Vitamin B2 for use in improving intestinal health

Abstract

The present invention relates to vitamin B2 (riboflavin) for use in improving intestinal health in animals and humans. In particular, it has been found that upon delivery to the large intestine, vitamin B2 increases the abundance of the beneficial bacterium Lactobacillus rhamnosus in the intestinal tract.

Description

Detailed Description of the Invention

[0001] [Field of the Invention] The present invention relates to vitamin B2 (riboflavin) for use in improving intestinal health in animals and humans. It has been found that upon delivery to the large intestine, vitamin B2 increases the abundance of the beneficial bacterium Lactobacillus rhamnosus in the intestinal tract.

[0002] [Background of the Invention] Increasing evidence indicates that an imbalance in the human gut microbiota (also referred to as "gut 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. Public 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 beneficial effects on human health. Lactobacillus rhamnosus GG (L. rhamnosus GG) is the first strain of the genus Lactobacillus to be patented in 1989, as a result of its ability to survive and grow in media containing gastric acid pH and bile and to adhere to intestinal cells. Furthermore, L. rhamnosus GG can produce a biofilm that can mechanically protect the intestine by enhancing intestinal crypt survival, reducing apoptosis of the intestinal epithelium, and maintaining the integrity of the cytoskeleton, thereby being beneficial to the intestine and various soluble factors. L. rhamnosus GG inhibits some pathogens such as Salmonella species due to its lectin-like proteins 1 and 2. In addition, L. rhamnosus GG can promote type 1 immunoreactivity by reducing the expression of some activation and inflammatory markers on monocytes and by increasing the production of interleukin-10, interleukin-12, and tumor necrosis factor-α in macrophages. A large amount of research data on L. rhamnosus GG has proven its beneficial role in human health (Capurso, J Clin Gastroenterol. 2019 S1 Mar;53 Suppl 1:S41). Lactobacillus rhamnosus has recently been renamed Lacticaseibacillus rhamnosus.

[0004] 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 enhance certain beneficial bacteria. In particular, it would be desirable to increase the abundance of Lactobacillus rhamnosus 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 for use in increasing the abundance of Lactobacillus (Lacticaseibacillus) rhamnosus in the intestine of an animal, preferably a human, said use comprising delivery of vitamin B2 to the large intestine, vitamin B2 for use. 2) Vitamin B2 according to item 1, wherein the vitamin B2 is delivered to the large intestine by a delayed release formulation, vitamin B2 for use according to item 1. 3) Vitamin B2 for use according to item 1 or 2, said use comprising administering to the animal a vitamin B2 dose of up to 200 mg / day, preferably 5 - 100 mg / day, more preferably 10 - 50 mg / day. 4) Vitamin B2 for use according to any one of items 1 - 3, wherein Lactobacillus (Lacticaseibacillus) rhamnosus is co-administered with the vitamin B2. 5) Vitamin B2 for use according to any one of items 1 - 4, wherein the animal, including a human, is experiencing a condition selected from the group consisting of gastrointestinal infections and diarrhea, diarrhea associated with antibiotics and Clostridium difficile, irritable bowel syndrome, inflammatory bowel disease, respiratory infections, allergies, cardiovascular disease, non-alcoholic fatty liver disease, non-alcoholic steatohepatitis, cystic fibrosis, and cancer. 6) Vitamin B2 for use according to any one of items 1 to 5, wherein Lactobacillus (Lacticaseibacillus) rhamnosus is Lactobacillus (Lacticaseibacillus) rhamnosus GG, preferably Lactobacillus rhamnosus DSM 32550. 7) An oral preparation containing vitamin B2 and Lactobacillus (Lacticaseibacillus) rhamnosus. 8) The oral preparation according to item 7, which is a delayed-release preparation. 9) A method for increasing the abundance of Lactobacillus (Lacticaseibacillus) rhamnosus 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. 10) The method according to item 9, wherein the animal is a human and vitamin B2 is delivered to the large intestine. 11) The method according to item 9 or 10, wherein vitamin B2 is delivered by a delayed-release preparation. 12) The method according to any one of claims 9 to 11, wherein Lactobacillus (Lacticaseibacillus) rhamnosus is co-administered with vitamin B2. 13) A method for treating, preventing and / or reducing the symptoms of gastrointestinal infections and diarrhea, diarrhea associated with antibiotics and Clostridium difficile, irritable bowel syndrome, inflammatory bowel disease, respiratory infections, allergies, cardiovascular diseases, non-alcoholic fatty liver disease, non-alcoholic steatohepatitis, cystic fibrosis, and cancer in animals including humans in need thereof, the method according to any one of items 9 to 12. 14) A method according to any one of items 9 to 13, wherein Lactobacillus (Lacticaseibacillus) rhamnosus is Lactobacillus (Lacticaseibacillus) rhamnosus GG, preferably Lactobacillus rhamnosus DSM 32550.

