Combination comprising vitamin C and Lactobacillus rhamnosus

Abstract

The present invention relates to a combination comprising vitamin C and Lactobacillus rhamnosus for improving the health of the intestines of animals and humans, and to its use. It has been found that the combination of vitamin C and Lactobacillus rhamnosus increases the concentration of butyrate 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 C and Lactobacillus rhamnosus for improving the health of the intestines of animals and humans, and to its use. It has been found that the combination of vitamin C and Lactobacillus rhamnosus increases the concentration of butyrate 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 "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 regulation of the human gut microbiota aimed at restoring the imbalance has emerged as a potential therapeutic and preventive strategy, attracting the attention of not only researchers but also people engaged in various industries. Public awareness and acceptance of substances that regulate the human gut microbiota continue to increase. 【0003】 There is a common understanding that certain live microorganisms exert beneficial effects on human health by producing metabolites such as butyrate. Butyrate is a short-chain fatty acid (SCFA) and an essential metabolite produced by the gut microbiota. Butyrate has many health benefits, being an energy source for intestinal epithelial cells, promoting the production of glutathione, being a natural antioxidant, suppressing intestinal inflammation, and supporting a healthy intestinal epithelium. Butyrate prevents cancer by preventing the occurrence of cancer cells and promotes the production of hormones for healthy metabolism (Rinninella et al., What is the Healthy Gut Microbiota Composition? A Changing Ecosystem across Age, Environment, Diet, and Diseases (2019); Hamer HM et al., The role of butyrate on colonic function (2008)). Also, butyrate is effective against obesity and obesity-related diseases (Coppola S.et al., The Protective Role of Butyrate against Obesity and Obesity-Related Diseases (2021)). 【0004】 The human gut microbiota produces hundreds of different metabolites, and it is desirable to be able to selectively enhance specific metabolites. In particular, it is desirable to increase the concentration of butyrate, an SCFA in the intestine, in order to enhance health, improve the health status, and support the immune system. 【0005】 [Summary of the Invention] The present invention relates to the following items: 1) A combination comprising vitamin C and Lactobacillus (Lacticaseibacillus) rhamnosus. 2) The combination according to item 1, wherein the combination comprises vitamin C 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 simultaneous delivery or simultaneous ingestion, preferably a fixed combination. 4) The combination according to item 1 or item 2, wherein the combination is for sequential administration or sequential delivery or sequential ingestion, preferably 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 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 medicament, 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 patients in need of increasing the concentration of butyrate in the large intestine. 9) The combination for use according to item 8, wherein the patient suffers from at least one of irritable bowel syndrome, inflammatory bowel disease, colorectal cancer, graft-versus-host disease, diabetes, obesity, and neurodegenerative disorders. 10) A combination comprising vitamin C and Lactobacillus (Lacticaseibacillus) rhamnosus for use in increasing the concentration of butyrate in the large intestine of an animal, preferably a human, wherein the use comprises delivering or administering vitamin C and Lactobacillus rhamnosus to the large intestine. 11) A combination for use according to item 10, comprising vitamin C and Lactobacillus rhamnosus, wherein vitamin C and Lactobacillus rhamnosus are delivered or administered to the large intestine by a delayed-release formulation. 12) A combination for use according to item 10 or item 11, comprising vitamin C and Lactobacillus rhamnosus, wherein said use comprises simultaneously and / or sequentially delivering or administering vitamin C and Lactobacillus rhamnosus to an animal, preferably a human. 13) A combination for use according to any one of items 10 to 12, comprising vitamin C and Lactobacillus rhamnosus, wherein the animal, including a human, is experiencing a symptom selected from irritable bowel syndrome, inflammatory bowel disease, colorectal cancer, graft-versus-host disease, diabetes, obesity, and neurodegenerative disorders. 