Vitamin B2 for improving intestinal health

Abstract

The present invention relates to vitamin B2 (riboflavin) for improving intestinal health in animals and humans. Specifically, it is found that after the vitamin B2 is delivered to the large intestine, the abundance of the beneficial bacterium lactobacillus rhamnosus in the intestinal tract can be increased.

Description

Technical Field

[0001] The present invention relates to vitamin B2 (riboflavin) for improving intestinal health in animals and humans. Vitamin B2 has been found to increase the abundance of the beneficial bacteria Lactobacillus rhamnosus in the intestine after delivery to the large intestine. Background Art

[0002] There is growing evidence that an imbalance in the human gut microbiota (also known as "microenvironmental dysbiosis") may be associated with Western diseases, including obesity and type 2 diabetes, as well as cardiovascular disease, autoimmune disease, and intestinal inflammatory diseases. Therefore, targeted regulation of the human gut microbiome aimed at restoring the imbalance is a potential therapeutic and preventive strategy and has attracted the attention of academia and people from all walks of life. Public awareness and acceptance of substances that regulate the human gut microbiome continues to increase.

[0003] It is generally agreed that certain live microorganisms, so-called probiotics, have beneficial effects on human health. Lactobacillus rhamnosus GG (L. rhamnosus GG) is the first strain of Lactobacillus to be patented in 1989 for its ability to survive and proliferate and adhere to intestinal epithelial cells at gastric acid pH and in a medium containing bile. In addition, Lactobacillus rhamnosus GG is able to produce a biofilm that mechanically protects the mucosa and different soluble factors that are beneficial to the intestine by enhancing intestinal crypt survival, reducing intestinal epithelial cell apoptosis and maintaining cytoskeletal integrity. Lactobacillus rhamnosus GG inhibits some pathogens, such as Salmonella species, due to its lectin-like proteins 1 and 2. In addition, Lactobacillus rhamnosus GG can promote type 1 immune reactivity by reducing the expression of several 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 Lactobacillus rhamnosus GG demonstrates its beneficial effects on human health (Capurso, J Clin Gastroenterol. 2019 Mar;53 Suppl 1:S1-S41). Lactobacillus rhamnosus was recently renamed Lactobacillus rhamnosus.

[0004] It has been shown that vitamins can modulate the human gut microbiome. WO2020 / 043797 discloses that vitamins can be used to increase the growth of certain beneficial bacteria in the gut. However, the human gut is inhabited by hundreds of different microorganisms, and it would be desirable to be able to increase specific beneficial bacteria. In particular, it would be desirable to increase the abundance of Lactobacillus rhamnosus in the gut to enhance health, improve digestive health, and support the immune system. Summary of the invention

[0005] The present invention relates to the following items:

[0006] 1) Vitamin B2 for use in increasing the abundance of Lactobacillus rhamnosus (Lactobacillus rhamnosus) in the intestine of an animal, preferably a human, wherein the use comprises delivering the vitamin B2 to the large intestine.

[0007] 2) Vitamin B2 for use according to item 1, wherein the vitamin B2 is delivered to the large intestine via a delayed-release formulation.

[0008] 3) Vitamin B2 for use according to item 1 or 2, wherein the use comprises administering to the animal a preparation comprising vitamin B2 in a dose of up to 200 mg / day, preferably 5-100 mg / day, more preferably 10-50 mg / day.

[0009] 4) Vitamin B2 for use according to any one of items 1 to 3, wherein Lactobacillus rhamnosus (Lactobacillus rhamnosus) is co-administered with vitamin B2.

[0010] 5) Vitamin B2 for use according to any one of items 1 to 4, wherein the animal, including humans, is experiencing a condition selected from the group consisting of gastrointestinal infection and diarrhea, antibiotic and Clostridium difficile associated diarrhea, irritable bowel syndrome, inflammatory bowel disease, respiratory tract infection, allergic reaction, cardiovascular disease, non-alcoholic fatty liver disease, non-alcoholic steatohepatitis, cystic fibrosis and cancer.