Brief Description of the Drawings

[0006]

Figure 1

[0007] [Detailed Description of the Invention] Lactobacillus (Lacticaseibacillus) rhamnosus is a bacterial strain known for its beneficial effects on human health, particularly intestinal health. The inventors have found that vitamin B2 can promote the growth of Lactobacillus rhamnosus and lead to an increase in the level of Lactobacillus rhamnosus in the intestine.

[0008] Accordingly, in a first aspect, the present invention relates to vitamin B2 for use in improving intestinal health in an animal. The improvement comprises or consists of increasing the abundance of Lactobacillus (Lacticaseibacillus) rhamnosus in the intestine of the animal. Specifically, vitamin B2 is for use in increasing the abundance of Lactobacillus rhamnosus in the large intestine (colon) of an animal, and preferably, the use comprises delivering vitamin B2 to the large intestine. Preferably, the animal is a human.

[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] The most common Lactobacillus (Lacticaseibacillus) rhamnosus strain is Lactobacillus rhamnosus GG. This can be purchased, for example, from Chr. Hansen, Denmark, as LGG®. Lactobacillus (Lacticaseibacillus) rhamnosus DSM 32550 (Biocare Copenhagen, Denmark) has a genomic sequence that is 99.99% identical to the genomic sequence of LGG®. Therefore, for practical purposes, L. rhamnosus DSM 32550 can be considered identical or equivalent to LGG®. Thus, L. rhamnosus DSM 32550 is referred to herein as "Lactobacillus rhamnosus GG".

[0011] Alternative Lactobacillus rhamnosus strains include, among others, Lactobacillus rhamnosus HN001 (Howaru; Danisco / DuPont), Lactobacillus rhamnosus GR-1® (Chr. Hansen, Denmark), and Lactobacillus rhamnosus Rosell-11 (Lallemand, Canada).

[0012] Lactobacillus (Lacticaseibacillus) rhamnosus DSM 32550 (Biocare Copenhagen) is a preferred strain according to the present invention. It has been deposited in accordance with the Budapest Treaty on July 6, 2017, with the 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 32550.

[0013] To achieve an increase in the abundance of Lactobacillus rhamnosus in the large intestine, vitamin B2 is preferably delivered directly to the large intestine. That is, the vitamin is delivered / administered in such a way that the vitamin is not absorbed in the stomach and / or small intestine; the vitamin is delivered / administered to the distal gastrointestinal tract, preferably the large intestine (colon). This is preferably done by delivering / administering vitamin B2 in a delayed - release formulation. Oral administration is preferred.

[0014] Preferably, the dosage 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 about 0.005 g / L to about 0.2 g / L, preferably about 0.01 to about 0.02 g / L.

[0015] The probiotic Lactobacillus rhamnosus can be co-administered with vitamin B2. Accordingly, the present invention also relates to vitamin B2 for use in increasing the abundance of Lactobacillus rhamnosus in the intestine of an animal, preferably a human, said use comprising delivering both vitamin B2 and Lactobacillus rhamnosus to the large intestine. This can be done, for example, when Lactobacillus rhamnosus has not yet colonized the intestine or only a small number is present. Lactobacillus rhamnosus GG, particularly Lactobacillus rhamnosus DSM 32550, is preferred for co-administration.

