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 abundance of Bifidobacterium bifidum 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 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 abundance of beneficial bacteria in the intestinal tract when delivered to the large intestine.

[0002] [Background of the Invention] Increasing evidence indicates that imbalances 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 aimed at restoring balance is a potential therapeutic and preventive strategy that has attracted the attention of scholars and those engaged in various industries. Public awareness and acceptance of substances that modulate the human gut microbiome continue to grow.

[0003] There is a consensus that certain live microorganisms have beneficial effects on human health. Bifidobacteria are one of the major genera of bacteria that make up the gut microbiota of mammals, and some are used as probiotics. As human gut inhabitants, bifidobacteria create a bacteria-host, symbiotic-like relationship that provides many health benefits to the human host, including, but not limited to: reestablishment of normal gut microflora, improvement of intestinal and immune function, prevention of nutrient loss, prevention of constipation, prevention of the initiation of colon cancer, treatment of liver damage, and reduction of cholesterol levels in the blood. Bifidobacteria have also been found to have antibiotic activity and to assist in antitumor activity in the host.

[0004] Bifidobacterium bifidum has shown promise in the treatment of the following conditions: infections caused by Helicobacter pylori (H. pylori), irritable bowel syndrome (IBS), reduction of intestinal bacteria after chemotherapy, constipation, lung infections, ulcerative colitis, certain types of diarrhea, necrotizing enterocolitis, a type of intestinal membrane infection caused by harmful bacteria, cystitis, complications of ulcerative colitis, and eczema (Guglielmetti S. et al., Randomised clinical trial: Bifidobacterium bifidum MIMBb75 significantly alleviates irritable bowel syndrome and improves quality of life - a double-blind, placebo-controlled study (2011); Turroni F. et al., Bifidobacterium bifidum PRL2010 modulates the host innate immune response (2014); and Kim JY et al., Effect of probiotic mix in the primary prevention of eczema: a double-blind, randomized, placebo-controlled trial (2010)).

[0005] Recently, it has been demonstrated that vitamins can modulate the human gut microbiota. WO 2020 / 043797 pamphlet discloses that vitamins may be useful for increasing the growth of certain beneficial bacteria in the intestine. However, WO 2020 / 043797 does not describe that vitamins can be used in combination with probiotics to increase the abundance of other beneficial bacteria. Furthermore, the human intestine is a habitat for hundreds of different microorganisms, and it would be desirable to be able to enhance certain beneficial bacteria. In particular, it would be desirable to increase the abundance of Bifidobacterium bifidum in the intestine in order to improve health status, improve health, and support the immune system.

[0006] [Summary of the Invention] The present invention relates to the following items: 1) A combination comprising vitamin 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 or delivery or consumption, preferably the combination is a fixed combination. 4) The combination according to item 1 or item 2, wherein the combination is for sequential administration or delivery or consumption, preferably the combination is a free combination. 5) The combination according to any one of items 1 to 4, wherein the combination is in an oral dosage form, more preferably in a solid oral dosage form. 6) The combination according to any one of items 1 to 5, 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 patients in need of an increase in the abundance of Bifidobacterium bifidum in the large intestine. 9) The combination for use according to item 8, wherein the patient suffers from one or more of the following: Helicobacter pylori (H. pylori) infection, irritable bowel syndrome (IBS), reduction of intestinal bacteria after chemotherapy, constipation, pulmonary infection, ulcerative colitis, diarrhea, necrotizing enteritis, cystitis, and eczema. 10) A combination comprising vitamin C and Bifidobacterium animalis ssp. lactis for use in increasing the abundance of Bifidobacterium bifidum 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. 11) The combination comprising vitamin C and Bifidobacterium animalis ssp. lactis for use according to item 10, wherein vitamin C and Bifidobacterium animalis ssp. lactis are administered or delivered to the large intestine by a sustained release formulation. 12) A combination comprising vitamin C and Bifidobacterium animalis ssp. lactis for use according to item 10 or item 11, wherein said use comprises administering or delivering vitamin C and Bifidobacterium animalis ssp. lactis simultaneously and / or sequentially to an animal, preferably a human. 13) A combination comprising vitamin C and Bifidobacterium animalis ssp. lactis according to any one of items 10 to 12, wherein the animal, including a human, is experiencing one or more conditions selected from the following: Helicobacter pylori (H. pylori) infection, irritable bowel syndrome (IBS), reduction of intestinal bacteria after chemotherapy, constipation, lung infection, ulcerative colitis, diarrhea, necrotizing enterocolitis, cystitis, and eczema. 14) A combination comprising vitamin C and Bifidobacterium animalis ssp. lactis for use according to any one of items 10 to 13, wherein 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

