Weissella confusa wilac d001 strain having intestinal health-improving efficacy and composition for improving intestinal health comprising same

Abstract

The present invention relates to a Weissella confusa WIKIM51 (Wilac D001) strain having intestinal health-improving efficacy, and a composition for improving intestinal health or a composition for preventing or treating intestinal health-related diseases comprising same. In the present invention, it was confirmed that the Weissella confusa WIKIM51 (Wilac D001) strain, which is a kimchi-derived strain, has an effect of improving bowel movements in a constipation-induced mouse model, has an effect of recovering from damage to large intestine and small intestine tissues, and has an effect of increasing beneficial intestinal bacteria. Therefore, according to the present invention, the composition comprising the kimchi-derived Weissella confusa WIKIM51 strain as an active ingredient is expected to be usefully employed as a pharmaceutical and functional food composition for improving intestinal health or preventing or treating intestinal health-related diseases.

Description

Weissella Confusa WILAC D001 strain having intestinal health improvement efficacy and a composition for improving intestinal health containing the same

[0001] The present invention relates to a Weissella confusa WIKIM51 (Wilac D001) strain having an effect on improving intestinal health and a composition for improving intestinal health containing the same, and more specifically, to a Weissella confusa WIKIM51 (Wilac D001) strain having an effect on improving intestinal health and a composition for improving intestinal health or preventing or treating intestinal health-related diseases containing the same.

[0002] Recently, due to the Westernization of dietary habits, the intake of dietary fiber among modern people has decreased, and the consumption of high-calorie foods such as meat and instant foods containing high fat and protein has increased. Furthermore, as lack of exercise and the accumulation of stress become more frequent, intestinal function is deteriorating and the number of patients with constipation is increasing.

[0003] The large intestine plays the role of breaking down food that has passed through the small intestine using a bacterial flora to absorb nutrients such as water, short-chain fatty acids, and vitamins, and to excrete the remaining waste as feces. In other words, the regulation of large intestine function refers to a series of actions that maintain and promote intestinal motility and bowel movements in the human body, as well as improve digestion and absorption. When the function of the large intestine deteriorates, diseases such as abdominal pain, difficulty defecating, constipation, diarrhea, malnutrition, ulcerative colitis, diverticulitis, or hemorrhoids may occur due to reduced intestinal motility, damage or inflammation of the intestinal mucosa, and an imbalance of intestinal microorganisms (dysbiosis).

[0004] Constipation refers to a condition where normal bowel movements do not occur. It describes a state where stools become hard and dry compared to when one is healthy, and the frequency and volume of bowel movements decrease, accompanied by discomfort or physiological disorders. Generally, constipation can be diagnosed if bowel movements occur two times or fewer per week or if the daily stool volume is 30 g or less. In cases of constipation, not only is the abdomen constantly distended, but toxins from unexcreted stool are absorbed into the intestines and then into the bloodstream. This accelerates skin aging and can lead to headaches, acne, and skin rashes. In severe cases, it can cause anal fissures and hemorrhoid prolapse during defecation. Furthermore, since constipation is a cause of various serious secondary diseases such as bad breath, colorectal diseases like colon cancer, arteriosclerosis, hypertension, stroke, and immunodeficiency, active prevention and treatment are necessary.

[0005] The intestinal microbiome within the human body is a collective of intestinal microorganisms and is a biologically important component of the body. It consists of hundreds of different types of microorganisms, and individual microorganisms can cause harm or benefit to the host through their ability to produce or decompose substances. Since the intestinal microbiome as a whole is involved in vitamin supply, infection prevention, and bowel function (peristalsis and absorption), the composition of the intestinal microbiome is known to be closely related to constipation and the occurrence of intestinal-related diseases.

[0006] Recently, various pharmaceuticals and functional foods designed to improve gut microbiota or relieve constipation are being sold, but there are issues with their temporary effects and the various side effects they cause depending on the type. For example, when using irritant agents containing anthraquinone derivatives such as senna and rhubarb, side effects such as abdominal pain and diarrhea occur, and problems regarding their use during pregnancy or continuous consumption have been pointed out.