14) A combination for use according to any one of items 10 to 13, comprising vitamin C and Lactobacillus rhamnosus, wherein Lactobacillus rhamnosus is Lactobacillus rhamnosus GG, preferably Lactobacillus rhamnosus DSM 32550. 【Brief Description of the Drawings】 【0006】 【Figure 1】Butyrate production (mM) (± standard deviation) at 0 - 48 hour time intervals of short - term colon cultures under different test conditions, compared to corresponding controls, averaged across 6 different human donors (n = 6). Results of the mean values in the case of Lactobacillus rhamnosus GG alone (L. rhamnosus (L. rham)) or when co - supplemented with vitamin C (L. rhamnosus (L. rham)+vitamin C) are included. Statistical significant differences between test conditions and controls are indicated by "*" (p < 0.05). 【0007】 [Detailed Description of the Invention] Butyrate is a metabolite known to have beneficial effects on human health. The inventors have found that vitamin C in combination with Lactobacillus rhamnosus can enhance the concentration of butyrate in the large intestine, leading to an increase in the level of butyrate in the intestine. 【0008】 Accordingly, in a first aspect, the present invention relates to a combination comprising vitamin C and Lactobacillus (Lacticaseibacillus) rhamnosus. Preferably, Lactobacillus (Lacticaseibacillus) rhamnosus is the Lactobacillus rhamnosus GG strain, more preferably Lactobacillus rhamnosus DSM 32550. The combination is for simultaneous and / or sequential administration. 【0009】 The claims regarding the "combination" are product claims. The product of the present invention contains two active ingredients, a vitamin (vitamin C) and a probiotic (Lactobacillus rhamnosus). For simultaneous and / or sequential administration, refer to the following definitions and embodiments. 【0010】 Vitamin C, also known as L-ascorbic acid, is a water-soluble vitamin required for the biosynthesis of collagen, L-carnitine, and certain neurotransmitters. Vitamin C is also involved in protein metabolism. Furthermore, vitamin C is an important physiological antioxidant. Vitamin C plays an important role in immune function and improves nutrient absorption. Vitamin C can be purchased from DSM GmbH. Alternative suppliers include, for example, TER Chemicals Distribution Group, BIOCHEM Bernburg GmbH, DVA International GmbH, Falken Trade GmbH, and Neupert Ingredients GmbH. 【0011】 The most common Lactobacillus (Lacticaseibacillus) rhamnosus strain is Lactobacillus rhamnosus GG. This strain 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, L. rhamnosus DSM 32550 can be considered practically identical or equivalent to LGG®. Therefore, in this specification, L. rhamnosus DSM 32550 is referred to as "Lactobacillus rhamnosus GG". 【0012】 Alternative Lactobacillus rhamnosus strains include, in particular, Lactobacillus rhamnosus HN001 (Howaru; Danisco / DuPont), Lactobacillus rhamnosus GR-1® (Chr. Hansen, Denmark), and Lactobacillus rhamnosus Rosell-11 (Lallemand, Canada). 【0013】 According to the present invention, a preferred strain is Lactobacillus (Lacticaseibacillus) rhamnosus DSM 32550 (Biocare Copenhagen). This strain was deposited on July 6, 2017, with the Leibniz Institute DSMZ - German Collection of Microorganisms and Cell Cultures (Inhoffenstr. 7B, D - 38124 Braunschweig, Germany) under the Budapest Treaty. The accession number assigned by the international depository authority is DSM 32550. 【0014】 In one embodiment, the combination of the present invention is for simultaneous administration. Preferably, the combination for simultaneous administration is a fixed combination. However, a free combination can also be used for simultaneous administration. 【0015】 In another embodiment, the combination is for sequential administration. The combination for sequential administration is a free combination. 【0016】 Preferably, the combination is in an oral dosage form, more preferably a solid oral dosage form. 【0017】 The combination of the present invention can be, for example, a pharmaceutical combination or composition, a health supplement, or a food supplement. 【0018】 In another aspect, the present invention relates to vitamin C and Lactobacillus rhamnosus (i.e., a combination of vitamin C and Lactobacillus rhamnosus) for use as a medicament. 【0019】 Preferably, the combination of the present invention (e.g., a pharmaceutical combination) is used for the treatment of patients in need of increasing the concentration of butyrate in the large intestine. In one embodiment, the patient suffers from irritable bowel syndrome, inflammatory bowel disease, colorectal cancer, graft-versus-host disease, diabetes, obesity, or a neurodegenerative disorder. 