[0008] [Detailed Description of the Invention] Bifidobacterium bifidum is a bacterial species known for its beneficial effects on human health. The inventors have found that the combination of vitamin C with Bifidobacterium animalis ssp. lactis can promote the growth of Bifidobacterium bifidum bacteria in the large intestine and increase the levels of Bifidobacterium bifidum in the intestine.

[0009] Accordingly, in a first aspect, the present invention relates to a combination of vitamin C and Bifidobacterium animalis ssp. lactis. Preferably, the Bifidobacterium animalis ssp. lactis is the Bifidobacterium animalis ssp. lactis BB-12 strain, more preferably Lactobacillus rhamnosus DSM 32550. The combination is for simultaneous and / or sequential administration.

[0010] The claims regarding "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 or equivalent to B. animalis ssp. lactis BB-12®. Accordingly, 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 food, or a nutritional supplement food.

[0019] In another aspect, the present invention relates to a combination of 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 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 Bifidobacterium bifidum in the large intestine. In one embodiment, the patient suffers from one or more of the following conditions: an infection by Helicobacter pylori (H. pylori), irritable bowel syndrome (IBS), a decrease in intestinal bacteria after chemotherapy, constipation, a lung infection, ulcerative colitis, diarrhea, necrotizing enterocolitis (a type of infection of the intestinal lining caused by harmful bacteria), pouchitis (a complication of ulcerative colitis), and eczema.

[0021] In a further aspect, the 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 abundance of Bifidobacterium bifidum in the intestine of the animal. Specifically, vitamin C and Bifidobacterium animalis ssp. lactis are for use in increasing the abundance of Bifidobacterium bifidum 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.

[0022] To achieve an increase in the abundance of Bifidobacterium bifidum 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.

[0023] In a preferred embodiment, the animal (including humans) has experienced one or more conditions selected from the group consisting of: infection by Helicobacter pylori (H. pylori), irritable bowel syndrome (IBS), reduction of intestinal bacteria after chemotherapy, constipation, lung infection, ulcerative colitis, diarrhea, necrotizing enterocolitis (a type of infection of the intestinal lining caused by harmful bacteria), pouchitis (a complication of ulcerative colitis), and eczema.

[0024] Preferably, the Bifidobacterium animalis ssp. lactis used is Bifidobacterium animalis ssp. lactis BB-12. Bifidobacterium animalis ssp. lactis DSM 32269 is particularly preferred.

[0025] In another aspect, the present invention relates to a method of increasing the abundance of Bifidobacterium bifidum 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 including increasing the abundance of Bifidobacterium bifidum 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.

[0026] The method of the present invention can be used to treat, prevent, and / or reduce the symptoms of Helicobacter pylori (H. pylori) infections, irritable bowel syndrome (IBS), reduction of intestinal bacteria after chemotherapy, constipation, lung infections, ulcerative colitis, diarrhea, necrotizing enterocolitis (a type of infection of the intestinal lining caused by harmful bacteria), pouchitis (a complication of ulcerative colitis), and eczema in animals including humans in need thereof.

[0027] 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 abundance of Bifidobacterium bifidum 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: an infection by Helicobacter pylori (H. pylori), irritable bowel syndrome (IBS), reduction of intestinal bacteria after chemotherapy, constipation, lung infection, ulcerative colitis, diarrhea, necrotizing enterocolitis (a type of infection of the intestinal lining caused by harmful bacteria), pouchitis (a complication of ulcerative colitis), and eczema.

[0028] 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 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.

[0029] The dosage of Bifidobacterium animalis ssp. lactis can be up to 5E+10 cfu / day. Preferably, the dosage range is 1E+08 to 1E+10 cfu / day, more preferably 1E+09 to 5E+10 cfu / day. Preferably, Bifidobacterium animalis ssp. lactis is Bifidobacterium animalis ssp. lactis BB-12. Bifidobacterium animalis ssp. lactis DSM 32269 is particularly preferred.