[0007] Accordingly, through the present invention, we aim to obtain data evaluating the gut health improvement efficacy of the Weissella confusa WIKIM51 (Wilac D001) strain, and based on this, conduct human clinical trials to achieve commercialization through recognition as a health functional food ingredient.

[0008] This research was conducted with support from the Ministry of Trade, Industry and Energy and the Korea Institute for Industrial Technology Advancement (KIAT) under the "Global Competitiveness Enhancement Project for Bioactive Agents" (Project No.: B0080207002074), and is filed with the support of the Korea Bio-Specialized Center Association.

[0009] Therefore, the technical problem to be solved by the present invention is to provide a strain derived from fermented food that has the efficacy of improving intestinal health.

[0010] In addition, another technical problem to be solved by the present invention is to provide a composition for improving intestinal health comprising the strain having the above-mentioned efficacy for improving intestinal health as an active ingredient.

[0011] Another technical problem to be solved by the present invention is to provide a composition for improving constipation that includes the strain as an active ingredient.

[0012] Another technical problem to be solved by the present invention is to provide a composition for repairing damage to large and small intestine tissues comprising the above-mentioned strain as an active ingredient.

[0013] In addition, another technical problem to be solved by the present invention is to provide a composition for increasing beneficial intestinal bacteria comprising the above strain as an active ingredient.

[0014] In addition, another technical problem to be solved by the present invention is to provide a composition for the prevention or improvement of intestinal health-related diseases comprising the above-mentioned strain as an active ingredient.

[0015] Another technical problem to be solved by the present invention is to provide a method for improving intestinal health using a composition containing the strain.

[0016] Another technical problem to be solved by the present invention is to provide a method for improving constipation using a composition containing the strain.

[0017] Another technical problem to be solved by the present invention is to provide a method for restoring damage to large and small intestine tissues using a composition containing the strain.

[0018] Another technical problem to be solved by the present invention is to provide a method for increasing beneficial bacteria in the intestines using a composition.

[0019]

[0020] In order to solve the above-mentioned technical problem, the present invention provides a strain Weissella confusa WIKIM51 (accession number: KCCM11914P) having an effect on improving gut health.

[0021] The above strain Weissella Confusa WIKIM51 is named Wilac D001 and is characterized by being derived from kimchi.

[0022] In order to solve the other technical problems mentioned above, the present invention provides a composition for improving gut health characterized by including the above Weissella Confusah WIKIM51 as an active ingredient.

[0023] In order to solve the other technical problems mentioned above, the present invention provides a composition for the prevention or treatment of intestinal health-related diseases characterized by including the above Weissella Confusah WIKIM51 as an active ingredient.

[0024] The above composition may be a pharmaceutical composition, a functional food composition, or a feed composition.

[0025] As such, the present invention confirmed that the kimchi-derived strain Weissella Confusa WIKIM51 (Wilac D001) has an effect of improving bowel movements in a mouse model of constipation induction, and also has an effect of restoring damage to large and small intestine tissues and increasing beneficial bacteria in the intestines. Therefore, according to the present invention, a composition containing the kimchi-derived Weissella Confusa WIKIM51 (Wilac D001) strain as an active ingredient is expected to be useful as a pharmaceutical and functional food composition for improving intestinal health or preventing or treating intestinal health-related diseases.

[0026] The following drawings attached to this specification illustrate preferred embodiments of the present invention and serve to further enhance understanding of the technical concept of the present invention together with the aforementioned description; therefore, the present invention should not be interpreted as being limited only to the matters described in such drawings.

[0027] Figure 1 shows the results of a cytotoxicity test of the Weissella Confusa WIKIM51 (Wilac D001) strain.

[0028] Figure 2 shows the change in mRNA expression levels of Human β-defensin 1 (HBD-1) and HBD-2 by the Weissella Confusa WIKIM51 (Wilac D001) strain.

