Combination containing vitamin B2 and Lactobacillus rhamnosus

Abstract

The present invention relates to a combination comprising vitamin B2 and Lactobacillus rhamnosus, and its use for improving intestinal health in animals and humans. The combination of vitamin B2 and Lactobacillus rhamnosus has been found to increase the abundance of beneficial Bifidobacteriaceae and Bifidobacterium bacteria in the intestinal tract when delivered to the large intestine.

Description

Detailed Description of the Invention

[0001] [Field of the Invention] The present invention relates to a combination comprising vitamin B2 and Lactobacillus rhamnosus, and its use for improving intestinal health in animals and humans. The combination of vitamin B2 and Lactobacillus rhamnosus has been found to increase the abundance of certain beneficial bacteria in the intestinal tract when delivered to the large intestine.

[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. Thus, targeted modulation of the human gut microbiome aimed at 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] The family Bifidobacteriaceae is a family of bacteria known to exert many beneficial effects on human health. Bifidobacterium is a genus belonging to the family Bifidobacteriaceae. As inhabitants of the human intestine, bifidobacteria create a bacteria-host symbiotic-like relationship and provide the human host with many health benefits, including: they re-establish normal gut microbiota, improve intestinal and immune function, prevent nutrient loss, prevent constipation, prevent the onset of colon cancer, treat liver damage, and lower cholesterol levels in the blood. Bifidobacteria have also been found to have antibiotic activity and assist in antitumor activity in the host (Bottacini et al, Diversity, ecology and intestinal function of bifidobacteria (2014)). For example, in atopic diseases, irritable bowel syndrome, inflammatory bowel disease, colorectal cancer, and celiac disease, abnormalities in certain bifidobacterial flora, such as decreased numbers or atypical species composition, have been identified. Furthermore, it has been reported that intestinal endotoxemia of bifidobacteria precedes obesity (Tojo R., Intestinal microbiota in health and disease: Role of bifidobacteria in gut homeostasis (2014)).

[0004] Bifidobacterium adolescentis has been shown to produce folic acid in the colon and may be used to prevent folic acid deficiency in colonic epithelial cells and more efficiently protect the colon from inflammation and cancer (Pompei A., Folate Production by Bifidobacteria as a Potential Probiotic Property (2020)). Experiments and tests have shown that Bifidobacterium adolescentis therapy can be an important intervening factor in the process of inflammatory bowel disease (Shah Shinil., Dietary Factors in the Modulation of Inflammatory Bowel Disease Activity (2007)). Furthermore, Bifidobacterium adolescentis inhibits tumor growth and is an appropriate and specific means of gene cancer therapy (Xi Li, et al., Bifidobacterium adolescentis as a delivery system of endostatin for cancer gene therapy: Selective inhibitor of angiogenesis and hypoxic tumor growth (2003)).

[0005] 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, WO 2020 / 043797 pamphlet does not describe that vitamins can be used in combination with probiotics to increase the abundance of other beneficial bacteria. Furthermore, 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 Bifidobacteriaceae and / or Bifidobacterium bacteria in the intestine in order to improve health status, improve health, and support the immune system.