[0030] [Definitions and Embodiments] When used throughout, the following definitions apply.

[0031] The claims regarding "combination" or "pharmaceutical 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).

[0032] "Combination for co - administration" or "combination for co - consumption" are combinations suitable for co - administration or co - consumption respectively. "Co - administration" or "co - 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 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 at the same time.

[0033] "Combinations for continuous administration or consumption" are combinations 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.

[0034] "Fixed combinations" are combinations that deliver both active substances (i.e., vitamins and probiotics) to the patient simultaneously. Solid oral dosage forms (e.g., tablets or capsules) containing both vitamins and probiotics are an example of fixed combinations. Liquid oral dosage forms (e.g., oral drops) containing both vitamins and probiotics are another example of fixed combinations.

[0035] "Free combinations" are combinations in which both active substances (i.e., vitamins and probiotics) can be administered / consumed separately, i.e., one at a time. Treatment regimens in which vitamins and probiotics are not administered / consumed via the same route and / or are not administered / consumed simultaneously require free combinations.

[0036] Simultaneous administration / consumption can be achieved by using both fixed combinations and free combinations. Continuous administration / consumption requires free combinations, 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.

[0037] "Separate administration / consumption" means that vitamins and probiotics are administered / consumed 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 at a time - and can also refer to simultaneous administration / consumption.

[0038] "Administering" or "administration" means imparting or delivering an active substance to a human or animal. Similarly, a human or animal can ingest (consume) the active substance.

[0039] 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.

[0040] "Increasing the abundance of" Bifidobacterium bifidum means increasing the level (or amount, or number, or population size) of Bifidobacterium bifidum compared to each control (i.e., the level / amount / number / population size of Bifidobacterium bifidum when the combination of vitamin C and Bifidobacterium animalis ssp. lactis was not added).

[0041] 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".

[0042] "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 (commonly obtained via diet and conventional vitamin supplementation). For human use, the preferred method according to the invention is via a form that delays release until reaching 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).

[0043] 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 delivery of the vitamin (and / or probiotic) to the large intestine (colon) after oral administration is delayed 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 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.

[0046] An "oral formulation" means that the vitamin and / or probiotic is formulated for oral administration / consumption.

[0047] "Co-administer" or "co-administration" means that the 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.

[0048] [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 daily dosage can range up to 2000 mg / day, preferably 100 - 2000 mg / day; more preferably 200 - 1000 mg / day.

[0049] 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.

[0050] [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 probiotic) available in the large intestine.

[0051] Oral formulations are preferred. Other formulations include those via non-oral routes such as suppositories or injections.

[0052] 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.

[0053] Delayed-release formulations are known in the art. Preferably, the delayed-release formulation has an enteric coating (also referred to as an enteric layer).

[0054] In one embodiment of the invention, the vitamins and / or probiotics, preferably both, are present in a formulation comprising enteric-coated capsules filled with a composition comprising the vitamins and / or probiotics. The enteric capsules confer resistance to the acidic environment of the stomach. For example, soft gel formulations deliver the active agent in solution but can still provide the advantages of a solid dosage form.

[0055] In another embodiment, the formulation is a tablet comprising (i) a core comprising vitamins and / or probiotics and (ii) a delayed-release coating such as an enteric coating. This can be a hard gel capsule.

[0056] Alternatively, for direct colon delivery, a matrix-based delivery system can be used. A matrix-based system does not have individual layers of coating material, but the active agent (i.e., vitamins and / or probiotics) is more or less homogeneously distributed in the matrix. Further, there is a colon release system in which the active agent is embedded in a fiber matrix (enzymatically triggered) and has an enteric coating on top.

[0057] 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).

[0058] In a preferred embodiment for humans, the vitamins and / or probiotics are formulated into 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 release of the active agent before the small intestine, preferably before the colon.

[0059] Coating or matrix materials for delayed release of vitamins and / or probiotics, particularly 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 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 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.