【0020】 In a further aspect, the present invention relates to vitamin C and Lactobacillus rhamnosus (i.e., a combination of vitamin C and Lactobacillus rhamnosus) for use in improving the intestinal health of an animal. The improvement comprises or consists of increasing the concentration of butyrate in the large intestine of the animal. In particular, vitamin C and Lactobacillus rhamnosus are used to increase the concentration of butyrate in the large intestine (colon) of an animal, said use preferably comprising delivering vitamin C and Lactobacillus rhamnosus to the large intestine. Preferably, the animal is a human. 【0021】 To achieve an increase in the concentration of butyrate in the large intestine, it is preferred that vitamin C and Lactobacillus rhamnosus be delivered directly to the large intestine. That is, rather than the vitamins and probiotics being absorbed in the stomach and / or small intestine, the vitamins are delivered / administered in such a way that the vitamins and probiotics are delivered to the distal intestinal tract, preferably the large intestine (colon). This delivery is preferably done by delivering / administering vitamin C and Lactobacillus rhamnosus in a delayed-release formulation. Oral administration is preferred. 【0022】 In a preferred embodiment, the animal (including humans) is experiencing a condition selected from the group consisting of irritable bowel syndrome, inflammatory bowel disease, colorectal cancer, graft-versus-host disease, diabetes, obesity, and neurodegenerative disorders. 【0023】 Preferably, the Lactobacillus rhamnosus used is Lactobacillus rhamnosus GG. Lactobacillus rhamnosus DSM 32550 is particularly preferred. 【0024】 In another aspect, the present invention relates to a method for increasing the concentration of butyrate in the intestine, preferably the large intestine, the method comprising administering to an animal an effective dose of vitamin C and Lactobacillus rhamnosus (preferably Lactobacillus rhamnosus GG; particularly Lactobacillus rhamnosus DSM 32550). The method is for improving the health of the intestine of an animal, including a human, said improvement including increasing the concentration of butyrate in the large intestine. Preferably, the animal is a human. Preferably, vitamin C and Lactobacillus rhamnosus are delivered directly to the large intestine. Delivery to the large intestine can be achieved by administering vitamin C and Lactobacillus rhamnosus as a delayed release formulation. 【0025】 The method of the present invention can be used to treat, prevent, and / or alleviate the symptoms of irritable bowel syndrome, inflammatory bowel disease, colorectal cancer, graft-versus-host disease, diabetes, obesity, and neurodegenerative disorders in animals, including humans, in need thereof. 【0026】 In a further aspect, the present invention relates to the use of vitamin C and Lactobacillus rhamnosus for increasing the concentration of butyrate in the large intestine of an animal, preferably a human, said use including delivering vitamin C and Lactobacillus rhamnosus to the large intestine. Preferably, the use includes delivering vitamin C and Lactobacillus rhamnosus to the large intestine by a delayed release formulation. Preferably, the animal, including a human, is experiencing at least one symptom selected from the group consisting of irritable bowel syndrome, inflammatory bowel disease, colorectal cancer, graft-versus-host disease, diabetes, obesity, and neurodegenerative disorders. 【0027】 In the combinations, uses, and methods of the present invention, preferably, the dosage of vitamin C (ascorbic acid) is at most 2000 mg / day, preferably 100 - 2000 mg / day; more preferably 200 - 1000 mg / day. In one embodiment, vitamin C is dosed / administered in an amount such that the local concentration in the colon is at least 0.05 g / L, preferably at least 0.1 g / L, most preferably at least 0.33 g / L. The preferred local concentration in the colon ranges from about 0.05 g / L to about 1.5 g / L, more preferably from about 0.1 g / L to about 1 g / L, and most preferably from about 0.2 g / L to about 0.5 g / L. 【0028】 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. 【0029】 [Definitions and Embodiments] When used throughout, the following definitions apply. 【0030】 Claims regarding "combinations" or "pharmaceutical combinations" are product claims. The product of the present invention contains two active ingredients, namely a vitamin (vitamin C) and a probiotic (Lactobacillus rhamnosus). 