[0029] Figure 3 shows the change in stool pH caused by the Weissella Confusa WIKIM51 (Wilac D001) strain in loperamide-induced mice.

[0030] Figure 4 shows the changes in stool parameters caused by the Weissella Confusa WIKIM51 (Wilac D001) strain in loperamide-induced mice.

[0031] Figure 5 shows the change in colon transit time by the Weissella Confusa WIKIM51 (Wilac D001) strain in loperamide-induced mice.

[0032] Figure 6 shows the gastrointestinal migration rate of the Weissella Confusa WIKIM51 (Wilac D001) strain in loperamide-induced mice.

[0033] Figure 7 shows the results of histopathological analysis of the small intestine by the Weissella Confusa WIKIM51 (Wilac D001) strain in loperamide-induced mice.

[0034] Figure 8 shows the results of the pathological analysis of the colon by the Weissella Confusa WIKIM51 (Wilac D001) strain in loperamide-induced mice.

[0035] Figure 9 shows the results of microbial community analysis by the Weissella Confusa WIKIM51 (Wilac D001) strain in loperamide-induced mice.

[0036] Figure 10 shows the differential abundance of loperamide-induced mouse taxa.

[0037] The present invention will be explained in more detail below.

[0038] The present invention provides a strain Weissella confusa WIKIM51 (Wilac D001) having intestinal health benefits.

[0039] The above Weissella confusa WIKIM511 (Wilac D001) strain is characterized by being derived from kimchi, and the above Weissella confusa WIKIM51 strain was deposited with the Korean Culture Collection of Microorganisms on October 12, 2016, and was assigned deposit number KCCM11914P.

[0040] As a result of 16S rDNA sequencing analysis for the identification and classification of the above Weissella confusa WIKIM51 (Wilac D001) strain, it was found to have the nucleic acid sequence of SEQ ID NO. 1.

[0041] Table 1 below shows the 16S rDNA sequence (1432 bp) of the Weissella confusa WIKIM51 (Wilac D001) strain.

[0042]

[0043] The Weissella Confusa WIKIM51 (Wilac D001) of the present invention is a Gram-positive bacterium and a facultative anaerobe capable of growing under both aerobic and anaerobic conditions; it does not form spores, is non-motile, and takes the form of a rod-shaped bacterium. In the present invention, it was confirmed that the Weissella Confusa WIKIM51 (Wilac D001) strain, which is a kimchi-derived strain, has an effect of improving bowel movements in a mouse model of constipation induction, an effect of restoring damage to large and small intestine tissues, and an effect of increasing beneficial bacteria in the intestines.

[0044] The present invention provides a composition for improving gut health characterized by including the above-mentioned Weissella Confusah WIKIM51 (Wilac D001) as an active ingredient.

[0045] In this invention, the term "improvement of intestinal health" refers to enhancing the functions of the intestines, such as digestion, absorption, excretion, and immune regulation. The intestines are responsible for the digestion of food and the absorption of nutrients, and as an important organ where a significant portion of the body's immune cells reside, they have a major impact on overall health. When intestinal health is improved, digestion and bowel movements become smooth, and the balance of intestinal microorganisms is maintained, allowing beneficial bacteria to proliferate while harmful bacteria are suppressed. This creates a healthy intestinal environment, strengthens immunity, and reduces inflammatory responses, thereby helping to prevent or manage various diseases. Improving intestinal health plays a crucial role in promoting overall physical health and preventing diseases, extending beyond mere digestive health.

[0046] According to one embodiment of the present invention, the composition for improving intestinal health of the present invention is characterized by having an effect of improving constipation, restoring damage to large and small intestine tissues, and also increasing beneficial bacteria in the intestines.

[0047] In addition, the present invention provides a composition for preventing or treating intestinal health-related diseases, characterized by including the above-mentioned Weissella Confusa WIKIM51 (Wilac D001) strain as an active ingredient.

