A combination comprising 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 Verrucomicrobia, Akkermansiaceae, and Akkermansia 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 "gut dysbiosis") may be associated with Western diseases including obesity and type 2 diabetes, as well as cardiovascular diseases, autoimmune diseases, and intestinal inflammatory diseases. Therefore, targeted modulation of the human gut microbiome with the intention of restoring the imbalance is a potential therapeutic and preventive strategy that has attracted the attention of scholars and those engaged in various industries. The public's awareness and acceptance of substances that modulate the human gut microbiome continue to grow. 【0003】 There is a consensus that certain live microorganisms have a beneficial effect on human health. 【0004】 Verrucomicrobia are mucin-degrading bacteria present in the intestinal mucosa that contribute to intestinal health and glucose homeostasis and function as an interface between the human gut microbiome and host tissues. A higher proportion of Verrucomicrobia is associated with better sleep quality and cognitive ability in adults (Anderson, J.R. et al. A preliminary examination of gut microbiota, sleep, and cognitive flexibility in healthy older adults (2017). 【0005】 The family Akkermansiaceae is a dominant family in the phylum Verrucomicrobia. The abundance of the family Akkermansiaceae has been found to increase significantly in mice with prostate cancer, suggesting its role in prostate cancer (Pin-Yu H. et al., Int. J Molecular Sciences. 2021:9626). 【0006】 Akkermansia belongs to the bacterial family Akkermansiaceae and produces butyrate and propionate. Akkermansia has been shown to have an anti-obesity protective effect in previous studies and can be used as a promising probiotic (Zhou Q et al, Gut bacteria Akkermansia is associated with reduced risk of obesity: evidence from the American Gut Project 2020). 【0007】 Among the symbiotic bacteria present in the intestine, Akkermansia muciniphila has been attracting increasing interest for its health-promoting effects. Akkermansia muciniphila is very abundant in lean and non-diabetic individuals and is associated with a lower prevalence of obesity and related metabolic disorders (Dao MC. Akkermansia muciniphila and improved metabolic health during a dietary intervention in obesity: relationship with gut microbiome richness and ecology (2016)). In contrast, several studies have reported reduced levels of Akkermansia muciniphila in various disorders and diseases in humans, including intestinal diseases such as inflammatory bowel disease, and extra-intestinal diseases such as autism, atopy or obesity and related diseases. Akkermansia muciniphila has also been found to be lower in several conditions such as diabetes, enteritis, liver disease, or chronic alcohol consumption. This is associated with altered gut barrier function, which ultimately induces mild inflammation and metabolic disorders. The presence of Akkermansia muciniphila is associated with a healthier state in humans. Indeed, many studies have found that Akkermansia muciniphila is positively associated with a healthy intestinal wall, a reduction in metabolic disorders, and a reduction in mild inflammation. 【0008】 In the context of obesity-related disorders, Akkermansia muciniphila has been systematically found to be inversely correlated with cardiometabolic risk factors. These factors include insulin resistance, serum lipids, BMI and hyperlipidemia. In contrast, it is positively correlated with protective markers such as high-density lipoprotein (HDL, or "good" cholesterol). 【0009】 Akkermansia muciniphila functions as a guardian of the intestinal barrier function through its unique mucolytic ability and its position within the mucus layer. It stimulates mucin production via mucin degradation and regenerates the mucus layer. In a healthy human gut, these bacteria maintain mucin production and thickness, thereby preventing the invasion of pathogenic bacteria (Zhai Q. et al., A next generation probiotic, Akkermansia muciniphila (2019) and Derrien M. et al. Akkermansia muciniphila and its role in regulating host functions (2017)). 【0010】 Recently, it has been demonstrated that vitamins can modulate the human gut microbiome. WO 2020 / 043797 discloses that vitamins may be useful for increasing the growth of certain beneficial bacteria in the intestine. However, WO 2020 / 043797 does not describe using vitamins in combination with probiotics to increase the abundance of other beneficial bacteria. Furthermore, the human gut is a habitat for hundreds of different microorganisms, and it would be desirable to be able to enhance specific beneficial bacteria. Specifically, it would be desirable to increase the abundance of Verrucomicrobia, Akkermansiaceae, and Akkermansia bacteria in the intestine to improve health status, enhance health, and support the immune system. 【0011】 [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, 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, 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) The combination according to any one of items 1 to 6, for use as a drug, a health supplement or a nutritional supplement. 8) The combination according to any one of items 1 to 7, for use in the treatment of a patient in need of an increase in the abundance of Verrucomicrobia, Akkermansiaceae, and / or Akkermansia in the large intestine. 