【0031】 "Combination for co - administration" or "combination for co - ingestion" refers to a combination suitable for co - administration or co - ingestion respectively. "Co - administration" or "co - ingestion" means that vitamins and probiotic bacteria are administered / ingested on the same day (i.e., within 24 hours). The two active ingredients can be administered / ingested simultaneously (in the case of a fixed combination), or one by one at a time (in the case of a free combination). For example, while vitamins are administered / ingested with one pill or tablet, probiotics can be administered / ingested with another pill or tablet, but both pills / tablets should be administered / ingested within 24 hours. In another example, vitamins and probiotics are formulated in the same composition and are administered / ingested exactly at the same time. 【0032】 "Combination for sequential administration or sequential ingestion" refers to a combination suitable for sequential administration or sequential ingestion respectively. "Sequential administration" or "sequential ingestion" means that only one of vitamins and probiotics is administered / ingested on any given day during a period of continuous treatment for two or more days. As an example, vitamins can be administered / ingested on the first day, and probiotics can be administered / ingested the next day (i.e., after more than 24 hours) or thereafter. The active ingredients can be administered / ingested in any order. 【0033】 "Fixed combination" refers to 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 drops) containing both vitamins and probiotics is another example of a fixed combination. 【0034】 "Free combination" refers to a combination that enables both active substances (i.e., vitamins and probiotics) to be administered / ingested individually, i.e., one by one at a time. A treatment regimen in which vitamins and probiotics are not administered / ingested via the same route and / or are not administered / ingested simultaneously requires a free combination. 【0035】 Simultaneous administration / simultaneous ingestion can be achieved by using both fixed combinations and free combinations. Sequential administration / sequential ingestion requires a free combination, and fixed combinations are not suitable for sequential administration / sequential ingestion. Therefore, free combinations are more versatile and suitable for sequential administration / sequential ingestion, and are also suitable for simultaneous administration / simultaneous ingestion if both active substances are to be administered / ingested on the same day. Fixed combinations are only suitable for simultaneous administration / simultaneous ingestion when both components (i.e., vitamins and probiotics) are to be administered / ingested simultaneously on the same day. However, fixed combinations are not suitable when vitamins and probiotics are to be administered / ingested individually on the same day. 【0036】 "Individual administration / individual ingestion" means administering vitamins and probiotics one by one at a time. Therefore, individual administration / individual ingestion means both sequential administration / sequential ingestion, and can also mean simultaneous administration / simultaneous ingestion when it is mentioned that both active substances are to be administered / ingested one by one on the same day. 【0037】 "To administer" or "administration" means to give or deliver an active substance to a human or animal, and similarly means that a human or animal can take in (ingest) the active substance. 【0038】 The term "vitamin C" can be used interchangeably with "ascorbic acid", and also includes its pharmacologically acceptable salts (e.g., sodium ascorbate and calcium ascorbate), its pharmacologically acceptable esters (especially ascorbyl palmitate), and other pharmacologically acceptable forms. 【0039】 "SCFA" (abbreviation for "short-chain fatty acid") refers to fatty acids having less than 6 carbon atoms. SCFAs are derived from the intestinal microbial fermentation of indigestible foods. The three most common SCFAs are acetate, propionate, and butyrate. Butyrate, also known as butyric acid or butanoic acid, functions as a major energy source for colon cells and is an SCFA that maintains intestinal homeostasis through its anti-inflammatory effects. 【0040】 "Increasing the concentration of butyrate" means that the level (or amount) of butyrate has increased as compared to the corresponding control (i.e., the level / amount of butyrate when the combination of vitamin C and Lactobacillus rhamnosus is not added). 【0041】 As used herein, the term "intestine" (or "gut") refers to the portion of the gastrointestinal tract consisting of the small intestine and the large intestine. The "large intestine" (intestinum crassum) is located in the lower part of the gastrointestinal tract and is also referred to herein as the "colon". 