[0060] 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, curdlan, levan, amylopectin, starch, amylose, resistant starch and azo compounds decomposed by bacteria that cleave azo bonds.

[0061] The following non-limiting examples are presented to explain the present invention in more detail.

[0062] [Examples] The purpose of this study was to examine the effect of the combination of vitamin C and Bifidobacterium animalis ssp. lactis on the composition of the intestinal microbiota in a long-term continuous fermentation experiment.

[0063] [Materials and Methods] [Design of the long-term SHIME fermentation experiment (colonic model)] The typical reactor configuration of SHIME® representing the gastrointestinal tract of adult humans was described by Molly et al. (1993) Applied Microbiology and Biotechnology 39(2):254-258. The inoculum preparation, retention time, pH, temperature setting, 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 colonic reactor mimicking the transverse colon (pH 6.15 - 6.4; retention time = 32 hours; volume 800 mL).

[0064] The SHIME® experiment for this study consisted of three stages: 1. Stabilization period: After inoculating the appropriate fecal sample into the colonic 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 SHIME to support the maximum diversity of the intestinal flora 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 the samples during this period made it possible to determine the baseline microbiota composition and activity in different reactors, which was used as a reference for the results obtained during the treatment. 3. Treatment: During this 3-week period, 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 C (ascorbic acid, DSM) was added to the reactors at a dose of 200 mg / day.

[0065] The probiotic strain used in this experiment was Bifidobacterium animalis ssp. lactis BB-12 equivalent Bifidobacterium animalis ssp. lactis DSM 32269 (Biocare Copenhagen).

[0066] [Quantitative microbiota analysis by 16S rRNA gene sequencing and flow cytometry] Samples for quantitative 16S-targeted Illumina sequencing were collected three times a 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 an Illumina MiSeq platform using v3 chemistry. The 341F (50-CCTACGGGNGGCWGCAG-30) and 785R (50-GACTACHVGGGTATCTAAKCC-30) 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) in the dark at 37 °C for 15 min. 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.

[0067] [Result] [Supplementation with a combination of Bifidobacterium animalis ssp. lactis and vitamin C increased the abundance of Bifidobacterium bifidum.] As can be understood from FIG. 1, supplementation with Bifidobacterium animalis ssp. lactis DSM 32269 alone did not significantly change the abundance of Bifidobacterium bifidum in the lumen compared to the control. In contrast, the combination of Bifidobacterium animalis ssp. lactis DSM 32269 and vitamin C significantly increased the abundance of Bifidobacterium bifidum in the lumen compared to the control.

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 who require an increase in the amount of Bifidobacterium bifidum in the colon.

9. The combination for use according to claim 8, wherein the patient suffers from one or more of the following: Helicobacter pylori (H. pylori) infection, irritable bowel syndrome (IBS), decreased intestinal bacteria after chemotherapy, constipation, lung infection, ulcerative colitis, diarrhea, necrotizing enterocolitis, cystitis, and eczema.

10. A combination comprising vitamin C and Bifidobacterium animalis ssp. lactis for use in increasing the abundance of Bifidobacterium bifidum 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.

11. A combination of vitamin C and Bifidobacterium animalis ssp. lactis for use according to claim 10, wherein the vitamin C and Bifidobacterium animalis ssp. lactis are administered or delivered to the large intestine by a delayed-release formulation.

12. A combination comprising vitamin C and Bifidobacterium animalis ssp. lactis for use according to claim 10 or 11, wherein the use comprises administering or delivering the vitamin C and Bifidobacterium animalis ssp. lactis to the animal simultaneously and / or sequentially.

13. A combination comprising vitamin C for use according to claim 10 or 11 and Bifidobacterium animalis ssp. lactis for use according to claim 10 or 11, wherein the animal, including a human, is experiencing one or more conditions selected from: Helicobacter pylori (H. pylori) infection, irritable bowel syndrome (IBS), decreased intestinal bacteria after chemotherapy, constipation, lung infection, ulcerative colitis, diarrhea, necrotizing enterocolitis, cystitis, and eczema.

14. Vitamin C and / or Bifidobacterium animalis ssp. lactis for use according to claim 10 or 11, wherein the Bifidobacterium animalis ssp. lactis is Bifidobacterium animalis ssp. lactis BB-12.

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