9) The combination for use according to item 8, wherein the patient is in need of an increase in the abundance of Akkermansiaceae and has prostate cancer. 10) The combination according to any one of items 1 to 7, for use in the treatment of a patient in need of an increase in the abundance of Akkermansia muciniphila in the large intestine. 11) The combination for use according to item 10, wherein the patient suffers from one or more of the following conditions: obesity, diabetes, enteritis, liver disease, autism, atopy, metabolic disorders, and chronic alcohol consumption. 12) A combination for use in increasing the abundance of Verrucomicrobia, Akkermansiaceae, and / or Akkermansia in the large intestine of an animal, preferably a human, comprising vitamin B2 and Lactobacillus (Lacticaseibacillus) rhamnosus, wherein said 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 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 said use comprises administering 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 state selected from the following: prostate cancer, obesity, diabetes, enteritis, liver disease, autism, atopy, metabolic disorder, and chronic alcohol consumption. 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】 【0012】 【Figure 1】 Relative abundance (%) of bacteria of the Verrucomicrobia phylum in the mucus of the transverse colon after treatment with Lactobacillus rhamnosus GG in the presence or absence of vitamin B2 at the end of the control period (n = 3) and at the end of the treatment period (n = 3). Statistically significant differences are indicated by '*' (p < 0.05). 【Figure 2】 Relative abundance (%) of bacteria of the Akkermansiaceae family in the mucus of the transverse colon after treatment with Lactobacillus rhamnosus GG in the presence or absence of vitamin B2 at the end of the control period (n = 3) and at the end of the treatment period (n = 3). Statistically significant differences are indicated by '*' (p < 0.05). 【Figure 3】 Relative abundance (%) of the Akkermansia muciniphila species in the mucus of the transverse colon after treatment with Bifidobacterium animalis ssp. lactis in the presence or absence of vitamin B2 at the end of the control period (n = 3) and at the end of the treatment period (n = 3). Statistically significant differences are indicated by '*' (p < 0.05). 【0013】 [Detailed Description of the Invention] Verrucomicrobia, Akkermansiaceae, and Akkermansia 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 Verrucomicrobia, Akkermansiaceae, and Akkermansia muciniphila bacteria in the large intestine, resulting in an increase in the levels of Verrucomicrobia, Akkermansiaceae, and Akkermansia muciniphila in the intestine. 【0014】 Accordingly, in a first aspect, the present invention relates to a combination of vitamin C, vitamin B2, and Lactobacillus (Lacticaseibacillus) rhamnosus. Preferably, Lactobacillus (Lacticaseibacillus) rhamnosus is Lactobacillus rhamnosus GG strain, more preferably Lactobacillus rhamnosus DSM 32550. The combination is for simultaneous and / or sequential administration. 【0015】 The claims regarding the "combination" are product claims. The product of the present invention contains two active ingredients: vitamin (vitamin B2) and probiotics (Lactobacillus rhamnosus). For simultaneous and / or sequential administration, see the following definitions and embodiments. 【0016】 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 BernburGGmbH, DVA International GmbH, Falken Trade GmbH, and Neupert Ingredients GmbH. 【0017】 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®. Therefore, L. rhamnosus DSM 32550 is referred to herein as "Lactobacillus rhamnosus GG". 【0018】 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). 【0019】 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. 【0020】 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. 【0021】 In another embodiment, the combination is for sequential administration. The combination for sequential administration is a free combination. 【0022】 Preferably, the combination is in an oral dosage form, more preferably in a solid oral dosage form. 【0023】 The combination of the present invention is, for example, a pharmaceutical combination or composition, a health supplement food, or a nutritional supplement food. 【0024】 In another aspect, the present invention relates to vitamin B2 and Lactobacillus rhamnosus (i.e., a combination of vitamin B2 and Lactobacillus rhamnosus) for use as a drug. 【0025】 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 Verrucomicrobia, Akkermansiaceae, and / or Akkermansia in the large intestine. In one embodiment, the patient is in need of an increase in the abundance of Akkermansiaceae and suffers from prostate cancer. 【0026】 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 Akkermansia muciniphila in the large intestine. Preferably, the patient suffers from at least one condition selected from the following: obesity, diabetes, enteritis, liver disease, autism, atopy, metabolic disorders, and chronic alcohol consumption. 