[0048] The above-mentioned intestinal health-related diseases include irritable bowel syndrome, inflammatory bowel disease, leaky gut syndrome, intestinal bacterial imbalance, constipation, diarrhea, small intestinal bacterial overgrowth, colorectal cancer, intestinal obstruction, food allergies and intolerances, diverticulitis, lactose intolerance, etc.

[0049] The aforementioned Irritable Bowel Syndrome is accompanied by abdominal pain and bowel dysfunction, with stress and dietary habits cited as major factors. Inflammatory Bowel Disease (IBD) is a condition characterized by chronic inflammation of the intestinal wall, such as Crohn's disease and ulcerative colitis. Leaky Gut Syndrome involves abnormally high permeability of the intestinal wall, allowing toxins to enter the bloodstream, which can trigger inflammation and autoimmune diseases. Gut bacterial imbalance disrupts the balance between beneficial and harmful bacteria, leading to indigestion or weakened immunity. Constipation and diarrhea refer to abnormal bowel movements, manifesting as reduced frequency or loose stools, respectively. Small intestinal bacterial overgrowth causes indigestion and abdominal bloating due to the excessive proliferation of bacteria in the small intestine, while colorectal cancer is a malignant tumor occurring in the large intestine, with changes in bowel habits and weight loss being the primary symptoms. Intestinal obstruction occurs when the intestine becomes blocked, making the passage of food impossible and accompanied by severe abdominal pain and vomiting. Food allergies and intolerances refer to conditions where the intestines overreact or fail to digest specific foods upon consumption. Diverticulitis is a condition in which inflammation occurs in small pouches formed in the intestinal wall, causing abdominal pain and fever, while lactose intolerance causes abdominal pain and diarrhea after consuming milk or dairy products due to a lack of the enzyme that breaks down lactose.

[0050] The above composition may be a pharmaceutical composition, a functional food composition, or a feed composition.

[0051] The Weissella Confusa WIKIM51 (Wilac D001) strain included in the composition according to the present invention may exist as a live cell or a dead cell, and may also exist in a dried or freeze-dried form. Forms of strains suitable for inclusion in various compositions and methods of formulation are well known to those skilled in the art.

[0052] The composition of the present invention may further include a suitable carrier, excipient, or diluent commonly used in addition to the active ingredient.

[0053] The pharmaceutical composition according to the present invention may include a pharmaceutically acceptable carrier in addition to the Weissella Confusa WIKIM51 (Wilac D001) strain. The pharmaceutically acceptable carrier included in the pharmaceutical composition of the present invention is one that is commonly used in formulations and includes, but is not limited to, lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia gum, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methylcellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and mineral oil. In addition to the above components, the pharmaceutical composition of the present invention may further include a lubricant, a wetting agent, a sweetener, a flavoring agent, an emulsifier, a suspending agent, a preservative, etc. Suitable pharmaceutically acceptable carriers and formulations are described in detail in Remington's Pharmaceutical Sciences (19th ed., 1995).

[0054] The pharmaceutical composition of the present invention may be administered orally or parenterally, preferably by parenteral administration, and more preferably by topical application.

[0055] Suitable dosages of the pharmaceutical composition of the present invention may be prescribed in various ways depending on factors such as the formulation method, mode of administration, patient's age, body weight, gender, pathological condition, diet, time of administration, route of administration, excretion rate, and response sensitivity. The dosage of the pharmaceutical composition of the present invention is generally within the range of 0.001 to 1000 mg / kg, preferably 0.05 to 200 mg / kg, and more preferably 0.1 to 100 mg / kg for adults. In addition, in the case of a topical preparation, it is preferable to apply an amount of 1.0 to 3.0 ml once to five times a day for at least one month for adults. However, the above dosages do not limit the scope of the present invention.