[0016] For co-administration, including co-administration, delivery, or consumption, the vitamin and the probiotic can be present in the same compartment or in separate compartments. For example, the vitamin and the bacterium can be present in the same tablet / pill or in the same sachet. Alternatively, the vitamin and the bacterium can be present in separate tablets / pills or in different sachets. In the latter case, administration / delivery / consumption within 24 hours is still considered co-administration.

[0017] Lactobacillus rhamnosus is known to improve digestive health and actively regulate the immune system. Many studies have shown that the population of Lactobacillus rhamnosus in the gut microbiota decreases when an animal, preferably a human, is 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 suggest how to increase the population of Lactobacillus rhamnosus 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 to the large intestine of an animal, preferably a human, can increase the population of Lactobacillus rhamnosus in the large intestine.

[0018] In a preferred embodiment, the animal (including humans) is experiencing a condition selected from the group consisting of gastrointestinal infections and diarrhea, diarrhea associated with antibiotics and Clostridium difficile, irritable bowel syndrome, inflammatory bowel disease, respiratory infections, allergies, cardiovascular disease, non-alcoholic fatty liver disease, non-alcoholic steatohepatitis, cystic fibrosis, and cancer.

[0019] Preferably, the Lactobacillus rhamnosus to be enhanced is Lactobacillus rhamnosus GG (e.g., Lactobacillus rhamnosus DSM 32550). Thus, in one embodiment, vitamin B2 is for use in increasing the abundance of Lactobacillus rhamnosus GG (preferably Lactobacillus rhamnosus DSM 32550) in the large intestine of an animal.

[0020] In another aspect, the present invention relates to an oral formulation comprising vitamin B2 and Lactobacillus rhamnosus. Preferably, the oral formulation is a delayed-release formulation.

[0021] In yet another aspect, the present invention relates to a method for increasing the abundance of Lactobacillus rhamnosus in the intestine of an animal, preferably the large intestine, the method comprising administering to the animal an effective dose of vitamin B2. This method is for improving the health of the animal's intestine, and said improvement comprises increasing the abundance of Lactobacillus rhamnosus GG, preferably Lr GG (in particular, Lactobacillus rhamnosus DSM 32550) in the large intestine of said animal. Preferably, the animal is a human.

[0022] Preferably, vitamin B2 is delivered directly to the large intestine. Delivery to the large intestine can be achieved by administering vitamin B2 as a delayed-release formulation.

[0023] In one embodiment, Lactobacillus rhamnosus is co-administered with vitamin B2. Preferably, an effective dose of Lactobacillus rhamnosus GG (such as Lactobacillus rhamnosus DSM 32550) is co-administered.

[0024] The method of the present invention can be used to treat, prevent and / or reduce the symptoms of gastrointestinal infections and diarrhea, diarrhea associated with antibiotics and Clostridium difficile, irritable bowel syndrome, inflammatory bowel disease, respiratory infections, allergies, cardiovascular diseases, non-alcoholic fatty liver disease, non-alcoholic steatohepatitis, cystic fibrosis, and cancer.

[0025] In a further aspect, the invention relates to the use of vitamin B2 for increasing the abundance of Lactobacillus rhamnosus in the intestine of an animal, preferably a human, said use comprising delivering vitamin B2 to the large intestine. Preferably, the use comprises delivering / administering vitamin B2 to the large intestine by means of a delayed-release formulation. In one embodiment, the use comprises administering to an 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. Vitamin B2 can be co-administered with Lactobacillus rhamnosus. Preferably, the animal, including a human, is experiencing a condition selected from the group consisting of gastrointestinal infections and diarrhea, diarrhea associated with antibiotics and Clostridium difficile, irritable bowel syndrome, inflammatory bowel disease, respiratory infections, allergies, cardiovascular diseases, non-alcoholic fatty liver disease, non-alcoholic steatohepatitis, cystic fibrosis, and cancer. Preferably, the Lactobacillus rhamnosus used is Lactobacillus rhamnosus GG. Lactobacillus rhamnosus DSM 32550 is particularly preferred.

[0026] The preferred (co-)administration method is oral administration.

[0027] [Definitions] When used throughout, the following definitions apply.

[0028] The term "vitamin B2", which is used interchangeably with "riboflavin", includes riboflavin and its esters, in particular riboflavin-5'-phosphate and other pharmaceutically acceptable forms.

