomodulates Human Cells. J. Agric. Food Chem. 2018, 66, 1121-1130). At the start of the short-term colonic incubations, fresh fecal material from six healthy human donors was collected and after preparation of an anaerobic fecal slurry, this slurry was inoculated at 10 vol% into SHIME nutrient medium containing basic nutrients including 3.5 g / L K2HPO4, 10.9 g / L KH2PO4, 2 g / L NaHCO3 (Chem-lab NV, Zedelgem, Belgium), 2 g / L yeast extract, 2 g / L peptone (Oxoid, Aalst, Belgium), 1 g / L mucin (Carl Roth, Karlsruhe, Germany), 0.5 g / L L-cysteine and 2 mL / L Tween80 (Sigma-Aldrich, Bornem, Belgium). All test components (i.e., probiotic strains, vitamins B2, C, or D3) were also added to the SHIME medium.Furthermore, as previously described (Van den Abbeele, P., et al. (2013). Butyrate-producing Clostridium cluster XIVa species specifically colonize mucins in an in vitro gut model. The ISME Journal 7(5), 949-961), the M-SHIME® technology was incorporated into the current experiment by adding an incubation of a microecosystem covered with mucin (modeling the mucus of the colon). The incubation was carried out at 37°C for 48 hours under shaking (90 rpm) and anaerobic conditions.

[0061] In this study, Bifidobacterium animalis ssp. lactis BB-12 equivalent Bifidobacterium animalis ssp. lactis DSM 32269 (Biocare Copenhagen) was added to the colon model containing donor samples either alone or in combination with vitamins B2, C, or D (see Table 1). After 48 hours of incubation, the abundance of the added strain was analyzed using primers specific to Bifidobacterium animalis ssp. lactis BB-12 / Bifidobacterium animalis ssp. lactis DSM 32269. The experiment was conducted in one replicate.

[0062] [Table 1]

[0063] Bifidobacterium animalis ssp. lactis BB-12 equivalent strain Bifidobacterium animalis ssp. lactis DSM 32269 was added to the SHIME medium as an overnight growth culture at a concentration of 1*10 9 cfu. The probiotic strain was supplemented alone or in combination with vitamin B2, vitamin C, or vitamin D3 (DSM). Vitamin B2, C, and D3 were added at concentrations of 0.01667 g / L, 0.333 g / L, and 0.000025 g / L, respectively, which are equivalent to doses of 10 mg, 200 mg, and 5 mg, respectively, assuming a colonic volume of approximately 600 ml.

[0064] [Analysis of Bifidobacterium animalis ssp. lactis level] As previously described (Junick et al. (2012), Quantification of Human Fecal Bifidobacterium Species by Use of Quantitative Real-Time PCR Analysis Targeting the groEL Gene), qPCR was performed on the groEL gene (NCBI reference CP003497) to monitor the level of the supplemented Bifidobacterium animalis ssp. lactis DSM 32269 strain in the gut microbiota. Samples were analyzed from the lumen and mucus environment after 48 hours of incubation.

[0065] [Statistics] Statistical analysis was performed to examine the mean effect of the test product. For this purpose, the mean of six donors was calculated for each endpoint. A paired t-test was performed to evaluate the potential effect of the test product compared to the control and also to compare different test products with each other. In the case of the microbiota composition, the statistical tests were performed on log-transformed data (to make it normally distributed). A difference was considered statistically significant if the p-value was less than 0.05.

[0066] [Results] [Supplementation with vitamins B2, C, and D increased the levels of Bifidobacterium animalis ssp. lactis in the colonic lumen and mucus] As shown in Figure 1A, when vitamin B2, vitamin C, or vitamin D was co-administered with Bifidobacterium animalis ssp. lactis, a greater number of copies of the Bifidobacterium animalis ssp. lactis DSM 32269-specific groEL gene were detected in the luminal environment by qPCR analysis. Thus, the addition of vitamins B2, C, or D significantly increased the levels of Bifidobacterium animalis ssp. lactis in the colonic model compared to their respective controls.