[0011] 6) Vitamin B2 for use according to any one of items 1 to 5, wherein the Lactobacillus rhamnosus (Lactobacillus rhamnosus) is Lactobacillus rhamnosus (Lactobacillus rhamnosus) GG, preferably Lactobacillus rhamnosus DSM 32550.

[0012] 7) An oral preparation comprising vitamin B2 and Lactobacillus rhamnosus (Lactobacillus rhamnosus).

[0013] 8) The oral preparation according to item 7, which is a delayed-release preparation.

[0014] 9) A method of increasing the abundance of Lactobacillus rhamnosus (Lactobacillus rhamnosus) in the intestine of an animal, preferably a human, the method comprising administering to the animal a preparation comprising an effective dose of vitamin B2.

[0015] 10) The method according to item 9, wherein the animal is a human and the vitamin B2 is delivered to the large intestine.

[0016] 11) The method according to item 9 or 10, wherein the vitamin B2 is delivered via a delayed release formulation.

[0017] 12) The method according to any one of claims 9 to 11, wherein the Lactobacillus rhamnosus (Lactobacillus rhamnosus) is co-administered with vitamin B2.

[0018] 13) A method according to any one of items 9 to 12, which is a method for treating, preventing or alleviating the symptoms of gastrointestinal infections and diarrhea, antibiotic and Clostridium difficile associated diarrhea, irritable bowel syndrome, inflammatory bowel disease, respiratory infections, allergic reactions, cardiovascular disease, non-alcoholic fatty liver disease, non-alcoholic steatohepatitis, cystic fibrosis and / or cancer in animals in need thereof, including humans.

[0019] 14) Method according to any of items 9 to 13, wherein the Lactobacillus rhamnosus (Lactobacillus rhamnosus) is Lactobacillus rhamnosus (Lactobacillus rhamnosus) GG, preferably Lactobacillus rhamnosus DSM 32550. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 Mean Lactobacillus rhamnosus GG levels (putative phage tail protein gene copies / mL) after 48 h of colonic incubation for six different human donors are shown (± standard deviation). Mean results are shown for Lactobacillus rhamnosus GG alone or Lactobacillus rhamnosus GG co-supplemented with vitamin B2. Statistically significant differences between the test conditions and the control are indicated with '*' (p<0.05). DETAILED DESCRIPTION

[0021] Lactobacillus rhamnosus (Lactobacillus rhamnosus) is a bacterial strain well known for its beneficial effects on human health, especially intestinal health. The present inventors have found that vitamin B2 can promote the growth of Lactobacillus rhamnosus, resulting in increased levels of Lactobacillus rhamnosus in the intestine.

[0022] Therefore, in a first aspect, the present invention relates to vitamin B2 for improving the intestinal health of an animal. The improvement comprises or consists of increasing the abundance of Lactobacillus rhamnosus (Lactobacillus rhamnosus) in the intestinal tract of the animal. Specifically, vitamin B2 is used to increase the abundance of Lactobacillus rhamnosus in the large intestine (colon) of the animal, and preferably the use comprises delivering vitamin B2 to the large intestine. Preferably, the animal is a human.

[0023] Vitamin B2 (also known as riboflavin) is one of the water-soluble B vitamins and an essential component of two major coenzymes, flavin mononucleotide (FMN; also known as riboflavin-5'-phosphate) and flavin adenine dinucleotide (FAD). These coenzymes play important roles 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, BIOCHEMBernburg GmbH, DVA International GmbH, Falken Trade GmbH, and Neupert IngredientsGmbH.

[0024] The most common strain of Lactobacillus rhamnosus (Lactobacillus rhamnosus) is Lactobacillus rhamnosus GG. It can be obtained, for example, from Chr. Hansen, Denmark as The genome sequence of Lactobacillus rhamnosus (Lactobacillus rhamnosus) DSM 32550 (Biocare Copenhagen, Denmark) was compared with The genome sequence of Lactobacillus rhamnosus DSM 32550 is 99.99% identical to that of Therefore, Lactobacillus rhamnosus DSM 32550 will be referred to herein as "Lactobacillus rhamnosus GG".