[0006] [Summary of the Invention] The present invention relates to the following items: 1) A combination comprising vitamin B2 and Lactobacillus (Lacticaseibacillus) rhamnosus. 2) The combination according to item 1, wherein the combination comprises vitamin B2 and Lactobacillus rhamnosus GG, preferably Lactobacillus rhamnosus DSM 32550. 3) The combination according to item 1 or item 2, wherein the combination is for simultaneous administration or delivery or consumption, preferably the combination is a fixed combination. 4) The combination according to item 1 or item 2, wherein the combination is for sequential administration or delivery or consumption, preferably the combination is a free combination. 5) The combination according to any one of items 1 to 4, wherein the combination is in an oral dosage form, more preferably the combination is in a solid oral dosage form. 6) The combination according to any one of items 1 to 5, wherein the combination is for administration or delivery to the large intestine. 7) A combination according to any one of items 1 to 6 for use as a drug, a health supplement or a nutritional supplement. 8) A combination according to any one of items 1 to 7 for use in the treatment of patients in need of an increase in the abundance of Bifidobacteriaceae and / or Bifidobacterium in the large intestine. 9) A combination for use according to item 8, wherein the patient is in need of an increase in the abundance of Bifidobacterium and suffers from at least one of the following conditions: constipation, colon cancer, liver damage, high cholesterol levels, atopic diseases, irritable bowel syndrome, inflammatory bowel disease, colorectal cancer, celiac disease, and obesity. 10) A combination according to any one of items 1 to 7 for use in the treatment of patients in need of an increase in the abundance of Bifidobacterium adolescentis in the large intestine. 11) A combination for use according to item 10, wherein the patient suffers from one or more of the following conditions: folate deficiency, inflammatory bowel disease, and colorectal cancer. 12) A combination comprising vitamin B2 and Lactobacillus (Lacticaseibacillus) rhamnosus for use in increasing the abundance of Bifidobacteriaceae and / or Bifidobacterium adolescentis in the large intestine (colon) of an animal, preferably a human, wherein the use comprises delivering vitamin B2 and Lactobacillus rhamnosus to the large intestine. 13) A combination comprising vitamin B2 and Lactobacillus rhamnosus for use according to item 12, wherein the vitamin B2 and Lactobacillus rhamnosus are delivered to the large intestine by a delayed-release formulation. 14) A combination comprising vitamin B2 and Lactobacillus rhamnosus for use according to item 12 or item 13, wherein the use comprises administering the vitamin B2 and Lactobacillus rhamnosus to an animal, preferably a human, simultaneously and / or sequentially. 15) A combination comprising vitamin B2 and Lactobacillus rhamnosus for use according to any one of items 12 to 14, wherein the animal including a human is experiencing at least one condition selected from constipation, colon cancer, liver damage, high cholesterol levels, folate deficiency, atopic diseases, irritable bowel syndrome, inflammatory bowel disease, colorectal cancer, celiac disease, and obesity. 16) A combination comprising vitamin B2 and Lactobacillus rhamnosus for use according to any one of items 12 to 15, wherein the Lactobacillus rhamnosus is Lactobacillus rhamnosus GG, preferably Lactobacillus rhamnosus DSM 32550.

Brief Description of the Drawings

[0007]

Figure 1

Figure 2

[0008] [Detailed Description of the Invention] Bifidobacteriaceae and Bifidobacterium are bacteria known for their beneficial effects on human health. The inventors have found that the combination of vitamin B2 and Lactobacillus rhamnosus can promote the growth of Bifidobacteriaceae and Bifidobacterium adolescentis bacteria in the large intestine, resulting in an increase in the levels of Bifidobacteriaceae and Bifidobacterium adolescentis in the intestine.

[0009] Accordingly, in a first aspect, the present invention relates to a combination of vitamin B2 and Lactobacillus (Lacticaseibacillus) rhamnosus. Preferably, the Lactobacillus (Lacticaseibacillus) rhamnosus is Lactobacillus rhamnosus GG strain, more preferably Lactobacillus rhamnosus DSM 32550. The combination is for simultaneous and / or sequential administration.

[0010] The claims regarding the "combination" are product claims. The product of the present invention contains two active ingredients: a vitamin (vitamin B2) and a probiotic (Lactobacillus rhamnosus). For simultaneous and / or sequential administration, see the following definitions and embodiments.

[0011] 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, for example, TER Chemicals Distribution Group, BIOCHEM Bernburg GmbH, DVA International GmbH, Falken Trade GmbH, and Neupert Ingredients GmbH.

[0012] 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".

[0013] 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).

[0014] 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, 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 32550.

[0015] In one embodiment, the combination of the present invention is for simultaneous administration. Preferably, the combination for simultaneous administration is a fixed combination. However, for simultaneous administration, a free combination can also be used.

[0016] In another embodiment, the combination is for sequential administration. The combination for sequential administration is a free combination.

[0017] Preferably, the combination is in an oral dosage form, more preferably in a solid oral dosage form.

[0018] The combination of the present invention is, for example, a pharmaceutical combination or composition, a health supplement food, or a nutritional supplement food.

