Combination containing 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 abundance of Actinobacteria, Bifidobacteriaceae, and Bifidobacterium bacteria in the intestinal tract when delivered to the large intestine.

Description

Detailed Description of the Invention

[0001] [Field of the Invention] The present invention relates to a combination comprising vitamin 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 abundance of beneficial bacteria in the intestinal tract when delivered to the large intestine.

[0002] [Background of the Invention] Increasing evidence indicates that an imbalance in the human gut microbiota (also referred to as "dysbiosis") may be associated with Western diseases including obesity and type 2 diabetes, as well as cardiovascular diseases, autoimmune diseases, and intestinal inflammatory diseases. Therefore, targeted modulation of the human gut microbiota intended to restore the imbalance represents potential therapeutic and preventive strategies and has attracted the attention of not only researchers but also people engaged in various industries. Public awareness and acceptance of substances that modulate the human gut microbiota have been continuously increasing.

[0003] There is a common understanding that certain live microorganisms have beneficial effects on human health. Actinobacteria is one of the four major phyla of the gut microbiota, present in only a small proportion but important for maintaining gut homeostasis. Over the past decade, many studies have focused on Actinobacteria, particularly their roles in both gastrointestinal and systemic diseases, as well as their potential therapeutic uses. In fact, classes of this phylum, particularly Bifidobacteria, are widely used as probiotics that show beneficial effects on many pathological conditions (Binda C., Actinobacteria: A relevant minority for the maintenance of gut homeostasis (2018)).

[0004] Bacteria of the Bifidobacteriaceae family belong to the phylum Actinobacteria. Bifidobacteriaceae exert beneficial effects on human health, including increasing the absorption of human milk proteins by removing casein in human milk, preventing nutrient loss by suppressing the growth of competing bacteria in the intestinal tract, preventing constipation in the host and promoting normal bowel movements by producing acids that promote peristalsis; suppressing the growth of pathogens in the host organism; treating liver damage; preventing the onset of colon cancer; reducing blood cholesterol levels; and improving intestinal function by stimulating the host's immune response. (Bottacini et al, Diversity, ecology and intestinal function of bifidobacteria (2014)). For example, in atopic diseases, irritable bowel syndrome, inflammatory bowel disease, colorectal cancer, and celiac disease, abnormalities in the specific bifidobacterial flora, such as a decrease in number or an atypical species composition, have been confirmed. In addition, dysbiosis of bifidobacteria has been reported to precede obesity (Tojo R., Intestinal microbiota in health and disease: Role of bifidobacteria in gut homeostasis (2014)).

[0005] Bifidobacterium is a genus belonging to the family Bifidobacteriaceae. As an inhabitant of the human intestine, Bifidobacteria form a symbiotic relationship between the bacteria and the host, providing the human host with many health benefits including: reconstructing the normal intestinal flora, improving intestinal and immune functions, preventing nutrient loss, preventing constipation, preventing the onset of colon cancer, treating liver damage, and reducing blood cholesterol levels. Bifidobacteria also have antibiotic activity and have been found to assist the host's anti-tumor activity (Bottacini et al, Diversity, ecology and intestinal function of bifidobacteria (2014)).

[0006] Bifidobacterium adolescentis has been shown to produce folic acid in the colon and can be used to prevent folic acid deficiency in colonic epithelial cells and more effectively protect the colon from inflammation and cancer (Pompei A., Folate Production by Bifidobacteria as a Potential Probiotic Property (2020)). Experiments and tests have shown that Bifidobacterium adolescentis therapy may be an important intervening factor in the process of inflammatory bowel disease (Shah Shinil., Dietary Factors in the Modulation of Inflammatory Bowel Disease Activity (2007)). Furthermore, Bifidobacterium adolescentis suppresses tumor growth and is a suitable and specific means of gene cancer therapy (Xi Li, et al., Bifidobacterium adolescentis as a delivery system of endostatin for cancer gene therapy: Selective inhibitor of angiogenesis and hypoxic tumor growth (2003)).

