A combination comprising vitamin C and Bifidobacterium animalis ssp. lactis

Abstract

The present invention relates to a combination comprising vitamin C and Bifidobacterium animalis ssp. lactis, and its use for improving intestinal health in animals and humans. The combination of vitamin C and Bifidobacterium animalis ssp. lactis has been found to increase the concentration of SCFAs including 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 Bifidobacterium animalis ssp. lactis, and its use for improving intestinal health in animals and humans. The combination of vitamin C and Bifidobacterium animalis ssp. lactis has been found to increase the concentration of SCFAs including 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 "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. 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 can have beneficial effects on human health through the production of metabolites such as short-chain fatty acids (SCFAs). SCFAs are the main metabolites produced by the gut microbiota. Short-chain fatty acids can play beneficial roles in human health such as intestinal barrier integrity, blood pressure control, energy intake and energy use, regulation of glucose and lipid metabolism, mediation of the immune system, and anti-inflammatory responses (Tan J. et al., The Role of Short-Chain Fatty Acids in Health and Disease (2014)).

[0004] SCFA butyrate has many health benefits: Butyrate is an energy source for cells in the intestinal wall, stimulates the production of glutathione, is a natural antioxidant, controls intestinal inflammation, and supports a strong intestinal wall. It helps prevent cancer by preventing the development of cancer cells and promoting 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)).

[0005] The human gut microbiota produces hundreds of different metabolites, and it would be desirable to selectively enhance certain metabolites. In particular, it would be desirable to increase the concentration of SCFAs, especially butyrate, 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 C and Bifidobacterium animalis ssp. lactis. 2) The combination according to item 1, wherein the combination comprises vitamin C and Bifidobacterium animalis ssp. lactis BB-12, preferably Bifidobacterium animalis ssp. lactis DSM 32269. 3) The combination according to item 1 or item 2, wherein the combination is for simultaneous administration, delivery or consumption, and 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, and 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, and 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, which is for use as a drug, a health food or a dietary supplement. 8) The combination according to any one of items 1 to 7, which is for use in the treatment of patients in need of an increase in the abundance of SCFAs in the large intestine. 9) The combination for use according to item 8, wherein the patient suffers from one or more of the following conditions: irritable bowel syndrome, inflammatory bowel disease, colorectal cancer, graft-versus-host disease, diabetes, obesity, allergy, inflammation, autoimmune disease, and neurodegenerative disorder. 10) The combination according to any one of items 1 to 7, which is for use in the treatment of patients in need of an increase in the abundance of butyrate 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: irritable bowel syndrome, inflammatory bowel disease, colorectal cancer, graft-versus-host disease, diabetes, obesity, neurodegenerative disorder. 12) A combination comprising vitamin C and Bifidobacterium animalis ssp. lactis for use in increasing the concentration of SCFAs and / or butyrate in the large intestine of an animal, preferably a human, wherein the use comprises administering or delivering vitamin C and Bifidobacterium animalis ssp. lactis to the large intestine. 13) A combination comprising vitamin C and Bifidobacterium animalis ssp. lactis for use according to item 12, wherein the vitamin C and Bifidobacterium animalis ssp. lactis are administered or delivered to the large intestine by a delayed release formulation. 14) A combination comprising vitamin C and Bifidobacterium animalis ssp. lactis for use according to item 12 or item 13, wherein the use comprises administering or delivering the vitamin C and Bifidobacterium animalis ssp. lactis to an animal, preferably a human, simultaneously and / or sequentially. 15) A combination comprising vitamin C and Bifidobacterium animalis ssp. lactis for use according to any one of items 12 to 14, wherein the animal including a human is experiencing one or more conditions selected from irritable bowel syndrome, inflammatory bowel disease, colorectal cancer, graft-versus-host disease, diabetes, obesity, allergy, inflammation, autoimmune disease, and neurodegenerative disorder. 16) A combination of vitamin C and / or Bifidobacterium animalis ssp. lactis for use according to any one of items 12 to 15, wherein the Bifidobacterium animalis ssp. lactis is Bifidobacterium animalis ssp. lactis BB-12, preferably Bifidobacterium animalis ssp. lactis DSM 32269.

Brief Description of the Drawings

[0007]

Figure 1

Figure 2

[0008] [Detailed Description of the Invention] SCFAs such as butyrate are metabolites known for their beneficial effects on human health. The inventors have found that the combination of vitamin C with Bifidobacterium animalis ssp. lactis can increase the concentration of SCFAs including butyrate in the large intestine, resulting in an increase in intestinal SCFAs and butyrate.