[0056] The pharmaceutical composition of the present invention may be prepared in a unit dose form or contained in a multi-dose container by formulation using a pharmaceutically acceptable carrier and / or excipient according to a method that can be easily carried out by a person skilled in the art. The formulation may be in the form of a solution, suspension, syrup, or emulsion in an oil or aqueous medium, or may be in the form of an excipient, powder, powder, granule, tablet, or capsule, and may additionally include a dispersant or a stabilizer.

[0057] The term "health functional food" as used in the present invention refers to a food manufactured and processed in the form of tablets, capsules, powders, granules, liquids, pills, etc., using raw materials or ingredients that have functional properties useful to the human body. Here, "functional properties" means obtaining effects useful for health purposes, such as regulating nutrients or physiological actions regarding the structure and function of the human body. The health functional food of the present invention can be manufactured by methods commonly used in the industry, and can be manufactured by adding raw materials and ingredients commonly added in the industry. Furthermore, unlike general pharmaceuticals, it has the advantage of being made from food ingredients, thus avoiding side effects that may occur with long-term use of pharmaceuticals, and can be highly portable.

[0058] The food composition according to the present invention may additionally include not only the Weissella Confusa WIKIM51 strain as an active ingredient, but also ingredients that are typically added during food manufacturing, such as protein, carbohydrates, fats, nutrients, seasonings, and flavorings.

[0059] Examples of the above carbohydrates are monosaccharides, e.g., glucose, fructose, etc.; disaccharides, e.g., maltose, sucrose, oligosaccharides, etc.; and polysaccharides, e.g., dextrin, cyclodextrin, etc., which are conventional sugars and sugar alcohols such as xylitol, sorbitol, erythritol, etc. As flavoring agents, natural flavoring agents [taumatin, stevia extract (e.g., rebaudioside A, glycyrrhizin, etc.)] and synthetic flavoring agents (saccharin, aspartame, etc.) may be used.

[0060] For example, when the food composition of the present invention is prepared as a drink, in addition to the Weissella Confusa WIKIM51 (Wilac D001) strain of the present invention, citric acid, liquid fructose, sugar, glucose, acetic acid, malic acid, fruit juice, Eucommia ulmoides extract, jujube extract, licorice extract, etc. may be additionally included.

[0061] The composition of the present invention may be a feed composition for improving intestinal health characterized by including the Weissella Confusa WIKIM51 (Wilac D001) strain as an active ingredient.

[0062] In the present invention, the term "feed" refers to any natural or artificial prescribed food, single meal, etc., or the components of said single meal, intended for or suitable for animals to eat, consume, and digest.

[0063] The types of the above feed are not particularly limited, and feeds commonly used in the relevant technical field may be used. Non-limiting examples of the above feed include plant-based feeds such as grains, root vegetables, food processing by-products, algae, fibers, pharmaceutical by-products, oils and fats, starches, meal, or grain by-products; and animal-based feeds such as proteins, inorganic substances, oils and fats, minerals, oils and fats, single-cell proteins, zooplankton, or food waste. These may be used individually or in a mixture of two or more types.

[0064] The above feed composition may include a feed additive. The feed additive of the present invention corresponds to a supplementary feed under the Feed Management Act.

[0065] Another aspect of the present invention provides a method for improving intestinal health, comprising the step of administering a composition of the present invention to an individual.

[0066] The composition of the present invention is not particularly limited to any individual intended for improving intestinal health and is applicable to any individual. For example, any non-human animal such as monkeys, dogs, cats, rabbits, guinea pigs, rats, mice, cattle, sheep, pigs, goats, etc., as well as birds and fish, may be used. The composition may be administered parenterally, subcutaneously, intraperitoneally, intrapulmonaryly, and intranasally, and for local treatment, may be administered by a suitable method including administration into the lesion if necessary. The preferred dosage of the composition of the present invention depends on the individual's condition and body weight, the severity of the disease, the drug form, the route of administration, and the duration, but can be appropriately selected by a person skilled in the art. For example, it may be administered orally, rectally or intravenously, intramuscularly, subcutaneously, intrathecally, or intracerebrovascularly, but is not limited thereto.