[0025] Alternative Lactobacillus rhamnosus strains are particularly Lactobacillus rhamnosus HN001 (Howaru; Danisco / DuPont), Lactobacillus rhamnosus (Chr. Hansen, Denmark) and Lactobacillus rhamnosus Rosell-11 (Lallemand, Canada).

[0026] Lactobacillus rhamnosus (Lactobacillus rhamnosus) DSM 32550 (Biocare Copenhagen) is a preferred strain according to the present invention. It has been deposited at the Leibniz Institute DSMZ-German Collection of Microorganisms and Cell Cultures, Inhoffenstr. 7B, D-38124 Braunschweig, Germany on July 6, 2017 according to the Budapest Treaty. The accession number given by the international depository is DSM 32550.

[0027] In order to achieve an increased 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 a manner such that the vitamin is not absorbed in the stomach and / or small intestine; instead, the vitamin is delivered / administered to the distal intestinal tract, preferably the large intestine (colon). This is preferably done by delivering / administering vitamin B2 in a delayed release formulation. Oral administration is preferred.

[0028] Preferably, the vitamin B2 dosage administered to an animal is at most 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 range in the colon is 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 g / L to about 0.02 g / L.

[0029] The probiotic strain Lactobacillus rhamnosus can be co-administered with vitamin B2. Therefore, the present invention also relates to vitamin B2 for increasing the abundance of Lactobacillus rhamnosus in the intestine of an animal, preferably a human, wherein the use comprises delivering both vitamin B2 and Lactobacillus rhamnosus to the large intestine. This can be done, for example, if Lactobacillus rhamnosus has not yet colonized the intestine, or is present only in lower numbers. Lactobacillus rhamnosus GG, in particular Lactobacillus rhamnosus DSM 32550, is preferred for co-administration.

[0030] For co-administration, including simultaneous administration / delivery / consumption, the vitamins and probiotics can be in the same compartment, or in different compartments. For example, the vitamins and bacteria can be in the same tablet / pill, or in the same capsule. Alternatively, the vitamins and bacteria can be in different tablets / pills, or in different capsules. In the latter case, administration / delivery / consumption within 24 hours is still considered co-administration.

[0031] Lactobacillus rhamnosus is known to improve digestive health and positively regulate the immune system. Numerous studies have shown that when animals, preferably humans, suffer from a particular disease / illness, the population of Lactobacillus rhamnosus in the intestinal microbiota is reduced compared to the population present in animals that do not suffer from the particular disease / illness. However, none of these studies have suggested a method for increasing the population of Lactobacillus rhamnosus, thereby alleviating at least one of the symptoms of the disease / illness. According to the present invention, it has been found that delivering vitamin B2 directly to the large intestine of an animal, preferably a human, can increase the population of Lactobacillus rhamnosus in the large intestine.

[0032] In a preferred embodiment, the animal (including a human) is experiencing a condition selected from the group consisting of gastrointestinal infection and diarrhea, antibiotic and Clostridium difficile associated diarrhea, irritable bowel syndrome, inflammatory bowel disease, respiratory tract infection, allergic reaction, cardiovascular disease, non-alcoholic fatty liver disease, non-alcoholic steatohepatitis, cystic fibrosis and cancer.

[0033] Preferably, the Lactobacillus rhamnosus to be enhanced is Lactobacillus rhamnosus GG (eg Lactobacillus rhamnosus DSM 32550). Therefore, in one embodiment, vitamin B2 is used to increase the abundance of Lactobacillus rhamnosus GG (preferably Lactobacillus rhamnosus DSM 32550) in the large intestine of an animal.

[0034] 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.