[0029] "Increasing the abundance" of Lactobacillus rhamnosus means increasing the level (or amount, or number, or population size) of Lactobacillus rhamnosus as compared to each control (i.e., the level / amount / number / population size of Lactobacillus rhamnosus without the addition of vitamin B2).

[0030] 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".

[0031] "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 in the large intestine (colon), where it is available to the microbiota. The vitamin is administered in an amount in excess of a part of the normal daily nutritional requirements of a human (normally obtained via diet and conventional vitamin supplementation). For human use, a preferred method according to the invention is via a form that delays release until it reaches the large intestine (colon). Alternatively, a sufficiently high dose can be administered such that only a part of the administered vitamin is absorbed in the proximal small intestine and the remaining part, which is an effective dose, is available in the large intestine; although less preferred, the latter delivery method can also be used in humans.

[0032] 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 as compared to an immediate release formulation.

[0033] The "enteric layer" or "enteric coating" is a layer that surrounds the core, the core contains the active pharmaceutical ingredient, and the layer confers resistance to gastric juice.

[0034] "Prevent" may include reducing the risk of occurrence of adverse conditions, reducing the symptoms of adverse conditions, reducing the severity of adverse conditions, and prolonging the occurrence time of adverse conditions.

[0035] "Oral preparation" means that vitamins and / or probiotics are formulated for oral administration / consumption.

[0036] "Co-administer" or "co-administration" means that vitamins and / or probiotics are delivered / administered / consumed simultaneously (i.e., together), or separately but within a 24-hour time frame. The vitamins may be delivered / administered / consumed first; similarly, the probiotics may be delivered / administered / consumed first.

[0037] Lactobacillus rhamnosus has recently been renamed Lacticaseibacillus rhamnosus, and both names are used interchangeably herein and can both be abbreviated as L. rhamnosus.

[0038] [Dosage] Riboflavin 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.

[0039] The dosage of the probiotics can be up to 5E+10 cfu / day. Preferably, the dosage range of the probiotics is 1E+08 - 1E+10 cfu / day, more preferably 1E+09 - 5E+10 cfu / day.

[0040] [Formulation] Vitamins (when the vitamin is co-administered with the probiotics, preferably the probiotics as well) are preferably present in a formulation that preferentially makes the vitamins (and / or probiotics) available in the large intestine.

[0041] An oral formulation is preferred. Other formulations include parenteral routes such as suppositories or injections.

[0042] 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 portion, which is the effective dose, is available in the gastrointestinal tract; although not preferred, this delivery method can also be used in humans.

[0043] Delayed-release formulations are known in the art. Preferably, the delayed-release formulation has an enteric coating (also referred to as an enteric layer).

[0044] In one embodiment of the present invention, vitamins (when the vitamin is co-administered with the probiotics, preferably the probiotics as well) are present in a formulation comprising enteric-coated capsules filled with a composition containing the vitamins. The enteric 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.

[0045] 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.

[0046] 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.

[0047] The release of the drug can be delayed until the small intestine. In another embodiment, the release of the active agent is delayed until the distal small intestine. In yet another preferred embodiment, the release of the drug is delayed until the colon (large intestine).

[0048] In a preferred embodiment for humans, the vitamins are formulated into a solid dosage form for oral administration. The formulation can be in the form of capsules, pellets, beads, spheres, minispheres, tablets, minitablets, or granules, optionally coated with a delayed release coating that prevents the release of the active agent before the small intestine, preferably before the colon.

[0049] 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 an enzyme trigger specific to the microflora of a specific region 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 a coating material that disintegrates pH-dependently, a coating material that disintegrates time-dependently, a coating material that disintegrates by an enzyme trigger in the intestinal environment (e.g., in the intestinal environment of the ileum and large intestine), and combinations thereof.

[0050] 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.

[0051] The following non-limiting examples are presented to explain the present invention in more detail.

[0052] [Examples] The purpose of this study was to examine the effect of vitamin B2 on the bacterium Lactobacillus rhamnosus, a species in the human gut microbiota.