[0067] In the present invention, the term "individual" refers to all animals excluding humans, and the individual can be efficiently treated by administering the composition of the present invention.

[0068] In the present invention, the term "administration" means introducing the composition of the present invention into an individual by any appropriate method. The route of administration may be various oral or parenteral routes as long as it can reach the target tissue, as described above.

[0069] As such, the present invention confirmed that the kimchi-derived strain Weissella Confusa WIKIM51 (Wilac D001) has an effect of improving bowel movements in a mouse model of constipation induction, and also has an effect of restoring damage to large and small intestine tissues and increasing beneficial bacteria in the intestines. Therefore, according to the present invention, a composition containing the kimchi-derived Weissella Confusa WIKIM51 (Wilac D001) strain as an active ingredient is expected to be useful as a pharmaceutical and functional food composition for improving intestinal health or preventing or treating intestinal health-related diseases.

[0070]

[0071] The present invention will be described in more detail below through examples. These examples are merely illustrative of the present invention, and the scope of the present invention should not be interpreted as being limited by these examples.

[0072] <Example 1> Isolation and Identification of Weissella confusaWIKIM51 (Wilac D001) Strain

[0073] Single bacterial colonies obtained by plating the undiluted kimchi extract onto MRS medium were collected using a loop and cultured in MRS broth. DNA extraction was performed using the QIAamp DNA Mini Kit (QIAgen, Germany). The extracted DNA was verified using a 1% agarose gel, and PCR was conducted using the extracted genomic DNA as a template to amplify the 16S rDNA gene. The PCR conditions were set as follows: denaturation at 95°C for 1 minute, annealing at 45°C for 1 minute, and extension at 72°C for 1 minute and 30 seconds, for a total of 30 cycles. The sequences of the obtained PCR products were sent to Macrogen (Seoul, Korea) for analysis. Bacterial identification was performed by analyzing the 16S rDNA sequences using the Basic Local Alignment Search Tool (BLAST) of the National Center for Biotechnology Information (NCBI, www.ncbi.nlm.nih.gov).

[0074] The Weissella Confusa WIKIM51 (Wilac D001) of the present invention is a Gram-positive bacterium and a facultative anaerobe capable of growing under both aerobic and anaerobic conditions; it does not form spores, is non-motile, and takes the form of a rod-shaped bacterium.

[0075] The strain isolated through the embodiment of the present invention was found to have the nucleic acid sequence of SEQ ID NO. 1 as a result of 16S rDNA sequencing analysis for the identification of the microorganism.

[0076]

[0077] Accordingly, it was confirmed that the strain from which the microorganism of the present invention was obtained is a strain of the genus Weissella. The isolated strain was named Weissella confusa WIKIM51 and was deposited with the Korean Culture Collection of Microorganisms on October 12, 2016, and assigned deposit number KCCM11914P. Hereinafter, Weissella confusa WIKIM51 will be referred to as Wilac D001.

[0078] <Test Example>

[0079] Experimental method

[0080] 1. Cell experiment

[0081] 1) Caco-2 cell culture

[0082] The human colorectal adenocarcinoma cell line Caco-2, which proliferates similarly to human small intestinal epithelial cells, was purchased from ATCC (Manassas, USA) and cultured in DMEM (Corning, USA) medium containing 10% FBS (GIBCO, USA) and 1% Antibiotic-Antimycotic (GIBCO, USA).

[0083] 2) Measurement of cell viability

[0084] Caco-2 cells were seeded into a 96-well plate at a density of 1.5 × 10⁴ cells / mL and cultured for 24 hours in a 5% CO₂ incubator at 37°C. Wilac D001 was administered at various concentrations (1 × 10⁴). 5 , 5 × 10 5 , 1 × 10 6 , 5 × 10 6 , 1 × 10 7 Cell viability was measured using CCK-8 reagent after treatment with CFU / g for 24 hours.