[0035] In yet another aspect, the present invention relates to a method for increasing the abundance of Lactobacillus rhamnosus in the intestine, preferably in the large intestine, of an animal, the method comprising administering to the animal an effective dose of vitamin B2. The method is for improving the intestinal health of an animal, wherein the improvement comprises increasing the abundance of Lactobacillus rhamnosus, preferably Lactobacillus rhamnosus GG (particularly Lactobacillus rhamnosus DSM 32550) in the large intestine. Preferably, the animal is a human.

[0036] Preferably, the vitamin B2 is delivered directly to the large intestine. Delivery to the large intestine can be achieved by administering the vitamin B2 as a delayed release formulation.

[0037] In one embodiment, Lactobacillus rhamnosus is co-administered with vitamin B2. Preferably, an effective dose of Lactobacillus rhamnosus GG (eg, Lactobacillus rhamnosus DSM 32550) is co-administered.

[0038] The methods of the invention can be used to treat, prevent and / or alleviate the symptoms of gastrointestinal infections and diarrhea, antibiotic and Clostridium difficile associated diarrhea, irritable bowel syndrome, inflammatory bowel disease, respiratory tract infections, allergic reactions, cardiovascular disease, nonalcoholic fatty liver disease, nonalcoholic steatohepatitis, cystic fibrosis and cancer.

[0039] In a further aspect, the present invention relates to the use of vitamin B2 for increasing the abundance of Lactobacillus rhamnosus in the intestine of an animal, preferably a human being, wherein the use comprises delivering vitamin B2 to the large intestine. Preferably, the use comprises delivering / administering vitamin B2 to the large intestine via a delayed release formulation. In one embodiment, the use comprises administering to the animal a formulation comprising a dose of vitamin B2 of up to 200 mg / day, preferably 5-100 mg / day, more preferably 10-50 mg / day. Vitamin B2 may be co-administered with Lactobacillus rhamnosus. Preferably, the animal, including humans, is experiencing a condition selected from the group consisting of: gastrointestinal infections and diarrhea, antibiotic and Clostridium difficile-associated diarrhea, irritable bowel syndrome, inflammatory bowel disease, respiratory tract infections, allergic reactions, cardiovascular disease, non-alcoholic fatty liver disease, non-alcoholic fatty hepatitis, cystic fibrosis and cancer. Preferably, the Lactobacillus rhamnosus is Lactobacillus rhamnosus GG. Lactobacillus rhamnosus DSM 32550 is particularly preferred.

[0040] The preferred mode of (co)administration is oral administration.

[0041] definition

[0042] As used throughout this document, the following definitions apply:

[0043] The term "vitamin B2" is used interchangeably with "riboflavin" and includes riboflavin and its esters, particularly riboflavin-5'-phosphate, and other pharmaceutically acceptable forms.

[0044] "Increasing the abundance of Lactobacillus rhamnosus" means increasing the level (or amount, number or population size) of Lactobacillus rhamnosus compared to a corresponding control (ie the level / amount / number / population size of Lactobacillus rhamnosus without the addition of vitamin B2).

[0045] The term "intestine" (or "gut") as used herein refers to the portion of the gastrointestinal tract consisting of the small intestine and the large intestine. The "large intestine" (intestinum crassum) is the lower portion of the gastrointestinal tract and is also referred to herein as the "colon."

[0046] "Direct delivery" or "directly delivered" means that the vitamin is formulated in a manner such that the vitamin is not absorbed by the stomach and / or small intestine; rather, the vitamin becomes available in the distal intestinal tract, preferably the large intestine (colon), where it is utilized by the microbiota. The vitamin is not part of the daily nutritional requirements of the human body (usually obtained through diet and conventional vitamin supplementation) and is administered in excess. For human use, the preferred method according to the present invention is through a form that delays release until reaching the large intestine (colon). Alternatively, a sufficiently large dose can be administered so that only a portion of the administered vitamin is absorbed in the proximal small intestine, while the remainder (which is the effective dose) is available to the large intestine; although not preferred, the latter delivery method can also be used in humans.