[0009] Accordingly, in a first aspect, the present invention relates to a combination of vitamin C and Bifidobacterium animalis ssp. lactis. Preferably, Bifidobacterium animalis ssp. lactis is the Bifidobacterium animalis ssp. lactis BB-12 strain, and 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 C) and a probiotic (Bifidobacterium animalis ssp. lactis). For simultaneous and / or sequential administration, refer to the following definitions and embodiments.

[0011] Vitamin C, also known as L-ascorbic acid, is a water-soluble vitamin necessary for the integrity 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 are, for example, TER Chemicals Distribution Group, BIOCHEM Bernburg GmbH, DVA International GmbH, Falken Trade GmbH, and Neupert Ingredients GmbH.

[0012] The most common Bifidobacterium animalis ssp. lactis strain is Bifidobacterium animalis ssp. lactis BB-12. This can be purchased, for example, from Chr. Hansen as BB-12®. Bifidobacterium animalis ssp. lactis DSM 32269 (Biocare Copenhagen) has a genomic sequence that is 99.99% identical to the genomic sequence of BB-12®. Therefore, for practical purposes, B. animalis ssp. lactis DSM 32269 can be considered the same as or equivalent to B. animalis ssp. lactis BB-12®. Therefore, Bifidobacterium animalis ssp. lactis DSM 32269 (Biocare Copenhagen) is referred to herein as the Bifidobacterium animalis ssp. lactis BB-12 strain.

[0013] Alternative Bifidobacterium animalis ssp. lactis strains are, for example, Bifidobacterium lactis Bi-07® (Howaru; Danisco / IFF / DuPont), Bifidobacterium lactis Bl-04® (Howaru; Danisco / IFF / DuPont), and Bifidobacterium lactis HN019 (Howaru; IFF / DuPont).

[0014] Bifidobacterium animalis ssp. lactis DSM 32269 (Biocare Copenhagen) is a preferred strain according to the present invention. Bifidobacterium animalis ssp. lactis DSM 32269 has been deposited in accordance with the Budapest Treaty on February 26, 2016, 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 32269.

[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, or a nutritional supplement.

[0019] In another aspect, the present invention relates to vitamin C and Bifidobacterium animalis ssp. lactis (i.e., a combination of vitamin C and Bifidobacterium animalis ssp. lactis) for use as a medicament.

[0020] Preferably, the combination (e.g., pharmaceutical combination) of the present invention is for use in the treatment of patients in need of an increase in the concentration of SCFAs in the large intestine. In one embodiment, the patient suffers from one or more of the following conditions: irritable bowel syndrome, inflammatory bowel disease, colorectal cancer, graft-versus-host disease, diabetes, obesity, allergy, inflammation, autoimmune disease, and neurodegenerative disorder.

[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 butyrate in the large intestine. Preferably, the patient suffers from one or more of the following conditions: irritable bowel syndrome, inflammatory bowel disease, colorectal cancer, graft-versus-host disease, diabetes, obesity, neurodegenerative disorder.

[0022] In a further aspect, the present invention relates to vitamin C and Bifidobacterium animalis ssp. lactis (i.e., a combination of vitamin C and Bifidobacterium animalis ssp. lactis) for use in improving intestinal health in an animal. The improvement comprises or consists of increasing the concentration of SCFA and / or butyrate in the large intestine of the animal. Specifically, vitamin C and Bifidobacterium animalis ssp. lactis are for use in increasing the concentration of SCFA and / or butyrate in the large intestine (colon) of an animal, said use preferably comprising delivering vitamin C and Bifidobacterium animalis ssp. lactis to the large intestine. Preferably, the animal is a human.

[0023] To achieve an increase in the concentration of SCFA and / or butyrate in the large intestine, vitamin C and Bifidobacterium animalis ssp. lactis 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 the probiotics are delivered / administered to the distal gastrointestinal tract, preferably the large intestine (colon). This is preferably done by delivering / administering vitamin C and Bifidobacterium animalis ssp. lactis 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 irritable bowel syndrome, inflammatory bowel disease, colorectal cancer, graft-versus-host disease, diabetes, obesity, allergy, inflammation, autoimmune disease, and neurodegenerative disorder.

[0025] Preferably, the Bifidobacterium animalis ssp. lactis used is Bifidobacterium animalis ssp. lactis BB - 12. Bifidobacterium animalis ssp. lactis DSM 32269 is particularly preferred.