【0027】 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. The improvement comprises or consists of increasing the abundance of Verrucomicrobia, Akkermansiaceae, and / or Akkermansia in the intestine of the animal. Specifically, vitamin B2 and Lactobacillus rhamnosus are for use in increasing the abundance of Verrucomicrobia, Akkermansiaceae, and / or Akkermansia in the large intestine (colon) of an animal, and said use preferably comprises delivering vitamin B2 and Lactobacillus rhamnosus to the large intestine. Preferably, the animal is a human. 【0028】 To achieve an increase in the abundance of Verrucomicrobia, Akkermansiaceae, and / or Akkermansia 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. 【0029】 In another preferred embodiment, the Akkermansia bacteria to be enhanced are of the Akkermansia muciniphila species, and the animal (including humans) has experienced one or more conditions selected from the group consisting of obesity, diabetes, enteritis, liver disease, autism, atopy, metabolic disorders, and chronic alcohol consumption. 【0030】 Preferably, the Lactobacillus rhamnosus used is Lactobacillus rhamnosus GG. Lactobacillus rhamnosus DSM 32550 is particularly preferred. 【0031】 In another aspect, the present invention relates to a method for increasing the abundance of Verrucomicrobia, Akkermansiaceae, and / or Akkermansia 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, and said improvement includes increasing the abundance of Verrucomicrobia, Akkermansiaceae, and / or Akkermansia 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 delayed-release formulation. 【0032】 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: prostate cancer, obesity, diabetes, enteritis, liver disease, autism, atopy, metabolic disorders, and chronic alcohol consumption. 【0033】 In a further aspect, the present invention relates to the use of vitamin B2 and Lactobacillus rhamnosus to increase the abundance of Verrucomicrobia, Akkermansiaceae, and / or Akkermansia (preferably Akkermansia muciniphila) in the intestine of animals, preferably humans, 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 symptoms selected from the group consisting of prostate cancer, obesity, diabetes, enteritis, liver disease, autism, atopy, metabolic disorders, and chronic alcohol consumption. 【0034】 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 from about 0.005 g / L to about 0.2 g / L, preferably from about 0.01 to about 0.02 g / L. 【0035】 The dosage of Lactobacillus rhamnosus can be up to 5E+10 cfu / day. Preferably, the dosage range is 1E+08 - 1E+10 cfu / day, more preferably 1E+09 - 5E+10 cfu / day. Preferably, Lactobacillus rhamnosus is Lactobacillus rhamnosus GG. Lactobacillus rhamnosus DSM 32550 is particularly preferred. 【0036】 [Definitions and Embodiments] When used throughout, the following definitions apply. 【0037】 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). 【0038】 "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 is administered / consumed in another pill or tablet, and both the pill or tablet 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. 【0039】 "Combination for continuous administration or consumption" is a combination 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 the vitamins and probiotics is administered / consumed on any given day. As an example, the vitamin can be administered / consumed on the first day, and the probiotic 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. 【0040】 "Fixed combination" is a combination that delivers both active substances (i.e., vitamins and probiotics) to the patient simultaneously. A solid oral dosage form (e.g., tablet or capsule) containing both vitamins and probiotics is an example of a fixed combination. A liquid oral dosage form (e.g., oral drip) containing both vitamins and probiotics is another example of a fixed combination. 【0041】 "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. A treatment regimen in which vitamins and probiotics are not administered / consumed via the same route and / or not simultaneously requires a free combination. 【0042】 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, and 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) should be administered / consumed simultaneously on the same day, but are not suitable when vitamins and probiotics should be administered / consumed separately on the same day. 【0043】 "Separate administration / consumption" means that vitamins and probiotics are administered one at a time. Thus, separate administration / consumption refers to both sequential 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. 【0044】 "To administer" or "administration" means to impart or deliver an active substance to a human or animal, and similarly, a human or animal can ingest (consume) the active substance. 【0045】 The term "vitamin B2", used interchangeably with "riboflavin", includes riboflavin and its esters, particularly riboflavin-5'-phosphate and other pharmaceutically acceptable forms. 【0046】 "Increasing the abundance of" Verrucomicrobia, Akkermansiaceae, or Akkermansia means increasing the level (or amount or number or population size) of Verrucomicrobia, Akkermansiaceae, or Akkermansia compared to the respective control (i.e., the level / amount / number / population size of Verrucomicrobia, Akkermansiaceae, or Akkermansia when the combination of vitamin B2 and Lactobacillus rhamnosus was not added). 【0047】 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". 