[0047] As used herein, "delayed release" refers to the release of vitamins and / or probiotics at a certain time after administration, rather than immediately after administration. Preferably, "delayed release" means that after oral administration, vitamins (and / or probiotics) are delivered to the large intestine (colon) in a delayed manner relative to immediate release formulations.

[0048] An "enteric layer" or "enteric coating" is a layer surrounding a core, wherein the core comprises the active agent and which layer confers resistance to gastric fluids.

[0049] "Prevention" can include reducing the risk of an adverse condition occurring, alleviating the symptoms of an adverse condition, lessening the severity of an adverse condition, and prolonging the duration of an adverse condition.

[0050] "Oral formulation" means that the vitamins and / or probiotics are formulated for oral administration / consumption.

[0051] "Co-administered" means that the vitamins and / or probiotics are delivered / administered / consumed simultaneously (ie, 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.

[0052] Lactobacillus rhamnosus has recently been renamed Lactobacillus rhamnosus; both names are used interchangeably herein and may both be abbreviated to L. rhamnosus.

[0053] dose

[0054] 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 range in the colon is 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 g / L to about 0.02 g / L. The specific dosage per day may range from up to 200 mg / day, preferably 5-100 mg / day, more preferably 10-50 mg / day.

[0055] The dosage of the probiotic may be up to 5E+10 cfu / day. Preferably, the dosage of the probiotic ranges from 1E+08 cfu / day to 1E+10 cfu / day, more preferably from 1E+09 cfu / day to 5E+10 cfu / day.

[0056] preparation

[0057] The vitamins (and, where the vitamins are co-administered with probiotics, preferably also the probiotics) are preferably present in a formulation which allows the vitamins (and / or probiotics) to be available primarily in the large intestine.

[0058] Oral formulations are preferred. Other dosage forms include non-oral routes, such as via suppositories or injections.

[0059] For human use, the preferred method is by delayed release forms that delay delivery until the intestinal tract is reached. For non-human animals, the preferred delivery involves administering a dose large enough so that only a portion of the delivered vitamin is absorbed in the stomach and the remainder (which is the effective dose) is available to the intestinal tract; although not preferred, this method of delivery can also be used in humans.

[0060] Delayed release formulations are known in the art. Preferably, the delayed release formulation has an enteric coating (also referred to as an enteric layer).

[0061] In one embodiment of the invention, the vitamins (and, when the vitamins are co-administered with probiotics, preferably also with probiotics) are in a formulation comprising an enteric-coated capsule filled with a composition comprising the vitamins. The enteric-coated capsule confers resistance to the acidic environment of the stomach. For example, a soft gel formulation can deliver an active agent in a solution and also provides the advantages of a solid dosage form.

[0062] In another embodiment, the formulation is a tablet comprising (i) a core comprising the vitamins (and / or probiotics); and (ii) a delayed release coating, such as an enteric coating. This may be a hard gel capsule.

[0063] Alternatively, direct colonic delivery can also be performed using matrix-based delivery systems. Matrix-based systems do not have a discrete layer of coating material, but the active agent is more or less evenly distributed within the matrix. Additionally, there are colonic release systems that embed the active agent in, for example, a fiber matrix (enzyme-triggered) and add an enteric coating on top.

[0064] The release of the drug may be delayed until it reaches the small intestine. In another embodiment, the release of the drug is delayed until it reaches the distal small intestine. In another preferred embodiment, the release of the drug is delayed until it reaches the colon (large intestine).

[0065] In a preferred embodiment for humans, the vitamins are formulated as solid dosage forms for oral administration. The formulations may be in the form of capsules, pills, beads, spheres, microspheres, tablets, microtablets or granules, optionally coated with a delayed release coating that prevents the release of the active agent prior to the small intestine, preferably prior to the colon.