[0026] In another aspect, the present invention relates to a method for increasing the concentration of SCFA and / or butyrate in the intestine, preferably the large intestine, the method comprising administering to an animal an effective dose of vitamin C and Bifidobacterium animalis ssp. lactis (preferably Bifidobacterium animalis ssp. lactis BB - 12, particularly Bifidobacterium animalis ssp. lactis DSM 32269). The method is for improving intestinal health in animals including humans, said improvement comprising increasing the concentration of SCFA and / or butyrate in the large intestine. Preferably, the animal is a human. Preferably, vitamin C and Bifidobacterium animalis ssp. lactis 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.

[0027] The method of the present invention can be used to treat, prevent, and / or reduce the symptoms of irritable bowel syndrome, inflammatory bowel disease, colorectal cancer, graft - versus - host disease, diabetes, obesity, allergies, inflammation, autoimmune diseases, or neurodegenerative disorders in animals including humans in need thereof.

[0028] In a further aspect, the invention relates to the use of vitamin C and Bifidobacterium animalis ssp. lactis (i.e., a combination of vitamin C and Bifidobacterium animalis ssp. lactis) for increasing the concentration of SCFA and / or butyrate in the large intestine of an animal, preferably a human, said use comprising delivering vitamin C and Bifidobacterium animalis ssp. lactis to the large intestine. Preferably, the use comprises delivering vitamin C and Bifidobacterium animalis ssp. lactis 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 irritable bowel syndrome, inflammatory bowel disease, colorectal cancer, graft-versus-host disease, diabetes, obesity, allergy, inflammation, autoimmune disease, and neurodegenerative disorder.

[0029] In the combinations, uses, and methods of the invention, preferably, the vitamin C (ascorbic acid) dosage is up to 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 its 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, most preferably from about 0.2 g / L to about 0.5 g / L.

[0030] The dosage of Bifidobacterium animalis ssp. lactis 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, Bifidobacterium animalis ssp. lactis is Bifidobacterium animalis ssp. lactis BB-12. Bifidobacterium animalis ssp. lactis DSM 32269 is particularly preferred.

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

[0032] Claims related to "combination" or "pharmaceutical combination" are product claims. The product of the present invention contains two active ingredients: vitamin (vitamin C) and probiotics (Bifidobacterium animalis ssp. lactis).

[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 are administered / consumed exactly simultaneously.

[0034] A "combination for continuous administration or consumption" is a combination suitable for continuous administration or consumption respectively. "Continuous administration" or "continuous consumption" means that only one of vitamins and probiotics is administered / consumed on any given day during a period of two or more days of continuous treatment. 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] A "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., tablets or capsules) 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.

[0036] A "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.

[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, 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.

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

[0039] "To administer" or "administration" means to impart or deliver an active substance to a human or an animal. Similarly, a human or an animal can ingest (consume) the active substance.

[0040] The term "vitamin C", which can be used interchangeably with "ascorbic acid", also includes its pharmaceutically acceptable salts (e.g., sodium ascorbate and calcium ascorbate) and its pharmaceutically acceptable esters (especially ascorbyl palmitate) and other pharmaceutically acceptable forms.

[0041] "Increasing the concentration of" SCFA or butyrate means increasing the level (or amount) of SCFA or butyrate compared to each respective control (i.e., the level / amount of SCFA or butyrate when the combination of vitamin C and Bifidobacterium animalis ssp. lactis was not added).

[0042] "SCFA" (abbreviation for "short-chain fatty acid") is a fatty acid having fewer than 6 carbon atoms. They are derived from the intestinal microbial fermentation of indigestible foods. The three most common SCFAs are acetate, propionate, and butyrate. Butyrate, also known by the name butyric acid or butanoic acid, functions as a major energy source for colonocytes and is an SCFA that maintains intestinal homeostasis through its anti-inflammatory effects.

[0043] 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".

[0044] "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 not part of a person's normal daily nutritional requirements (normally obtained through diet and conventional vitamin supplementation) and is administered in excess. For human use, a preferred method according to the invention is via a form that delays release until it reaches the large intestine (colon). Alternatively, a sufficiently high dose can be administered such that only a 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 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).

[0045] 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.

[0046] "Enteric layer" or "enteric coating" is a layer surrounding a core that contains an active agent and imparts resistance to gastric juice.

[0047] "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.

[0048] "Oral preparation" means that vitamins and / or probiotics are formulated for oral administration / consumption.

[0049] "Co-administering" or "co-administration" means that vitamins and / or probiotics are delivered / administered / consumed either 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.

[0050] [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, and more preferably 200 - 1000 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, and more preferably 1E+09 - 5E+10 cfu / day.