[0067] As shown in Figure 1B, when vitamin B2 was co-administered with Bifidobacterium animalis ssp. lactis, approximately six-fold more copies of the Bifidobacterium animalis ssp. lactis DSM 32269-specific groEL gene were detected in the mucus environment by qPCR analysis. Therefore, the addition of vitamin B2 significantly increased the level of Bifidobacterium animalis ssp. lactis in the colon model compared to each control.

[0068] [Example 2] The aim of this study was to investigate the effect of vitamin B2 on Bifidobacterium animalis ssp. lactis in the human gut microbiota in a long-term continuous fermentation experiment.

[0069] [Materials and Methods] [Design of the Long-Term SHIME Fermentation Experiment (Colon Model)] The typical reactor configuration of SHIME (registered trademark), which represents the gastrointestinal tract of an adult human, was described by Molly et al. (1993) Applied Microbiology and Biotechnology 39(2):254-258. The inoculum preparation, retention time, pH, temperature settings, and reactor feed composition were previously described by Possemiers et al. (2004) FEMS Microbiol Ecol. 49(3):495-507. Compared to the typical configuration of SHIME, the long-term SHIME experiment used in this example included several adjustments. In one reactor, initially, the conditions simulated the stomach and then changed to conditions that simulated the small intestine by computer. Then, the suspension was added to a colon reactor that mimicked the transverse colon (pH 6.15 - 6.4; retention time = 32 hours; volume 800 mL).

[0070] The SHIME® experiment for this study consisted of three stages: 1. Stabilization period: After inoculating the appropriate fecal sample into the colon reactor, a two-week stabilization period was used to differentiate the microbiota in different reactors according to the local environmental conditions. During this period, a basal nutrient matrix was provided to the SHIME to support the maximum diversity of the gut microbiota originally present in the fecal inoculum. 2. Control period: During this two-week reference period, the standard SHIME nutrient matrix was further administered to the model for 14 days. Analysis of the samples during this period made it possible to determine the baseline microbiota composition and activity in different reactors, which was used as a reference for the results obtained during the treatment. 3. Treatment: During this three-week period, the SHIME was operated under nominal conditions, but the appropriate probiotic strain and vitamin were supplemented to the appropriate reactor. The probiotic strain was added to the reactor at a concentration of 1*10 10 cfu / reactor. Vitamin B2 (riboflavin, DSM) was added to the reactor at a dose of 10 mg / day.

[0071] The probiotic strains used in this experiment were Bifidobacterium animalis ssp. lactis BB - 12 equivalent Bifidobacterium animalis ssp. lactis DSM 32269 (Biocare Copenhagen) alone or in combination with vitamin B2.

[0072] [Quantitative microbiota analysis by 16S rRNA gene sequencing and flow cytometry] Samples for quantitative 16S-targeted Illumina sequencing were collected three times per week during the control and final week of the treatment period. Next-generation 16S rRNA gene amplicon sequencing of the V3-V4 region was performed on samples from the mid-term SHIME experiment by LGC Genomics GmbH (Berlin, Germany). Library preparation and sequencing were performed on the Illumina MiSeq platform using v3 chemistry. The 341F (5′-CCTACGGGNGGCWGCAG-3′) and 785R (5′- GACTACHVGGGTATCTAAKCC-3′) primers were used as described by De Paepe et al. (2017), and the reverse primer was adapted to increase coverage. Quality control PCR was performed using Taq DNA polymerase with the Fermentas PCR kit according to the manufacturer's instructions (Thermo Fisher Scientific, Waltham, MA, USA). DNA quality was verified by electrophoresis at 100 V for 30 min on a 2% (w / v) agarose gel. Bioinformatics analysis of the amplicon data was performed as described by De Paepe et al. (2017). The resulting high-resolution proportional phylogenetic information (i.e., proportional abundances (%)) was combined with accurate quantification of total bacterial cells by flow cytometry to obtain quantitative data at the phylum, family, and species levels. This was done by multiplying the proportional abundances by the absolute cell numbers (cells / mL) obtained by flow cytometry. For flow cytometry analysis, 10-fold serial dilutions of all samples were prepared in Dulbecco's phosphate-buffered saline (DPBS) (Sigma-Aldrich, Bornem, Belgium) and stained with 0.01 mM SYTO24 (Life Technologies Europe, Merelbeke, Belgium) for 15 min at 37 °C in the dark. Samples were analyzed on a BD Facsverse (BD Biosciences, Erembodegem, Belgium) using a high flow rate setting, and bacteria were separated from media debris and signal noise by applying a threshold level of 200 to the SYTO channel.