【0042】 "Direct delivery" or "delivered directly" means that the vitamin is formulated in such a way that it is not absorbed in the stomach and / or small intestine, but rather is made available for utilization by the microbiota in the distal intestine, preferably the large intestine (colon). The vitamin is administered in an amount that exceeds, rather than being part of, the normal daily nutritional requirements of a human (which are generally obtained through diet and conventional vitamin supplementation). When used in humans, a preferred method according to the present invention is a method via a form that delays release until it reaches the large intestine (colon). Alternatively, a sufficiently large dose may 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 for utilization in the large intestine. Although less preferred, this latter delivery method can also be used in humans. With respect to probiotics, "direct delivery" or "delivered directly" means that the probiotic is formulated in such a way that it is not released in the stomach and / or small intestine, but rather is made available for utilization in the distal intestine, preferably the large intestine (colon). 【0043】 As used herein, "delayed release" refers to the release of the vitamin and / or probiotic at a time later than immediately after administration. Preferably, "delayed release" means that upon oral administration, the vitamin (and / or probiotic) is delivered to the large intestine (colon) with a delay as compared to an immediate release formulation. 【0044】 An "enteric layer" or "enteric coating" is a layer that surrounds a core, where the core contains an active agent and the layer confers resistance to gastric juice. 【0045】 "Prevent" can include reducing the risk of a harmful condition occurring, reducing the symptoms of a harmful condition, reducing the severity of a harmful condition, and prolonging the time when a harmful condition occurs. 【0046】 An "oral formulation" means that the vitamin and / or probiotic is formulated for oral administration / ingestion. 【0047】 "Co-administered" or "co-administration" means that the vitamins and / or probiotics are delivered / administered / ingested simultaneously (i.e., together), or individually but within a 24-hour time frame. The vitamins may be delivered / administered / ingested first. Similarly, the probiotics may be delivered / administered / ingested first. 【0048】 "Lactobacillus rhamnosus" has in recent years been renamed "Lacticaseibacillus rhamnosus", and both names are used interchangeably herein and both can be abbreviated as "L. rhamnosus". 【0049】 [Dosage] Preferably, vitamin C is administered in an amount such that its local concentration in the colon is at least 0.05 g / L, preferably at least 0.1 g / L, and most preferably at least 0.33 g / L. The preferred local concentration in the colon ranges from about 0.05 g / L to about 1.5 g / L, more preferably from about 0.1 g / L to about 1 g / L, and most preferably from about 0.2 g / L to about 0.5 g / L. The specific dosage per day can range up to 2000 mg / day, preferably 100 - 2000 mg / day; more preferably 200 - 1000 mg / day. 【0050】 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. 【0051】 [Formulation] Vitamins (vitamin C) and / or probiotics (Lactobacillus rhamnosus), preferably both, are preferably present in a formulation that enables the vitamins (and / or probiotics) to be mainly utilized in the large intestine. 【0052】 Oral formulations are preferred. Other formulations include those via parenteral routes such as suppositories or injections. 【0053】 When used in humans, the preferred method is via a delayed-release form that delays delivery until it reaches the intestinal tract. In the case of non-human animals, the preferred delivery includes administering a sufficiently large dose such that only a portion of the delivered vitamins and / or probiotics is absorbed in the stomach and the remaining effective dose is available in the intestinal tract. Although not preferred, this delivery method can also be used in humans. 【0054】 Delayed-release formulations are known in the art. Preferably, the delayed-release formulation has an enteric coating (also referred to as an enteric layer). 【0055】 In one embodiment of the present invention, vitamins and / or probiotics, preferably both, are present in a formulation comprising enteric capsules filled with a composition containing vitamins and / or probiotics. The enteric capsules confer resistance to the acidic environment of the stomach. For example, soft gel formulations can deliver the active agent in solution and yet provide the advantages of a solid dosage form. 