【0048】 "Direct delivery" or "delivered directly" means that the vitamin is not absorbed in the stomach and / or small intestine; the vitamin is formulated to be available in the distal gastrointestinal tract, preferably the large intestine (colon), where it is available to the microbiota. The vitamin is administered in excess, rather than as part of a person's normal daily nutritional requirements (typically obtained 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 relation to probiotics, "direct delivery" or "delivered directly" means that the probiotic is not absorbed in the stomach and / or small intestine; the probiotic is formulated to be available in the distal gastrointestinal tract, preferably the large intestine (colon). 【0049】 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. 【0050】 "Enteric layer" or "enteric coating" is a layer that surrounds a core, where the core contains the active agent and the layer confers resistance to gastric juice. 【0051】 "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. 【0052】 "Oral formulation" means that the vitamin and / or probiotic is formulated for oral administration / consumption. 【0053】 "Co-administer" or "co-administration" means that the 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. 【0054】 "Lactobacillus rhamnosus" has recently been re-named "Lacticaseibacillus rhamnosus", and both names are used interchangeably herein and both can be abbreviated as "L. rhamnosus". 【0055】 [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 about 0.005 g / L to about 0.2 g / L, preferably 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. 【0056】 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. 【0057】 [Formulation] The vitamin (vitamin B2) and / or the probiotic (Lactobacillus rhamnosus), preferably both, are preferably present in a formulation that preferentially makes the vitamin (and / or probiotic) available in the large intestine. 【0058】 Oral formulations are preferred. Other formulations include parenteral routes such as suppositories or injections. 【0059】 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 vitamins and / or probiotics 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. 【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 present invention, the vitamins and / or probiotics, preferably both, are present in a formulation comprising enteric capsule agents filled with a composition containing the vitamins and / or probiotics. The enteric capsule agents confer resistance to the acidic environment of the stomach. For example, soft gel formulations deliver the active drug in solution but can still provide the advantages of a solid dosage form. 【0062】 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 agent. 【0063】 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 drug (i.e., vitamins and / or probiotics) is more or less homogeneously distributed in the matrix. Furthermore, there is a colon release system in which the active drug is embedded in a fiber matrix (enzymatically triggered) and has an enteric coating on top. 【0064】 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). 【0065】 In a preferred embodiment for humans, the vitamins and / or probiotics are formulated in a solid dosage form for oral administration. The formulation can be in the form of capsules, pellets, beads, spheres, microspheres, 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. 【0066】 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 commonly used in combination. Different categories of coating materials targeting the large intestine are reviewed, 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 within the intestinal environment (e.g., within the intestinal environment of the ileum and large intestine), and combinations thereof. 【0067】 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. 【0068】 The following non-limiting examples are presented to explain the present invention in more detail. 【0069】 [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. 【0070】 [Materials and Methods] [Design of Long-Term SHIME Fermentation Experiment (Colon Model)] The configuration of a typical reactor 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). 【0071】 The SHIME® experiment for this study consisted of three stages: 1. Stabilization period: After inoculating the appropriate fecal sample into the colon reactor, a 2-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 2-week reference period, the standard SHIME nutrient matrix was further administered to the model for 14 days. Analysis of 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 3-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. 【0072】 The probiotic strain used in this experiment was Lactobacillus rhamnosus DSM 32550 (Biocare Copenhagen), an equivalent of Lactobacillus rhamnosus GG. 【0073】 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 the present invention, Lactobacillus rhamnosus DSM 32550 is referred to as the Lactobacillus rhamnosus GG strain. 