[0052] [Formulation] The vitamin (vitamin C) and / or the probiotic (Bifidobacterium animalis ssp. lactis), preferably both, are preferably present in a formulation that preferentially makes the vitamin (and / or the probiotic) available in the large intestine.

[0053] Oral preparations are preferred. Other formulations include non-oral routes such as suppositories or injections.

[0054] For human use, the preferred method is via a delayed-release form that delays delivery until reaching 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 less 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 present invention, the vitamins and / or probiotics, preferably both, are present in a formulation comprising enteric-coated capsules filled with a composition containing the 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 provide 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 drug (i.e., vitamins and / or probiotics) is more or less homogeneously distributed in the matrix. Further, 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.

[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 in a solid dosage form for oral administration. The formulation may 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 commonly used in combination. Different categories of coating materials for targeting the large intestine are reviewed, for example, by 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 coating materials that disintegrate pH - dependently, coating materials that disintegrate time - dependently, coating materials that disintegrate by enzyme triggers in the intestinal environment (e.g., within the intestinal environment of the ileum and large intestine), and combinations thereof.

[0062] Examples of coating materials that disintegrate in a pH-dependent manner include polyvinyl acetate phthalate, cellulose acetate trimellitate, hydroxypropyl methylcellulose phthalate HP-50, HP-55 or HP-55S, cellulose acetate phthalate, shellac, hydroxypropyl methylcellulose acetate succinate (HPMCAS), poly(methacrylic acid, ethyl acrylate) 1:1 (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 coating materials that disintegrate in a time-dependent manner include Eudragit® RL, Eudragit® RS, and ethyl cellulose. Examples of coating materials that disintegrate by enzyme triggers in the large intestine environment include chondroitin sulfate, pectin, guar gum, chitosan, inulin, lactulose, raffinose, stachyose, alginate, dextran, xanthan gum, locust bean gum, arabinogalactan, cyclodextrin, pullulan, carrageenan, scleroglucan, chitin, curdulan, levan, amylopectin, starch, amylose, resistant starch, and azo compounds decomposed by azo bond-decomposing bacteria.

[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 C and Bifidobacterium animalis ssp. lactis on the metabolic activity of the gut microbiota in a short-term continuous fermentation experiment.

[0065] [Materials and Methods] [Design of Batch Fermentation Experiments (Colon Model)] Short-term batch - Fermentation experiments were performed by ProDigest consisting of colonic incubations of representative doses of selected vitamins with a representative bacterial inoculum under conditions simulating the proximal colon. Under the current experiments, the bacterial inoculum was derived from fresh fecal samples of six different healthy adult donors. Incubations were performed as previously described (Van den Abbeele, P.; Taminiau, B.; Pinheiro, I.; Duysburgh, C.; Jacobs, H.; Pijls, L.; Marzorati, M. Arabinoxylo-Oligosaccharides and Inulin Impact Inter-Individual Variation on Microbial Metabolism and Composition, Which Immunomodulates Human Cells. J. Agric. Food Chem. 2018, 66, 1121 - 1130 At the start of the short-term colonic incubations, fresh fecal material from six healthy human donors was collected and after preparation of the anaerobic fecal slurry, this slurry was inoculated at 10 vol% into SHIME nutrient medium containing basic nutrients including 3.5 g / L K2HPO4, 10.9 g / L KH2PO4, 2 g / L NaHCO3 (Chem-lab NV, Zedelgem, Belgium), 2 g / L yeast extract, 2 g / L peptone (Oxoid, Aalst, Belgium), 1 g / L mucin (Carl Roth, Karlsruhe, Germany), 0.5 g / L L-cysteine and 2 mL / L Tween80 (Sigma-Aldrich, Bornem, Belgium). All test components (i.e., probiotic strains, vitamin C) were also added to the SHIME medium.Furthermore, as previously described (Van den Abbeele, P., et al. (2013). Butyrate-producing Clostridium cluster XIVa species specifically colonize mucins in an in vitro gut model. The ISME Journal 7(5), 949-961), the M-SHIME® technology was incorporated into the current experiment by adding an incubation with a mucus-covered microecosystem (modeling the mucus of the colon). The incubation was carried out at 37 °C for 48 hours with shaking (90 rpm) under anaerobic conditions.

[0066] In this study, Bifidobacterium animalis ssp. lactis BB-12 equivalent Bifidobacterium animalis ssp. lactis DSM 32269 (Biocare Copenhagen) was added alone or in combination with vitamin C to the colon model containing donor samples (see Table 1), and the levels of SCFA and butyrate were analyzed respectively. The experiment was conducted with one repetition.