[0019] In another aspect, the present invention relates to the use as a drug of vitamin B2 and Lactobacillus rhamnosus (i.e., the combination of vitamin B2 and Lactobacillus rhamnosus).

[0020] Preferably, the combination of the present invention (e.g., a pharmaceutical combination) is for use in the treatment of patients in need of an increase in the abundance of Bifidobacteriaceae and / or Bifidobacterium in the large intestine. In one embodiment, the patient is in need of an increase in the abundance of Bifidobacterium and suffers from constipation, colon cancer, liver damage, high cholesterol levels, atopic diseases, irritable bowel syndrome, inflammatory bowel disease, colorectal cancer, celiac disease, or obesity.

[0021] In another preferred embodiment, the (pharmaceutical) combination of the present invention is for use in the treatment of patients in need of an increase in the abundance of Bifidobacterium adolescentis in the large intestine. Preferably, the patient suffers from folate deficiency, inflammatory bowel disease, or colorectal cancer.

[0022] In a further aspect, the present invention relates to vitamin B2 and Lactobacillus rhamnosus (i.e., a combination of vitamin B2 and Lactobacillus rhamnosus) for use in improving intestinal health in an animal. Said improvement comprises or consists of increasing the abundance of Bifidobacteriaceae and / or Bifidobacterium (preferably Bifidobacterium adolescentis) in the intestine of said animal. Specifically, vitamin B2 and Lactobacillus rhamnosus are for use in increasing the abundance of Bifidobacteriaceae and / or Bifidobacterium in the large intestine (colon) of an animal, said use preferably including delivering vitamin B2 and Lactobacillus rhamnosus to the large intestine. Preferably, the animal is a human.

[0023] To achieve an increase in the abundance of Bifidobacteriaceae and / or Bifidobacterium in the large intestine, vitamin B2 and Lactobacillus rhamnosus are preferably delivered directly to the large intestine. That is, the vitamin is delivered / administered in such a manner that the vitamin is not absorbed in the stomach and / or small intestine; the vitamin and probiotics are delivered / administered to the distal gastrointestinal tract, preferably the large intestine (colon). This is preferably done by delivering / administering vitamin B2 and Lactobacillus rhamnosus in a delayed release formulation. Oral administration is preferred.

[0024] In a preferred embodiment, the animal (including humans) is experiencing one or more conditions selected from the group consisting of constipation, colon cancer, liver damage, high cholesterol levels, folate deficiency, atopic diseases, irritable bowel syndrome, inflammatory bowel disease, colorectal cancer, celiac disease, and obesity.

[0025] Preferably, the Lactobacillus rhamnosus used is Lactobacillus rhamnosus GG. Lactobacillus rhamnosus DSM 32550 is particularly preferred.

[0026] In another aspect, the present invention relates to a method of increasing the abundance of Bifidobacteriaceae and / or Bifidobacterium (preferably Bifidobacterium adolescentis) in the intestine, preferably the large intestine, the method comprising administering to the animal an effective dose of vitamin B2 and Lactobacillus rhamnosus (preferably Lactobacillus rhamnosus GG, particularly Lactobacillus rhamnosus DSM 32550). The method is for improving intestinal health in animals including humans, said improvement comprising increasing the abundance of Bifidobacteriaceae and / or Bifidobacterium in the large intestine. Preferably, the animal is a human. Preferably, vitamin B2 and Lactobacillus rhamnosus are delivered directly to the large intestine. Delivery to the large intestine can be achieved by administering vitamin B2 and Lactobacillus rhamnosus as a sustained release formulation.

[0027] The method of the present invention can be used to treat, prevent, and / or reduce one or more of the following symptoms in animals, including humans in need thereof: constipation, colon cancer, liver damage, high cholesterol levels, folate deficiency, atopic diseases, irritable bowel syndrome, inflammatory bowel disease, colorectal cancer, celiac disease, and obesity.