【0056】 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 may also be a hard gel capsule. 【0057】 Alternatively, a matrix-based delivery system can be used for direct delivery to the colon. In matrix-based systems, there are no individual layers of coating material, but the active agent (i.e., vitamins and / or probiotics) is distributed somewhat homogeneously within the matrix. Further, there are colon release systems in which the active agent is embedded in, for example, a fiber matrix (induced by an enzyme) and the outermost layer is an enteric coating. 【0058】 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). 【0059】 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. 【0060】 Coating materials, or matrix materials, for delaying the release of vitamins and / or probiotics, particularly for targeted release in the ileum or large intestine, upon oral administration, are known in the art. These can be finely divided into coating materials that disintegrate when a specific pH is exceeded, coating materials that disintegrate after a specific residence time in the gastrointestinal tract has elapsed, and coating materials that disintegrate by enzyme induction specific to the bacterial flora in a specific region of the intestine. Generally, different classifications of coating materials are used in combination. Different classifications 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 enzyme induction in the intestinal environment (e.g., the intestinal environment of the ileum and large intestine), and combinations thereof. 【0061】 Examples of pH-dependent disintegrating coating materials include polyvinyl acetate phthalate, cellulose acetate trimellitate, hydroxypropylmethylcellulose phthalate HP-50, HP-55 or HP-55S, cellulose acetate phthalate, shellac, hydroxypropylmethylcellulose 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 induction 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, curdlan, levan, amylopectin, starch, amylose, resistant starch, and azo compounds decomposed by bacteria that cleave azo bonds. 【0062】 To better illustrate the present invention, the following non-limiting examples are shown. 【0063】 [Examples] The purpose of this test was to examine the effect of the combination of vitamin C and Lactobacillus rhamnosus on the metabolic activity of the intestinal flora in a short-term batch fermentation experiment. 【0064】 [Materials and Methods] [Design of Batch Fermentation Experiment (Colon Model)] Short-term batch fermentation experiments were carried out by ProDigest. This experiment consisted of colon culturing representative doses of selected vitamins together with a representative bacterial inoculum under conditions simulating the proximal colon. In this experiment, the fecal inoculum was derived from fresh fecal samples of six different healthy adult donors. Culturing was performed as previously described (Van den Abbeele, P.; Taminiau, B.; Pinheiro, I.; Duysburgh, C.; Jacobs, H.; Pijls, L.; Marzorati, M. Arabinoxylo-Oligosaccharides and Inulin Impact Inter-Individual Variation on Microbial Metabolism and Composition, Which Immunomodulates Human Cells. J. Agric. Food Chem. 2018, 66, 1121-1130). At the start of the short-term colon culture, fresh fecal material was collected from six healthy human donors to prepare an anaerobic fecal slurry, which was then inoculated at 10% by volume into SHIME nutrient medium containing basal nutrients consisting of 3.5 g / L of K2HPO4, 10.9 g / L of KH2PO4, 2 g / L of NaHCO3 (Chem-lab NV, Zedelgem, Belgium), 2 g / L of yeast extract, 2 g / L of peptone (Oxoid, Aalst, Belgium), 1 g / L of mucin (Carl Roth, Karlsruhe, Germany), 0.5 g / L of L-cysteine and 2 mL / L of Tween 80 (Sigma-Aldrich, Bornem, Belgium). All test components (i.e., probiotic strains, vitamin C) were also added to the SHIME medium.Furthermore, as previously described, the M-SHIME® technology was incorporated into this experiment by adding a mucus-covered microecosystem (modeling colonic mucus) to the culture (Van den Abbeele, P., et al. (2013). Butyrate-producing Clostridium cluster XIVa species specifically colonize mucins in an in vitro gut model. The ISME Journal 7(5), 949-961). Cultivation was carried out at 37 °C for 48 h under shaking (90 rpm) and anaerobic conditions. 【0065】 In this study, Lactobacillus rhamnosus DSM 32550 (Biocare Copenhagen), an equivalent of Lactobacillus rhamnosus GG, was added alone or in combination with vitamin C to a colonic model containing donor samples (see Table 1), and the butyrate levels were analyzed. The experiment was carried out as a single replicate. 