[0067]

Table 1

[0068] Bifidobacterium animalis ssp. lactis BB-12 equivalent strain Bifidobacterium animalis ssp. lactis DSM 32269 was added to the SHIME medium as an overnight growth culture at a concentration of 1*10 9It was added in 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 is equivalent to a 200 mg dose assuming a colonic volume of about 600 ml.

[0069] [Analysis of SCFA levels] Short-chain fatty acids (SCFAs) such as acetate, propionate, and butyrate are an evaluation of microbial carbohydrate metabolism and can be compared with the typical fermentation patterns of the normal GI bacterial flora. Samples for SCFA analysis were analyzed after 0 and 48 hours of incubation. After adding 2-methylhexanoic acid as an internal standard, SCFAs were extracted from the samples using diethyl ether. The extract was analyzed using a GC-2014 gas chromatograph (Shimadzu, ‘s-Hertogenbosch, 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 min−1. The carrier gas was nitrogen, and the injector and detector temperatures were 100 and 220 °C, respectively. The production of non-branched and branched SCFAs was calculated by summing the molar concentrations of acetate, propionate, butyrate, isovalerate, and isocaproate, and separately summing the molar concentrations of isobutyrate, isovalerate, and isocaproate. Total SCFA production was defined as the sum of non-branched and branched SCFAs.

[0070] [Statistics] Statistical analysis was performed to examine the average effects 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 effects of the test products compared to the control and also to compare different test products with each other. A difference was considered statistically significant if the p-value was less than 0.05.

[0071] [Result] [Supplementation with a combination of Bifidobacterium animalis ssp. lactis and vitamin C increased the concentration of SCFAs] As can be understood from Figure 1, supplementation with a combination of vitamin C and Bifidobacterium animalis ssp. lactis DSM 32269 resulted in a significant increase in the concentration of SCFAs compared to their respective controls (Bifidobacterium animalis ssp. lactis DSM 32269 alone).

[0072] [Supplementation with a combination of Bifidobacterium animalis ssp. lactis and vitamin C increased the concentration of butyrate] As can be understood from Figure 2, supplementation with a combination of vitamin C and Bifidobacterium animalis ssp. lactis DSM 32269 resulted in a significant increase in the concentration of butyrate compared to their respective controls (Bifidobacterium animalis ssp. lactis DSM 32269 alone).

Claims

1. A combination containing vitamin C and Bifidobacterium animalis ssp. lactis.

2. The combination according to claim 1, wherein the combination comprises vitamin C and Bifidobacterium animalis ssp. lactis Bb-12.

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 requiring an increase in the amount of SCFA in the colon.

9. The combination for use according to claim 8, wherein the patient suffers from one or more of the following: irritable bowel syndrome, inflammatory bowel disease, colorectal cancer, graft-versus-host disease, diabetes mellitus, obesity, allergies, inflammation, autoimmune diseases, and neurodegenerative disorders.

10. The combination according to claim 1 or 2 for use in the treatment of patients requiring an increase in the amount of butyrate in the colon.

11. The combination for use according to claim 10, wherein the patient suffers from one or more of the following: irritable bowel syndrome, inflammatory bowel disease, colorectal cancer, graft-versus-host disease, diabetes mellitus, obesity, and neurodegenerative disorders.

12. A combination comprising vitamin C and Bifidobacterium animalis ssp. lactis for use in increasing the concentration of SCFA and / or butyrate in the large intestine of an animal, wherein the use comprises administering or delivering the vitamin C and Bifidobacterium animalis ssp. lactis to the large intestine.

13. A combination of vitamin C and Bifidobacterium animalis ssp. lactis for use according to claim 12, wherein the vitamin C and Bifidobacterium animalis ssp. lactis are administered or delivered to the large intestine by a delayed-release formulation.

14. A combination comprising vitamin C and Bifidobacterium animalis ssp. lactis for use according to claim 12 or 13, wherein the use comprises administering or delivering the vitamin C and Bifidobacterium animalis ssp. lactis to the animal simultaneously and / or sequentially.

15. A combination comprising vitamin C for use and Bifidobacterium animalis ssp. lactis according to claim 12 or 13, wherein the animal, including a human, is experiencing one or more conditions selected from irritable bowel syndrome, inflammatory bowel disease, colorectal cancer, graft-versus-host disease, diabetes mellitus, obesity, allergies, inflammation, autoimmune diseases, and neurodegenerative disorders.

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