[0028] In a further aspect, the present invention relates to the use of vitamin B2 and Lactobacillus rhamnosus to increase the abundance of Bifidobacteriaceae and / or Bifidobacterium (preferably Bifidobacterium adolescentis) in the intestine of an animal, preferably a human, said use comprising delivering vitamin B2 and Lactobacillus rhamnosus to the large intestine. Preferably, the use comprises delivering / administering vitamin B2 and Lactobacillus rhamnosus to the large intestine by means of a delayed release formulation. Preferably, the animal, including a human, is experiencing one or more conditions selected from the group consisting of constipation, colon cancer, liver damage, high cholesterol levels, folate deficiency, atopic diseases, irritable bowel syndrome, inflammatory bowel disease, colorectal cancer, celiac disease, and obesity.

[0029] In the combinations, uses, and methods of the present invention, preferably, the vitamin B2 dosage is up to 200 mg / day, preferably 5 - 100 mg / day, more preferably 10 - 50 mg / day. In one embodiment, vitamin B2 is administered / dosed 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.

[0030] The dosage of Lactobacillus rhamnosus can be up to 5E+10 cfu / day. Preferably, the dosage range is 1E+08 to 1E+10 cfu / day, more preferably 1E+09 to 5E+10 cfu / day. Preferably, Lactobacillus rhamnosus is Lactobacillus rhamnosus GG. Lactobacillus rhamnosus DSM 32550 is particularly preferred.

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

[0032] The claims regarding "combination" or "pharmaceutical combination" are product claims. The product of the present invention contains two active ingredients: vitamin (vitamin B2) and probiotics (Lactobacillus rhamnosus).

[0033] "Combination for simultaneous administration" or "combination for simultaneous consumption" are combinations suitable for simultaneous administration or consumption, respectively. "Simultaneous administration" or "simultaneous consumption" means that the vitamin and the probiotic bacteria are administered / consumed on the same day (i.e., within 24 hours). The two active ingredients can be administered / consumed simultaneously (in the case of a fixed combination) or one by one at a time (in the case of a free combination). For example, the vitamin can be administered / consumed in one pill or tablet, while the probiotic can be administered / consumed in another pill or tablet, and both pills or tablets are administered / consumed within 24 hours. In another example, the vitamin and the probiotic are formulated in the same composition and administered / consumed exactly simultaneously.

[0034] "Combination for continuous administration or consumption" refers to combinations that are suitable for continuous administration or consumption respectively. "Continuous administration" or "continuous consumption" means that during a period of two or more days of continuous treatment, only one of vitamins and probiotics is administered / consumed on any given day. As an example, vitamins can be administered / consumed on the first day, and probiotics can be administered / consumed the next day (i.e., after more than 24 hours), or even later. The therapeutic components can be administered / consumed in any order.

[0035] "Fixed combination" is a combination that delivers both active substances (i.e., vitamins and probiotics) to the patient simultaneously. Solid oral dosage forms (e.g., tablets or capsules) containing both vitamins and probiotics are an example of a fixed combination. Liquid oral dosage forms (e.g., oral drops) containing both vitamins and probiotics are another example of a fixed combination.

[0036] "Free combination" is a combination in which both active substances (i.e., vitamins and probiotics) can be administered / consumed separately, i.e., one at a time. Treatment regimens in which vitamins and probiotics are not administered / consumed via the same route and / or are not administered / consumed simultaneously require a free combination.

[0037] Simultaneous administration / consumption can be achieved by using both fixed combinations and free combinations. Continuous administration / consumption requires a free combination, and fixed combinations are not suitable for continuous administration / consumption. Therefore, free combinations are more versatile as they are suitable for both continuous administration / consumption and - when both active substances are administered / consumed on the same day - simultaneous administration / consumption. Fixed combinations are only suitable for simultaneous administration / consumption when both components (i.e., vitamins and probiotics) are to be administered / consumed simultaneously on the same day, but are not suitable when vitamins and probiotics are to be administered / consumed separately on the same day.

[0038] "Separate administration / consumption" means that vitamins and probiotics are administered one at a time. Thus, separate administration / consumption refers to both consecutive administrations / consumptions, - when both active substances are administered / consumed on the same day, but one at a time - and can also refer to simultaneous administration / consumption.

[0039] "Administering" or "administration" means imparting or delivering an active substance to a human or animal. Similarly, a human or animal can ingest (consume) the active substance.

[0040] 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.