[0053] [Materials and Methods] [Design of Batch Fermentation Experiments (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, vitamin B2) 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 mucins (modeling the mucus of the colon). The incubation was carried out at 37°C for 48 hours under shaking (90 rpm) and anaerobic conditions.

[0054] In this study, Lactobacillus rhamnosus GG equivalent Lactobacillus rhamnosus DSM 32550 (Biocare Copenhagen) was added alone or in combination with vitamin B2 to the colon model containing donor samples (see Table 1). After 48 hours of incubation, the abundance of the added strain was analyzed using primers specific to Lactobacillus rhamnosus GG and its equivalent Lactobacillus rhamnosus DSM 32550. The experiment was performed in one replicate.

[0055] Lactobacillus rhamnosus DSM 32550 has a genomic sequence that is 99.99% identical to the genomic sequence of LGG®. Therefore, for practical purposes, L. rhamnosus DSM 32550 can be considered identical or equivalent to LGG®. In the examples of the present invention, Lactobacillus rhamnosus DSM 32550 is referred to as the Lactobacillus rhamnosus GG strain.

[0056]

Table 1

[0057] Lactobacillus rhamnosus GG equivalent strain Lactobacillus rhamnosus DSM 32550 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 B2 (riboflavin TG, DSM) was added at a concentration of 0.01667 g / L, which is equivalent to a 10 mg dose assuming a colonic volume of approximately 600 ml.

[0058] [Analysis of Lactobacillus rhamnosus GG level] As previously described (Brandt, Alatossava (2003) - Specific identification of certain probiotic actobacillus rhamnosus strains with PCR primers based on phage-related sequences), qPCR was performed on the putative phage tail protein gene (NCBI reference NC_013198) to monitor the level of the supplemented probiotic Lactobacillus rhamnosus GG strain in the gut microbiota. Samples were analyzed from the mucus environment after 48 hours of incubation.

[0059] [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 to each other. In the case of the microbiota composition, the statistical test was 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.

[0060] [Results] [Supplementation with vitamin B2 increased the level of Lactobacillus rhamnosus GG] As shown in Figure 1B, when vitamin B2 was co-administered with Lactobacillus rhamnosus GG, approximately three-fold more copies of the Lactobacillus rhamnosus GG putative phage tail protein gene were detected in the mucus environment by qPCR analysis. Thus, the addition of vitamin B2 significantly increased the level of Lactobacillus rhamnosus GG in the colon model compared to each respective control.

Claims

1. A preparation comprising vitamin B2 for use in increasing the abundance of Lactobacillus rhamnosus in the intestines of animals, wherein the use includes the delivery of vitamin B2 to the large intestine.

2. The formulation for use according to claim 1, wherein the vitamin B2 is delivered to the large intestine by a delayed-release formulation.

3. The formulation for use according to claim 1 or 2, wherein the use comprises administering a vitamin B2 dose of up to 200 mg / day to the animal.

4. The formulation for use according to claim 1 or 2, wherein the formulation further comprises Lactobacillus rhamnosus.

5. A formulation for use according to claim 1 or 2, wherein the animal, including a human, has experienced at least one condition selected from the group consisting of gastrointestinal infection and diarrhea, antibiotic and Clostridium difficile-associated diarrhea, irritable bowel syndrome, inflammatory bowel disease, respiratory infection, allergy, cardiovascular disease, non-alcoholic fatty liver disease, non-alcoholic steatohepatitis, cystic fibrosis, and cancer.

6. The formulation for use according to claim 1 or 2, wherein the Lactobacillus rhamnosus is Lactobacillus rhamnosus GG.

7. An oral preparation containing vitamin B2 and Lactobacillus rhamnosus.

8. The oral formulation according to claim 7, which is a delayed-release formulation.

9. The formulation according to claim 1 or 2, wherein the animal is a human.

10. A formulation according to claim 1 or 7 for treating, preventing and / or reducing symptoms of gastrointestinal infections and diarrhea, antibiotic and Clostridium difficile-associated diarrhea, irritable bowel syndrome, inflammatory bowel disease, respiratory infections, allergies, cardiovascular disease, non-alcoholic fatty liver disease, non-alcoholic steatohepatitis, cystic fibrosis, and cancer in animals, including humans, for which such treatment is necessary.

Images