【0074】 [Quantitative microbiota analysis by 16S rRNA gene sequencing and flow cytometry] Samples for quantitative 16S-targeted Illumina sequencing were collected three times per week during the control and final week of the treatment period. Next-generation 16S rRNA gene amplicon sequencing of the V3-V4 region was performed on samples from the mid-term SHIME experiment by LGC Genomics GmbH (Berlin, Germany). Library preparation and sequencing were performed on the Illumina MiSeq platform using v3 chemistry. The 341F (5′-CCTACGGGNGGCWGCAG-3′) and 785R (5′- GACTACHVGGGTATCTAAKCC-3′) primers were used as described by De Paepe et al. (2017), and the reverse primer was adapted to increase coverage. Quality control PCR was performed using Taq DNA polymerase with the Fermentas PCR kit according to the manufacturer's instructions (Thermo Fisher Scientific, Waltham, MA, USA). DNA quality was verified by electrophoresis at 100 V for 30 min on a 2% (w / v) agarose gel. Bioinformatics analysis of the amplicon data was performed as described by De Paepe et al. (2017). The resulting high-resolution proportional phylogenetic information (i.e., proportional abundances (%)) was combined with accurate quantification of total bacterial cells by flow cytometry to obtain quantitative data at the phylum, family, and species levels. This was done by multiplying the proportional abundances by the absolute cell numbers (cells / mL) obtained by flow cytometry. For flow cytometry analysis, 10-fold serial dilutions of all samples were prepared in Dulbecco's phosphate-buffered saline (DPBS) (Sigma-Aldrich, Bornem, Belgium) and stained with 0.01 mM SYTO24 (Life Technologies Europe, Merelbeke, Belgium) for 15 min at 37 °C in the dark. Samples were analyzed on a BD Facsverse (BD Biosciences, Erembodegem, Belgium) using a high flow rate setting, and bacteria were separated from media debris and signal noise by applying a threshold level of 200 to the SYTO channel. 【0075】 [Result] [Supplementation with the combination of Lactobacillus rhamnosus GG and vitamin B2 increased the abundance of Bifidobacterium bifidum.] As can be understood from FIG. 1, supplementation with Lactobacillus rhamnosus GG alone did not significantly change the abundance of Verrucomicrobia compared to the control. In contrast, the combination of Lactobacillus rhamnosus GG and vitamin B2 significantly increased the abundance of Verrucomicrobia compared to the control. 【0076】 [Supplementation with the combination of Lactobacillus rhamnosus GG and vitamin B2 increased the abundance of Akkermansiaceae.] As can be understood from FIG. 2, supplementation with Lactobacillus rhamnosus GG alone did not significantly change the abundance of Akkermansiaceae in the lumen compared to the control. In contrast, the combination of Lactobacillus rhamnosus GG and vitamin B2 significantly increased the abundance of Akkermansiaceae compared to the control. 【0077】 [Supplementation with the combination of Lactobacillus rhamnosus GG and vitamin B2 increased the abundance of Akkermansia muciniphila.] As can be understood from Figure 3, supplementation with Lactobacillus rhamnosus GG alone did not significantly change the abundance of Akkermansia muciniphila in the lumen compared to the control. In contrast, the combination of Lactobacillus rhamnosus GG and vitamin B2 significantly increased the abundance of Akkermansia muciniphila compared to the control.

Claims

[Claim 1] A combination containing vitamin B2 and Lactobacillus rhamnosus. [Claim 2] The combination according to claim 1, wherein the combination comprises vitamin B2 and Lactobacillus rhamnosus GG. [Claim 3] The combination according to claim 1 or claim 2, wherein the combination is for simultaneous administration or consumption. [Claim 4] The combination according to claim 1 or claim 2, wherein the combination is for continuous administration or consumption. [Claim 5] The combination according to claim 1 or 2, wherein the combination is in the form of an oral dosage. [Claim 6] The combination according to claim 1 or 2, wherein the combination is for administration to the large intestine. [Claim 7] The combination according to claim 1 or 2, for use as a drug, health supplement, or nutritional supplement. [Claim 8] The combination according to claim 1 or 2 for use in the treatment of patients who require an increase in the abundance of Verrucomicrobia, Akkermansiaceae, and / or Akkermansia in the colon. [Claim 9] The combination for use according to claim 8, wherein the patient requires an increased abundance of Akkermansiae and suffers from prostate cancer. [Claim 10] The combination according to claim 1 or 2 for use in the treatment of patients who require an increase in the amount of Akkermansia muciniphila in the colon. [Claim 11] The combination for use according to claim 10, wherein the patient suffers from one or more of the following: obesity, diabetes, enteritis, liver disease, autism, atopic dermatitis, metabolic disorders, and chronic alcohol consumption. [Claim 12] A combination comprising vitamin B2 and Lactobacillus rhamnosus for use in increasing the abundance of Verrucomicrobia, Akkermansiaceae, and / or Akkermansia in the large intestine of an animal, wherein the use comprises delivering the vitamin B2 and Lactobacillus rhamnosus to the large intestine. [Claim 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. [Claim 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. [Claim 15] A combination comprising vitamin B2 and Lactobacillus rhamnosus for use according to claim 12 or 13, wherein the animal, including a human, is experiencing one or more of the following conditions: prostate cancer, obesity, diabetes, enteritis, liver disease, autism, atopic dermatitis, metabolic disorders, and chronic alcohol consumption.

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