[0007] In recent years, it has been demonstrated that vitamins may regulate the human gut microbiota. WO 2020 / 043797 pamphlet discloses that vitamins may be useful for increasing the growth of certain beneficial bacteria in the intestine. However, WO 2020 / 043797 pamphlet does not describe that it is possible to use vitamins 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 is desirable to be able to enhance certain beneficial bacteria. In particular, it is desirable to increase the abundance of Actinobacteria, Bifidobacteriaceae, and Bifidobacterium in the intestine in order to enhance health, improve the state of health, and support the immune system.

[0008] [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) Use of the combination according to any one of items 1 to 6 for use as a medicament, health supplement, or nutritional supplement. 8) Use of the combination according to any one of items 1 to 7 for use in the treatment of a patient in need of increasing the abundance of Actinobacteria, Bifidobacteriaceae, and / or Bifidobacterium in the large intestine. 9) The combination for use according to item 8, wherein the patient is in need of increasing the abundance of Bifidobacteriaceae and suffers from an atopic disease, irritable bowel syndrome, inflammatory bowel disease, colorectal cancer, celiac disease, or obesity. 10) The combination for use according to item 8, wherein the patient is in need of increasing the abundance of Bifidobacterium and suffers from constipation, colon cancer, liver injury, or high blood cholesterol. 11) Use of the combination according to any one of items 1 to 7 for use in the treatment of a patient in need of increasing the abundance of Bifidobacterium adolescentis in the large intestine. 12) The combination for use according to item 11, wherein the patient suffers from folate deficiency, inflammatory bowel disease, or colorectal cancer. 13) A combination comprising vitamin C and Lactobacillus (Lacticaseibacillus) rhamnosus for use in increasing the abundance of Actinobacteria, Bifidobacteriaceae, and / or Bifidobacterium in the large intestine of an animal, preferably a human, wherein the use comprises administering or delivering vitamin C and Lactobacillus rhamnosus to the large intestine. 14) A combination for use according to item 13, comprising vitamin C and Lactobacillus rhamnosus, wherein vitamin C and Lactobacillus rhamnosus are administered or delivered to the large intestine by a delayed-release formulation. 15) A combination for use according to item 13 or item 14, comprising vitamin C and Lactobacillus rhamnosus, wherein the use comprises administering or delivering vitamin C and Lactobacillus rhamnosus to an animal, preferably a human, simultaneously and / or sequentially. 16) A combination for use according to any one of items 13 to 15, comprising vitamin C and Lactobacillus rhamnosus, wherein the animal, including a human, is experiencing at least one symptom selected from folate deficiency, atopic diseases, irritable bowel syndrome, inflammatory bowel disease, colorectal cancer, celiac disease, obesity, constipation, liver damage, and high blood cholesterol. 17) A combination for use according to any one of items 13 to 16, comprising vitamin C and Lactobacillus rhamnosus, wherein Lactobacillus rhamnosus is Lactobacillus rhamnosus GG, preferably Lactobacillus rhamnosus DSM 32550.

Brief Description of the Drawings

[0009]

Figure 1

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[0010] [Detailed Description of the Invention] Actinobacteria, Bifidobacteriaceae, and Bifidobacterium are bacteria known to have beneficial effects on human health. The inventors have found that vitamin C combined with Lactobacillus rhamnosus can promote the growth of Actinobacteria, Bifidobacteriaceae, and Bifidobacterium adolescentis in the large intestine, leading to an increase in the levels of Actinobacteria, Bifidobacteriaceae, and Bifidobacterium adolescentis in the intestine.

[0011] Accordingly, in a first aspect, the present invention relates to a combination comprising vitamin C and Lactobacillus (Lacticaseibacillus) rhamnosus. Preferably, Lactobacillus (Lacticaseibacillus) rhamnosus is Lactobacillus rhamnosus GG strain, more preferably Lactobacillus rhamnosus DSM 32550. The combination is for simultaneous and / or sequential administration.

[0012] The claims regarding the "combination" are product claims. The product of the present invention contains two active ingredients, namely a vitamin (vitamin C) and a probiotic (Lactobacillus rhamnosus). For simultaneous and / or sequential administration, refer to the following definitions and embodiments.

[0013] 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.

[0014] 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".