[0041] "Increasing the abundance of" Bifidobacteriaceae, Bifidobacterium, or Bifidobacterium adolescentis means increasing the level (or amount or number or population size) of Bifidobacteriaceae, Bifidobacterium, or Bifidobacterium adolescentis compared to their respective controls (i.e., the levels / amounts / numbers / population sizes of Bifidobacteriaceae, Bifidobacterium, or Bifidobacterium adolescentis in the absence of the combination of vitamin B2 and Lactobacillus rhamnosus).

[0042] 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".

[0043] "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 (which are generally obtained through diet and conventional vitamin supplementation). For human use, the 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 portion of the administered vitamin 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. In the context of probiotics, "direct delivery" or "delivered directly" means that the probiotic is not released in the stomach and / or small intestine; the probiotic is formulated to be available in the distal gastrointestinal tract, preferably the large intestine (colon).

[0044] As used herein, "delayed release" refers to the release of a vitamin and / or probiotic being slower than immediately after administration. Preferably, "delayed release" means that the delivery of the vitamin (and / or probiotic) to the large intestine (colon) after oral administration is delayed compared to an immediate release formulation.

[0045] An "enteric layer" or "enteric coating" is a layer surrounding a core that contains an active agent and imparts resistance to gastric juice.

[0046] "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 prolonging the time of occurrence of an adverse condition.

[0047] "Oral preparation" means that vitamins and / or probiotics are formulated for oral administration / consumption.

[0048] "Co-administering" 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 can be delivered / administered / consumed first. Similarly, the probiotics can be delivered / administered / consumed first.

[0049] "Lactobacillus rhamnosus" has recently been renamed "Lacticaseibacillus rhamnosus", and both names are used interchangeably herein and can both be abbreviated as "L. rhamnosus".

[0050] [Dosage] Preferably, 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.

[0051] 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.

[0052] [Formulation] Vitamins (vitamin B2) and / or probiotics (Lactobacillus rhamnosus), preferably both, are preferably present in a formulation that preferentially makes the vitamins (and / or probiotics) available in the large intestine.

[0053] Oral formulations are preferred. Other formulations include parenteral routes such as suppositories or injections.

[0054] 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 and / or probiotic 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.

[0055] Delayed-release formulations are known in the art. Preferably, the delayed-release formulation has an enteric coating (also referred to as an enteric layer).

[0056] In one embodiment of the invention, vitamins and / or probiotics, preferably both, are present in a formulation comprising enteric-coated capsules filled with a composition containing vitamins and / or probiotics. 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.

[0057] 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.

[0058] Alternatively, for direct colon delivery, a matrix-based delivery system can be used. The matrix-based system does not have individual layers of coating material, but the active agent (i.e., vitamins and / or probiotics) is more or less homogeneously distributed within 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.

[0059] The release of vitamins and / or probiotics can be delayed until the small intestine. In another embodiment, the release is delayed until the distal small intestine. In yet another preferred embodiment, the release of vitamins and / or probiotics is delayed until the colon (large intestine).

[0060] In a preferred embodiment for humans, the vitamins and / or probiotics are formulated into a solid dosage form for oral administration. The formulation can be in the form of capsules, pellets, beads, spheres, minispheres, tablets, mini-tablets, 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.

[0061] 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 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., within the intestinal environment of the ileum and large intestine), and combinations thereof.

[0062] 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-degrading bacteria.

[0063] The following non-limiting examples are presented to illustrate the present invention in more detail.

[0064] [Examples] The purpose of this study was to examine the effect of the combination of vitamin B2 and Lactobacillus rhamnosus on the composition of the gut microbiota in a long-term continuous fermentation experiment.

[0065] [Materials and Methods] [Design of Long-Term SHIME Fermentation Experiment (Colon Model)] The typical reactor configuration of SHIME® representing the gastrointestinal tract of adult humans 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, first, after simulating gastric conditions, the conditions were changed by computer to simulate the small intestine. Then, the suspension was added to a colon reactor mimicking the transverse colon (pH 6.15 - 6.4; retention time = 32 h; volume 800 mL).

[0066] 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 appropriate probiotic strains and vitamins were supplemented to the appropriate reactors. The probiotic strains were added to the reactors at a concentration of 1*10 10 cfu / reactor. Vitamin B2 (riboflavin, DSM) was added to the reactors at a dose of 10 mg / day.