[0085] 3) RNA extraction and measurement of mRNA expression levels

[0086] The mRNA expression levels of the antimicrobial peptides HBD-1 and HBD-2 were measured using qRTPCR. Caco-2 cells were seeded in a 6-well plate at a ratio of 3 × 10⁶ 5 Cells were seeded at a density of cells / mL and cultured for 24 hours in a 37°C, 5% CO2 incubator. Wilac D001 (1 × 10⁶) was used in antibiotic-free medium. 5 , 1 × 10 6 , 1 × 10 7 (CFU / g) was treated. After incubation for 24 hours, total RNA was extracted from the cells using the Easy-spin™ total RNA extraction kit (iNtRON Biotechnology, Korea), and the RNA was quantified at a concentration of 50 ng / μL. cDNA was synthesized using GoScript Reverse Transcriptase (Promega, USA), and the target gene was amplified by reacting each primer with TB Green Premix Ex Taq II (TaKaRa Bio, Japan). mRNA expression levels were quantified and analyzed using the QuantStudio 3 real-time PCR instrument (Thermo Fisher Scientific, USA), and standardized to β-actin during quantification. The primer sequences used for qRT-PCR analysis are shown in Table 3 below.

[0087]

[0088] 2. Animal Experiment 1) Test Substance

[0089] Weissella Confusar WIKIM51 (Wilac D001) was provided by Palmsville Co., Ltd. and used in cell and animal experiments. Loperamide hydrochloride (Sigma-Aldrich, USA) was used as the negative control, and Bisacodyl (Sigma-Aldrich, USA) was used as the positive control. All samples were dissolved in sterile physiological saline before use.

[0090] 2) Construction of a mouse model for constipation induction using loperamide hydrochloride

[0091] The experimental animals were male 6-week-old ICR mice purchased from Saeron Bio Co., Ltd. After arrival, they were checked for any abnormalities and acclimatized to the rearing environment for one week. The rearing, management, and experiments of the experimental animals were conducted in accordance with regulations after submitting a plan to the Animal Ethics Committee of Gachon University for review and approval (Approval No.: GU1-2024-IA0022). During the rearing period, the animal housing room was maintained at a temperature of 20-25℃, 50-60% humidity, and a 12-hour light-dark cycle (07:00-19:00).

[0092] After confirming that no adverse reactions occurred in any individuals during the adaptation period, groups were separated using a randomized complete block design. The experimental groups consisted of a Normal Control (NC), a Loperamide-induced Control (LC), a Bisacodyl Control (PC), and a Wilac D001 low concentration (1 × 10⁻⁶). 9 CFU / mL) administration group (Wilac D001-Low dose, WD-L), high concentration (1 × 10⁻⁶ 10 The group was divided into a total of 5 groups, including the CFU / mL administration group (Wilac D001-High dose, WD-H).

[0093] A constipation model was established in experimental animals by orally administering 10 mg / kg bw of Loperamide hydrochloride (Sigma-Aldrich Co., USA), a constipation-inducing substance, dissolved in sterile physiological saline 11 days prior to the end of the experiment. Simultaneously, 100 mg / kg bw of Bisacodyl (Sigma-Aldrich Co., USA), a positive control, was orally administered in the same manner. For the NC group, a normal control group not induced with constipation, the same amount of sterile physiological saline was administered in the same manner instead of Loperamide hydrochloride. Wilac D001 (WD) was orally administered in 200 µL samples at each respective concentration for 4 weeks.

[0094] Mice in a total of 5 groups were orally administered once daily for 4 weeks. Except for the Wilac D001 group, the remaining groups were orally administered 200 µL of saline for 18 days, and 11 days before the end of the experiment, Loperamide hydrochloride and Bisacodyl were orally administered to the LC and PC groups according to the concentrations.

[0095] 3) Measurement of body weight, organ (liver, spleen) weight, and dietary intake

[0096] Body weight and food intake were measured once a week to observe changes.