[0015] Alternative Lactobacillus rhamnosus strains are, in particular, Lactobacillus rhamnosus HN001 (Howaru; Danisco / DuPont), Lactobacillus rhamnosus GR-1® (Chr. Hansen, Denmark), and Lactobacillus rhamnosus Rosell-11 (Lallemand, Canada).

[0016] According to the present invention, a preferred strain is Lactobacillus (Lacticaseibacillus) rhamnosus DSM 32550 (Biocare Copenhagen). This strain was deposited with the Leibniz Institute DSMZ - German Collection of Microorganisms and Cell Cultures (Inhoffenstr. 7B, D - 38124 Braunschweig, Germany) on July 6, 2017, under the Budapest Treaty. The accession number assigned by the international depository authority is DSM 32550.

[0017] 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.

[0018] In another embodiment, the combination is for sequential administration. The combination for sequential administration is a free combination.

[0019] Preferably, the combination is in an oral dosage form, more preferably in a solid oral dosage form.

[0020] The combination of the present invention can be, for example, a pharmaceutical combination or composition, a health supplement, or a food supplement.

[0021] 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.

[0022] Preferably, the combination of the present invention (e.g., a pharmaceutical combination) is used for the treatment of patients in need of increasing the abundance of Actinobacteria, Bifidobacteriaceae, and / or Bifidobacterium in the large intestine. In one embodiment, the patient is in need of increasing the abundance of Bifidobacteriaceae and suffers from an atopic disease, irritable bowel syndrome, inflammatory bowel disease, colorectal cancer, celiac disease, or obesity. In another embodiment, the patient is in need of increasing the abundance of Bifidobacterium and suffers from constipation, liver damage, or high blood cholesterol.

[0023] In another preferred embodiment, the (pharmaceutical) combination of the present invention is used for the treatment of patients in need of increasing the abundance of Bifidobacterium adolescentis in the large intestine. Preferably, the patient suffers from folate deficiency, inflammatory bowel disease, and / or colorectal cancer.

[0024] 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 abundance of Actinobacteria, Bifidobacteriaceae and / or Bifidobacterium in the large intestine of the animal. In particular, vitamin C and Lactobacillus rhamnosus are used to increase the abundance of Actinobacteria, Bifidobacteriaceae and / or Bifidobacterium 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. Preferably, the Bifidobacterium that is enhanced is Bifidobacterium adolescentis.

[0025] To achieve an increase in the abundance of Actinobacteria, Bifidobacteriaceae, and / or Bifidobacterium 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 vitamin being absorbed in the stomach and / or small intestine, the vitamin is delivered / administered in such a way that the vitamin and probiotics are delivered to the distal intestine, 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.

[0026] In a preferred embodiment, the animal (including humans) is experiencing a symptom selected from the group consisting of folate deficiency, atopic disease, irritable bowel syndrome, inflammatory bowel disease, colorectal cancer, celiac disease, obesity, constipation, colon cancer, liver damage, and high blood cholesterol.

[0027] Preferably, the Lactobacillus rhamnosus used is Lactobacillus rhamnosus GG. Lactobacillus rhamnosus DSM 32550 is particularly preferred.

[0028] In another aspect, the present invention relates to a method for increasing the abundance of Actinobacteria, Bifidobacteriaceae, and / or Bifidobacterium 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, and the improvement includes increasing the abundance of Actinobacteria, Bifidobacteriaceae, and / or Bifidobacterium 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. Preferably, the Bifidobacterium to be enhanced is Bifidobacterium adolescentis.

[0029] Using the method of the present invention, the symptoms of folate deficiency, atopic diseases, irritable bowel syndrome, inflammatory bowel disease, rectal colon cancer, celiac disease, obesity, constipation, colon cancer, liver damage, and high blood cholesterol in an animal including a human in need thereof can be treated, prevented, and / or alleviated.