[0067] The probiotic strain used in this experiment was Lactobacillus rhamnosus DSM 32550 (Biocare Copenhagen), an equivalent of Lactobacillus rhamnosus GG.

[0068] 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 and drawings of this specification, Lactobacillus rhamnosus DSM 32550 is referred to as the Lactobacillus rhamnosus GG strain.

[0069] [Quantitative microbiota analysis by 16S rRNA gene sequencing and flow cytometry] Samples for quantitative 16S-targeted Illumina sequencing were collected three times per week between the control and the 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 on a 2% (w / v) agarose gel at 100 V for 30 min. 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.

[0070] [Result] [Supplementation with a combination of Lactobacillus rhamnosus GG and vitamin B2 increased the abundance of Bifidobacteriaceae.] As can be understood from FIG. 1, supplementation with Lactobacillus rhamnosus GG alone did not significantly change the abundance of Bifidobacteriaceae compared to the control. In contrast, the combination of Lactobacillus rhamnosus GG and vitamin B2 significantly increased the abundance of Bifidobacteriaceae compared to the control.

[0071] [Supplementation with a combination of Lactobacillus rhamnosus GG and vitamin B2 increased the abundance of Bifidobacterium adolescentis.] As can be understood from FIG. 2, supplementation with Lactobacillus rhamnosus GG alone did not significantly change the abundance of Bifidobacterium adolescentis compared to the control. In contrast, the combination of Lactobacillus rhamnosus GG and vitamin B2 significantly increased the abundance of Bifidobacterium adolescentis compared to the control.

Claims

1. A combination containing vitamin B2 and Lactobacillus rhamnosus.

2. The combination according to claim 1, wherein the combination comprises vitamin B2 and Lactobacillus rhamnosus GG.

3. The combination according to claim 1 or claim 2, wherein the combination is for simultaneous administration or consumption.

4. The combination according to claim 1 or claim 2, wherein the combination is for continuous administration or consumption.

5. The combination according to claim 1 or 2, wherein the combination is in the form of an oral dosage.

6. The combination according to claim 1 or 2, wherein the combination is for administration to the large intestine.

7. The combination according to claim 1 or 2, for use as a drug, health supplement, or nutritional supplement.

8. The combination according to claim 1 or 2 for use in the treatment of patients who require an increase in the abundance of Bifidobacteriae and / or Bifidobacterium in the colon.

9. The combination for use according to claim 8, wherein the patient requires an increased abundance of Bifidobacterium and suffers from at least one of the following: constipation, colon cancer, liver injury, high cholesterol levels, atopic disease, irritable bowel syndrome, inflammatory bowel disease, colorectal cancer, celiac disease, and obesity.

10. The combination according to claim 1 or 2 for use in the treatment of patients who require an increase in the abundance of Bifidobacterium adolescentis in the colon.

11. The combination for use according to claim 10, wherein the patient suffers from at least one of the following: folate deficiency, inflammatory bowel disease, and colorectal cancer.

12. A combination comprising vitamin B2 and Lactobacillus rhamnosus for use in increasing the abundance of Bifidobacteriae and / or Bifidobacterium adolescentis in the large intestine of an animal, wherein the use comprises delivering the vitamin B2 and Lactobacillus rhamnosus to the large intestine.

13. A combination of vitamin B2 and Lactobacillus rhamnosus for use according to claim 12, wherein the vitamin B2 and Lactobacillus rhamnosus are delivered to the large intestine by a delayed-release formulation.

14. A combination comprising vitamin B2 and Lactobacillus rhamnosus for use according to claim 12 or 13, wherein the use comprises administering the vitamin B2 and Lactobacillus rhamnosus to the animal simultaneously and / or sequentially.

15. The combination of vitamin B2 and Lactobacillus rhamnosus for use according to claim 12 or 13, wherein the animal, including a human, is experiencing one or more conditions selected from: constipation, colon cancer, liver injury, high cholesterol levels, folic acid deficiency, atopic disease, irritable bowel syndrome, inflammatory bowel disease, colorectal cancer, celiac disease, and obesity.

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