[0073] [Results] [Supplementation with vitamin B2 increased the abundance of Bifidobacterium animalis ssp. lactis.] As can be understood from FIG. 2, supplementation with Bifidobacterium animalis ssp. lactis significantly increased the abundance of Bifidobacterium animalis ssp. lactis compared to the control. Similarly, supplementation with Bifidobacterium animalis ssp. lactis with vitamin B2 also significantly increased the abundance of Bifidobacterium animalis ssp. lactis compared to the control. Surprisingly, treatment with the combination of Bifidobacterium animalis ssp. lactis and vitamin B2 resulted in a significantly higher abundance of Bifidobacterium animalis ssp. lactis compared to treatment with Bifidobacterium animalis ssp. lactis alone.

Claims

1. Vitamin B2, vitamin C, and / or vitamin D for use in increasing the abundance of Bifidobacterium animalis subsp. lactis in the intestine of an animal, preferably a human, wherein said use comprises delivering said vitamin to the large intestine.

2. The vitamin B2, vitamin C, and / or vitamin D for use according to claim 1, wherein said vitamin is delivered to the large intestine by a delayed release formulation.

3. The use according to claim 1 or 2, wherein said use comprises administering to said animal a formulation comprising a vitamin B2 dosage of up to 200 mg / day, preferably 5 - 100 mg / day, more preferably 10 - 50 mg / day; a vitamin C dosage of up to 2000 mg / day, preferably 100 - 2000 mg / day; more preferably 200 - 1000 mg / day; and / or a vitamin D dosage of up to 200 μg / day, preferably 100 μg / day; more preferably 20 μg / day.

4. The vitamin B2, vitamin C, and / or vitamin D for use according to any one of claims 1 to 3, wherein said vitamin is co - administered with Bifidobacterium animalis subsp. lactis.

5. The animal, including a human, is experiencing at least one condition selected from the group consisting of irritable bowel syndrome, functional intestinal gas symptoms, diarrhea, constipation, abdominal pain and distension, upper airway symptoms, respiratory tract infections, colon inflammation, colon cancer, liver damage, nutrient loss, and folate deficiency, for the use of vitamin B2, vitamin C, and / or vitamin D according to any one of claims 1 to 4.

6. The vitamin B2, vitamin C, and / or vitamin D for use according to any one of claims 1 to 5, wherein said Bifidobacterium animalis subsp. lactis is Bifidobacterium animalis subsp. lactis BB - 12.

7. a) vitamin B2, vitamin C, and / or vitamin D, and b) Bifidobacterium animalis ssp. lactis An oral preparation containing the same.

8. The oral preparation according to claim 7, which is a sustained-release preparation.

9. A method for increasing the abundance of Bifidobacterium animalis ssp. lactis in the intestine of an animal, preferably a human, the method comprising administering to the animal a preparation containing an effective dose of vitamin B2, vitamin C, and / or vitamin D.

10. The method according to claim 9, wherein the animal is a human and the vitamin is delivered to the large intestine.

11. The method according to claim 9 or 10, wherein the vitamin is delivered by a sustained-release preparation.

12. The method according to any one of claims 9 to 11, wherein the vitamin is co-administered with Bifidobacterium animalis ssp. lactis.

13. A method for treating, preventing, or reducing the symptoms of irritable bowel syndrome, functional intestinal gas symptoms, diarrhea, constipation, abdominal pain and distension, upper airway symptoms, airway infections, colon inflammation, colon cancer, liver damage, nutrient loss, or folate deficiency in an animal, including a human in need thereof, the method according to any one of claims 9 to 12.

14. The method according to any one of claims 9 to 13, wherein the Bifidobacterium animalis ssp. lactis is Bifidobacterium animalis ssp. lactis BB-12.

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