[0030] In a further aspect, the present invention relates to the use of vitamin C and Lactobacillus rhamnosus (i.e., a combination of vitamin C and Lactobacillus rhamnosus) for increasing the abundance of Actinobacteria, Bifidobacteriaceae, and / or Bifidobacterium in the large intestine of an animal, preferably a human. Preferably, the use comprises delivering vitamin C and Lactobacillus rhamnosus to the large intestine by a delayed-release formulation. Preferably, the animal, including a human, is experiencing one or more symptoms selected from the group consisting of folate deficiency, atopic diseases, irritable bowel syndrome, inflammatory bowel disease, colorectal cancer, celiac disease, obesity, constipation, colon cancer, liver damage, and high blood cholesterol.

[0031] In the combinations, uses, and methods of the present invention, preferably, the dosage of vitamin C (ascorbic acid) 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 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, most preferably from about 0.2 g / L to about 0.5 g / L.

[0032] The dosage of Lactobacillus rhamnosus can be up to 5E+10 cfu / day. Preferably, the dosage range is 1E+08 to 1E+10 cfu / day, more preferably 1E+09 to 5E+10 cfu / day. Preferably, Lactobacillus rhamnosus is Lactobacillus rhamnosus GG. Lactobacillus rhamnosus DSM 32550 is particularly preferred.

[0033] [Definitions and Embodiments] When used throughout, the following definitions apply.

[0034] Claims related to "combination" or "pharmaceutical combination" are product claims. The product of the present invention contains two active ingredients, namely vitamin (vitamin C) and probiotics (Lactobacillus rhamnosus).

[0035] "Combination for simultaneous administration" or "combination for simultaneous ingestion" refers to a combination suitable for simultaneous administration or simultaneous ingestion respectively. "Simultaneous administration" or "simultaneous ingestion" means that the vitamin and the 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 the vitamin is administered / ingested with one pill or tablet, the probiotics can be administered / ingested with another pill or tablet, but both pills / tablets are administered / ingested within 24 hours. In another example, the vitamin and the probiotics are formulated in the same composition and are administered / ingested exactly at the same time.

[0036] "Combination for continuous administration or continuous intake" refers to a combination suitable for continuous administration or continuous intake respectively. "Continuous administration" or "continuous intake" means that during a period of continuous treatment for two or more days, only one of vitamins and probiotics is administered / intaken on any given day. As an example, vitamins can be administered / intaken on the first day, and probiotics can be administered / intaken only on the next day (i.e., after more than 24 hours) or thereafter. The active ingredients can be administered / intaken in any order.

[0037] "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.

[0038] "Free combination" refers to a combination that allows both active substances (i.e., vitamins and probiotics) to be administered / intaken individually, i.e., one by one at a time. A treatment regimen in which vitamins and probiotics are not administered / intaken via the same route and / or not simultaneously requires a free combination.

[0039] Simultaneous administration / simultaneous intake can be achieved by using both fixed combination and free combination. Continuous administration / continuous intake requires a free combination, and a fixed combination is not suitable for continuous administration / continuous intake. Therefore, a free combination is more versatile and suitable for continuous administration / continuous intake, and is also suitable for simultaneous administration / simultaneous intake if both active substances are to be administered / intaken on the same day. A fixed combination is only suitable for simultaneous administration / simultaneous intake when both components (i.e., vitamins and probiotics) are to be administered / intaken simultaneously on the same day. However, a fixed combination is not suitable when vitamins and probiotics are to be administered / intaken individually on the same day.

[0040] "Individual administration / individual intake" means administering / intaking vitamins and probiotics one by one at a time. Therefore, individual administration / individual intake means both continuous administration / continuous intake, and may also mean simultaneous administration / simultaneous intake when it is mentioned that both active substances are administered / intaken one by one on the same day although it is at the same time.

[0041] "To administer" or "administration" means giving or delivering an active substance to a human or an animal, and similarly means that a human or an animal can take in (intake) the active substance.

[0042] The term "vitamin C" can be used interchangeably with "ascorbic acid", and includes its pharmacologically acceptable salts (such as sodium ascorbate and calcium ascorbate), its pharmacologically acceptable esters (especially ascorbyl palmitate), and other pharmacologically acceptable forms.