【0066】 Lactobacillus rhamnosus DSM 32550 has a genomic sequence that is 99.99% identical to the genomic sequence of LGG®. Therefore, L. rhamnosus DSM 32550 can be considered to be identical or equivalent to LGG® in practical terms. Therefore, in the examples herein, Lactobacillus rhamnosus DSM 32550 is referred to as Lactobacillus rhamnosus GG. 【0067】 【Table 1】 【0068】 The probiotic strain to be examined, Lactobacillus rhamnosus DSM 32550, was added to the SHIME medium as a culture grown overnight at a concentration of 1*10 9 CFU. The probiotic strain was supplemented alone or in combination with vitamin C. Vitamin C (ascorbic acid, DSM) was added at a concentration of 0.333 g / L, which can be translated to a dose of 200 mg considering a colonic volume of approximately 600 ml. 【0069】 [Analysis of butyrate levels] Samples for butyrate analysis were analyzed at 0 h and 48 h of culture. After adding 2-methylhexanoic acid as an internal standard, butyrate was extracted from the samples with diethyl ether. The extract was analyzed using a GC-2014 gas chromatograph (Shimadzu, ‘s-Hertogenbosch, the Netherlands) equipped with a capillary fatty acid-free EC-1000 Econo-Cap column (dimensions: 25 mm × 0.53 mm, film thickness 1.2 mM; Alltech, Laarne, Belgium), a flame ionization detector, and a split injector. The injection volume was 1 mL, and the temperature profile was set from 110 to 160 °C with a temperature increase of 6 °C per minute. The carrier gas was nitrogen, and the injector and detector temperatures were 100 °C and 220 °C, respectively. 【0070】 [Statistics] Statistical analysis was performed to examine the average effect of the test products. For this purpose, the average of 6 donors was calculated for each endpoint. A paired t-test was performed to evaluate the potential effect of the test products compared to the control and to compare different test products with each other. A p-value less than 0.05 was considered a statistically significant difference. 【0071】 [Results] [Supplementation with a combination of Lactobacillus rhamnosus and vitamin C increased the concentration of butyrate] As can be read from Figure 1, the combination of vitamin C and Lactobacillus rhamnosus GG resulted in a significant increase in butyrate concentration compared to the corresponding control (Lactobacillus rhamnosus GG alone).

Claims

[Claim 1] i) Vitamin C, and ii) Combinations that include Lactobacillus rhamnosus. [Claim 2] The aforementioned combination is i) Vitamin C, and ii) The combination according to claim 1, comprising Lactobacillus rhamnosus GG. [Claim 3] The combination according to claim 1 or claim 2, wherein the combination is for simultaneous administration. [Claim 4] The combination according to claim 1 or claim 2, wherein the combination is for continuous administration. [Claim 5] The combination according to claim 1 or 2, wherein the combination is in an oral dosage form. [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 need to increase the concentration of butyrate in the colon. [Claim 9] The combination for use according to claim 8, wherein the patient suffers from irritable bowel syndrome, inflammatory bowel disease, colorectal cancer, graft-versus-host disease, diabetes mellitus, obesity, or neurodegenerative disorder. [Claim 10] A combination comprising vitamin C and Lactobacillus rhamnosus for use in increasing the concentration of butyrate in the large intestine of animals, wherein the use comprises administering or delivering the vitamin C and Lactobacillus rhamnosus to the large intestine. [Claim 11] A combination comprising vitamin C and Lactobacillus rhamnosus for use according to claim 10, wherein the vitamin C and Lactobacillus rhamnosus are administered or delivered to the large intestine by a delayed-release formulation. [Claim 12] A combination comprising vitamin C and Lactobacillus rhamnosus for use according to claim 10 or claim 11, wherein the use comprises administering or delivering the vitamin C and Lactobacillus rhamnosus to the animal simultaneously and / or sequentially. [Claim 13] A combination for use according to claim 10 or 11, comprising vitamin C and Lactobacillus rhamnosus, wherein the animal, including a human, is experiencing at least one condition selected from irritable bowel syndrome, inflammatory bowel disease, colorectal cancer, graft-versus-host disease, diabetes mellitus, obesity, and neurodegenerative disorders. [Claim 14] A combination for use according to claim 10 or 11, comprising vitamin C and Lactobacillus rhamnosus, wherein the Lactobacillus rhamnosus is Lactobacillus rhamnosus GG.

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