[0066] The coating or matrix material for delayed release of vitamins and / or probiotics, especially targeted release in the ileum or large intestine after oral administration is known in the art. They can be subdivided into coating materials that disintegrate at a specific pH value, coating materials that disintegrate after a specific residence time in the gastrointestinal tract, and coating materials that disintegrate due to enzyme triggers of microflora in a specific area of ​​the intestine. Coating materials from different categories are usually used in combination. For example, different categories of coating materials for targeting to the large intestine have been reviewed in Bansal et al. (Polim. Med. 2014, 44, 2, 109-118). In one embodiment of the present invention, the delayed release coating comprises at least one component selected from the following: a coating material that disintegrates in a pH-dependent manner, a coating material that disintegrates in a time-dependent manner, a coating material that disintegrates due to an enzyme trigger in the intestinal environment (e.g., in the intestinal environment of the ileum and large intestine), and a combination thereof.

[0067] Coating materials that disintegrate in a pH-dependent manner include polyvinyl acetate phthalate, cellulose acetate trimellitate, hydroxypropyl methylcellulose phthalate HP-50, HP-55 or HP-55S, cellulose acetate phthalate, shellac, hydroxypropyl methylcellulose acetate succinate (HPMCAS), poly(methacrylic acid, ethyl acrylate) 1:1 ( L100-55, L30D-55), poly (methacrylic acid, methyl methacrylate) 1:1 ( L-100, L12.5), poly (methacrylic acid, methyl methacrylate) 1:2 ( S-100, S12,5 and FS30D). Time-dependent disintegration coating materials include RL, RS and ethylcellulose. Coating materials that disintegrate in the large intestine environment due to enzyme triggers include chondroitin sulfate, pectin, guar gum, chitosan, inulin, lactulose, raffinose, stachyose, alginate, dextran, xanthan gum, locust bean gum, arabinogalactan, cyclodextrin, pullulan, carrageenan, scleroglucan, chitin, curdlan, fructan, pullulan, starch, amylose, resistant starch, and azo compounds degraded by azo bond cleaving bacteria.

[0068] The following non-limiting examples are provided to better illustrate the present invention.

[0069] Example

[0070] The aim of this study was to investigate the effect of vitamin B2 on the species Lactobacillus rhamnosus in the human gut microbiota.

[0071] Materials and methods

[0072] Design of batch fermentation experiments (colon model)

[0073] ProDigest conducted a short-term batch fermentation experiment consisting of colonic incubation of representative doses of selected vitamins with representative bacterial inocula under conditions simulating the proximal large intestine. In the current experiment, 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 beginning of the short-term colonic incubation, fresh fecal material from six healthy human donors was collected and, after preparing an anaerobic fecal slurry, this slurry was inoculated at 10% by volume into SHIME nutrient medium containing basal 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. In addition, the following methods were used to inoculate the SHIME medium: Techniques incorporated into the current experiments: Mucin-covered microcosms (simulating colonic mucus) were added to the incubation as previously described (Van den Abbeele, P. et al. (2013). Butyrate-producing Clostridium cluster XIV species specifically colonize mucins in an in vitro gut model. The ISME Journal 7(5), 949-961). Incubation was performed at 37°C, shaking (90 rpm) and anaerobic conditions for 48 h.

[0074] In this study, Lactobacillus rhamnosus GG equivalent Lactobacillus rhamnosus DSM 32550 (Biocare Copenhagen) was added to the colon model containing donor samples alone or in combination with vitamin B2 (see Table 1). After 48 h of incubation, the abundance of the added strains was analyzed using primers specific for Lactobacillus rhamnosus GG and its equivalent strain Lactobacillus rhamnosus DSM 32550. The experiment was performed in single replicate.

[0075] The genome sequence of Lactobacillus rhamnosus DSM 32550 and The genome sequence of Lactobacillus rhamnosus DSM 32550 is 99.99% identical to that of In the examples herein, Lactobacillus rhamnosus DSM 32550 will therefore be referred to as Lactobacillus rhamnosus strain GG.

[0076] Table 1: Experimental layout

[0077]

[0078] Lactobacillus rhamnosus GG equivalent strain Lactobacillus rhamnosus DSM 32550 at 1*10 9 The concentration of CFU was added to SHIME medium as an overnight growth culture. The probiotic strains can be supplemented alone or in combination with vitamin B2. Vitamin B2 (riboflavin TG, DSM) was added at a concentration of 0.01667 g / L, which translates to a dose of 10 mg given a colon volume of approximately 600 ml.