[0043] "Increasing the abundance of" Actinobacteria, Bifidobacteriaceae, Bifidobacterium, or Bifidobacterium adolescentis means increasing the level (or amount, or number, or population size) of Actinobacteria, Bifidobacteriaceae, Bifidobacterium, or Bifidobacterium adolescentis, respectively, compared to their respective controls (i.e., the levels / amounts / numbers / population sizes of Actinobacteria, Bifidobacteriaceae, Bifidobacterium, or Bifidobacterium adolescentis in the absence of the combination of vitamin C and Lactobacillus rhamnosus).

[0044] 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 the lower part of the gastrointestinal tract and is also referred to herein as the "colon".

[0045] "Direct delivery" or "delivered directly" means that the vitamin is formulated in such a way that it is made available not in the stomach and / or small intestine but rather in the distal intestine, preferably the large intestine (colon), where it can be utilized by the microbiota. The vitamin is administered in an amount that exceeds rather than being part of the normal daily human nutritional requirements (normally 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 in the large intestine, although this latter delivery method is less preferred but 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 made available not in the stomach and / or small intestine but rather in the distal intestine, preferably the large intestine (colon).

[0046] 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.

[0047] An "enteric layer" or "enteric coating" is a layer surrounding a core that contains an active agent and imparts resistance to gastric juice.

[0048] "Prevent" may 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.

[0049] An "oral formulation" means that the vitamin and / or probiotic is formulated for oral administration / oral intake.

[0050] "Co-administered" or "co-administration" means that vitamins and / or probiotics are delivered / administered / ingested simultaneously (i.e., together), or separately but within a 24-hour time frame. The vitamins may be delivered / administered / ingested first. Similarly, the probiotics may be delivered / administered / ingested first.

[0051] "Lactobacillus rhamnosus" has recently been renamed "Lacticaseibacillus rhamnosus", and both names are used interchangeably herein and can both be abbreviated as "L. rhamnosus".

[0052] [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.

[0053] 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.

[0054] [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.

[0055] Oral formulations are preferred. Other formulations include parenteral routes, such as by suppository or injection.

[0056] 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.

[0057] Delayed-release formulations are known in the art. Preferably, the delayed-release formulation has an enteric coating (also referred to as an enteric layer).

[0058] 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 can also provide the advantages of a solid dosage form.

[0059] 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 also be a hard gel capsule.

[0060] Alternatively, a matrix-based delivery system can be used to deliver directly to the colon. In matrix-based systems, there are no individual layers of coating material, but the active agents (i.e., vitamins and / or probiotics) are distributed somewhat homogeneously within the matrix. Further, there are colon release systems in which, for example, the active agents are embedded in a fiber matrix (induced by enzymes) and the outermost layer is an enteric coating.

[0061] 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).

[0062] 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 a capsule, pellet, bead, sphere, microsphere, tablet, mini-tablet or granule optionally coated with a delayed release coating that prevents the release of the active agent before the small intestine, preferably before the colon.

[0063] 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 flora of 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 coating materials that disintegrate pH-dependently, coating materials that disintegrate time-dependently, coating materials that disintegrate by enzyme induction in the intestinal environment (e.g., the intestinal environment of the ileum and large intestine), and combinations thereof.

[0064] 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 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.

[0065] To better explain the present invention, the following non-limiting examples are shown.

[0066] [Examples] The purpose of this test was to examine the effect of the combination of vitamin C and Lactobacillus rhamnosus on the composition of the intestinal flora in a long-term continuous fermentation experiment.

[0067] [Materials and Methods] [Design of Long-Term SHIME Fermentation Experiment (Colon Model)] The typical reactor settings of SHIME® representing the human adult gastrointestinal tract were described in Molly et al. (1993) Applied Microbiology and Biotechnology 39(2):254-258. The preparation of the inoculum, retention time, pH, temperature settings, and reactor feed composition were previously described in Possemiers et al. (2004) FEMS Microbiol Ecol. 49(3):495-507. Compared to the typical settings of SHIME®, several modifications were included in the long-term SHIME experiment used in this example. In one reactor, the gastric conditions were first simulated and then changed by the computer to conditions simulating the small intestine. Then, the suspension was added to the large intestine reactor mimicking the transverse colon (pH 6.15 - 6.4; retention time = 32 h; volume 800 mL).