[0079] Analysis of Lactobacillus rhamnosus GG levels

[0080] qPCR was performed for a putative phage tail protein gene (NCBI reference NC_013198) to monitor the levels of the supplemented probiotic Lactobacillus rhamnosus GG strain within the gut microbiota as previously described (Brandt, Alatossava (2003) - Specific identification of certain probiotic Lactobacillus rhamnosus strains with PCR primers based on phage-related sequences). Samples from the mucus environment after 48 h of incubation were analyzed.

[0081] statistics

[0082] Statistical analysis was performed to investigate the average effect of the test product. For this purpose, the mean value of six donors for each endpoint was calculated. Paired t-tests were performed to assess the potential effect of the test product compared to the control, and different test products were compared to each other. For microbial community composition, statistical tests were performed on logarithmically transformed data (so that the logarithmically transformed data were normally distributed). If the p value was less than 0.05, the difference was considered to be statistically significant.

[0083] result

[0084] Vitamin B2 supplementation increases levels of Lactobacillus rhamnosus GG

[0085] like Figure 1 As shown, when vitamin B2 was co-supplemented with Lactobacillus rhamnosus GG, approximately three times more copies of the Lactobacillus rhamnosus GG-specific putative phage tail protein gene were detected in the mucus environment by qPCR analysis. Thus, the addition of vitamin B2 significantly increased the levels of Lactobacillus rhamnosus GG in the colon model as compared to the corresponding control.

Claims

1. A vitamin B2 for use in increasing the abundance of Lactobacillus rhamnosus in the intestine of an animal, preferably a human, wherein the use comprises delivering the vitamin B2 to the large intestine.

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

3. Vitamin B2 for use according to claim 1 or 2, wherein the use comprises administering to the animal a preparation of vitamin B2 at a dosage 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 claims 1 to 3, wherein the vitamin B2 is co-administered with Lactobacillus rhamnosus.

5. Vitamin B2 for use according to any one of claims 1 to 4, wherein the animal, including humans, is experiencing at least one condition selected from the group consisting of gastrointestinal infections and diarrhea, antibiotic and Clostridium difficile associated diarrhea, irritable bowel syndrome, inflammatory bowel disease, respiratory infections, allergic reactions, cardiovascular disease, non-alcoholic fatty liver disease, non-alcoholic steatohepatitis, cystic fibrosis and cancer.

6. Vitamin B2 for use according to any one of claims 1 to 5, wherein the Lactobacillus rhamnosus is Lactobacillus rhamnosus GG.

7. An oral preparation comprising vitamin B2 and Lactobacillus rhamnosus. The oral preparation according to claim 7 , which is a delayed-release preparation.

9. A method of increasing the abundance of Lactobacillus rhamnosus in the intestinal tract of an animal, preferably a human, the method comprising administering to the animal a formulation comprising an effective amount of vitamin B2.

10. The method of claim 9, wherein the animal is a human and the vitamin B2 is delivered to the large intestine.

11. The method of claim 9 or 10, wherein the vitamin B2 is delivered via a delayed release formulation.

12. The method according to any one of claims 9-11, wherein the vitamin B2 is co-administered with Lactobacillus rhamnosus.

13. The method of any one of claims 9-12, wherein the method is a method of treating, preventing or alleviating the symptoms of gastrointestinal infections and diarrhea, antibiotic and Clostridium difficile associated diarrhea, irritable bowel syndrome, inflammatory bowel disease, respiratory infections, allergic reactions, cardiovascular disease, non-alcoholic fatty liver disease, non-alcoholic steatohepatitis, cystic fibrosis or cancer in animals in need thereof, including humans.

14. The method according to any one of claims 9 to 13, wherein the Lactobacillus rhamnosus is Lactobacillus rhamnosus GG.

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