[0068] The SHIME® experiment of this study consisted of the following three stages: 1. Stabilization period: After inoculating the colon reactor with an appropriate fecal sample, a 2-week stabilization period was used to differentiate the microbiota in different reactors according to the local environmental conditions. During this period, a basic nutrient matrix for SHIME was provided to assist in maximizing the diversity of the gut microbiota originally present in the fecal inoculum. 2. Control period: During this 2-week reference period, the standard SHIME nutrient matrix for this model was further administered for a 14-day period. By analyzing the samples during this period, it was possible to determine the composition and activity of the baseline microbiota in different reactors, which was used as a reference for the results obtained during treatment. 3. Treatment: During this 3-week period, SHIME was operated under normal 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 per reactor. Vitamin C (ascorbic acid, DSM) was added to the reactors at a dose of 200 mg / day.

[0069] The probiotic strain used in this experiment was an equivalent of Lactobacillus rhamnosus GG, Lactobacillus rhamnosus DSM 32550 (Biocare Copenhagen).

[0070] 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® for practical purposes. Thus, in the examples herein, Lactobacillus rhamnosus DSM 32550 is referred to as Lactobacillus rhamnosus GG.

[0071] [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 last week of both the control and treatment periods. 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 (5′-CCTACGGGNGGCWGCAG-3′) primer and 785R (5′-GACTACHVGGGTATCTAAKCC-3′) primer were used as described by De Paepe et al. (2017), along with a reverse primer 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 confirmed 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 quantitative data was obtained 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 for 15 min at 37 °C in the dark with 0.01 mM SYTO24 (Life Technologies Europe, Merelbeke, Belgium). Samples were analyzed on a BD Facsverse (BD Biosciences, Erembodegem, Belgium) using a high flow rate setting, and bacteria were separated from the medium, debris, and signal noise by applying a threshold level of 200 in the SYTO channel.

[0072] [Results] [The abundance of Actinobacteria increased by supplementing with a combination of Lactobacillus rhamnosus GG and vitamin C] As can be read from Figure 1, even when supplemented with only an equivalent of Lactobacillus rhamnosus GG, the abundance of Actinobacteria did not change significantly compared to the control. In contrast, with the combination of Lactobacillus rhamnosus GG and vitamin C, the abundance of Actinobacteria increased significantly compared to the control.

[0073] [The abundance of Bifidobacteriaceae increased by supplementing with a combination of Lactobacillus rhamnosus GG and vitamin C] As can be read from Figure 2, even when supplemented with only an equivalent of Lactobacillus rhamnosus GG, the abundance of Bifidobacteriaceae did not change significantly compared to the control. In contrast, with the combination of Lactobacillus rhamnosus GG and vitamin C, the abundance of Bifidobacteriaceae increased significantly compared to the control.

[0074] [The abundance of Bifidobacterium adolescentis increased by supplementing with a combination of Lactobacillus rhamnosus GG and vitamin C] As can be read from FIG. 3, even when only the equivalent of Lactobacillus rhamnosus GG was supplemented, the abundance of Bifidobacterium adolescentis did not change significantly compared to the control. In contrast, in the combination of Lactobacillus rhamnosus GG and vitamin C, the abundance of Bifidobacterium adolescentis increased significantly compared to the control.

Claims

1. i) Vitamin C and ii) Combinations that include Lactobacillus rhamnosus.

2. The aforementioned combination is i) Vitamin C and ii) The combination according to claim 1, comprising Lactobacillus rhamnosus GG.

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

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

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

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 need to increase the abundance of Bifidobacteriaceae and / or Bifidobacterium in the large intestine.

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

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

11. The combination for use according to claim 10, wherein the patient suffers from folic acid deficiency, inflammatory bowel disease, or colorectal cancer.

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

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

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

15. A combination for use according to claim 12 or 13, comprising vitamin C and Lactobacillus rhamnosus, wherein the animal, including a human, is experiencing at least one condition selected from folate deficiency, atopic disease, irritable bowel syndrome, inflammatory bowel disease, colorectal cancer, celiac disease, obesity, constipation, colon cancer, liver injury, and high blood cholesterol.

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