[0097] After the 5-week experiment was completed, the experimental animals were fasted for 16 hours prior to sacrifice, anesthetized with CO2, blood was collected, and then sacrificed. Tissues such as the liver and spleen were extracted, weighed, and stored in a -80℃ deep freezer for subsequent use in experiments.

[0098] 4) Measurement of intestinal stool pH

[0099] After the experiment was completed, the cecum was removed, and the feces inside the cecum were scraped out with a scraper and weighed. The feces were dispersed in PBS, and the pH was measured using pH paper; the pH was counted a total of three times.

[0100] 5) Measurement of fecal weight and moisture content

[0101] On the 9th day of constipation induction, stool samples were collected at 4-hour intervals and weighed. The measured stool samples were dried in a dry oven at 105°C for 24 hours, and the dry weight was measured. The moisture content was calculated using the following formula.

[0102] Fecal water content (%) = (wet weight - dry weight) / wet weight × 100

[0103] 6) Measurement of Gastrointestinal Transit Time

[0104] Intestinal motility was measured on day 10 of constipation induction after a 16-hour fast. After orally administering 200 µL of 6% Carmine Red (w / v) dissolved in 5% CMC solution, each mouse was placed in an individual cage, and the time to the first red fecal discharge was checked at 30-minute intervals. The difference between the time of oral Carmine Red administration and the time of the first red fecal discharge was defined as the colonic transit time.

[0105] 7) Measurement of Small Intestinal Transit Rate (SIT, %)

[0106] Before the end of the experiment, Loperamide and Carmine Red were orally administered at 30-minute intervals, and euthanasia was performed 30 minutes later by CO2 anesthesia. Subsequently, the small intestine was excised, and intestinal motility was measured by dividing the distance traveled by Carmine Red by the total length of the small intestine and converting it into a percentage.

[0107] Small Instestinal Transit rate (SIT, %) = Disatance traveled by Carmine Red / total length of the small intestine × 100

[0108] 8) Histopathological analysis

[0109] Large and small intestine tissues were fixed with 10% formaldehyde and then sectioned to a thickness of 4 μm using a paraffin embedding process. After preparing the section slides, Hematoxylin and eosin (H&E) staining was performed, and Alcian Blue (AB) staining was performed to identify acidic mucinous substances in the large intestine tissue.

[0110] 9) NGS Fecal Gut Microbiota Analysis

[0111] One day before the end of the experiment, fecal samples were collected, and a paired-end sequencing library was constructed by amplifying and indexing the 16S rRNA V3-V4 region. Sequencing data was generated from the constructed sequencing library, and the microbial community was analyzed using Quantitative Insights Into Microbial Ecology (QIIME2) software and the v128 SILVA ribosomal RNA database, which has 97% similarity.

[0112] 10) Statistical Analysis

[0113] All experimental results were expressed as mean ± standard deviation (SD), and Prism 10.2.2 (Graph Pad Software Inc., USA) was used to check for statistical differences between groups. One-way ANOVA was performed, followed by Turkey's multiple range test at the p < 0.05 level.

[0114] result

[0115] 1. Cell test results

[0116] 1) Cytotoxicity test of the test substance on Caco-2 cells

[0117] According to the above experimental method, Wilac D001 was treated to Caco-2 cells at various concentrations.

[0118] Figure 1 shows the results of the cytotoxicity test of the Weissella Confusa WIKIM51 strain (Wilac D001). As shown here, the maximum concentration treated was 1 × 10⁻⁶ 7 It was found that no cytotoxicity was observed with Wilac D001 up to CFU / g.

[0119] 2) mRNA expression levels of Human β-defensin 1 (HBD-1) and HBD-2

[0120] According to the above experimental method, Wilac D001 was treated to Caco-2 cells at different concentrations, and the mRNA expression levels of Human β-defensin 1 (HBD-1) and HBD-2 were measured.

[0121] Figure 2 shows the changes in mRNA expression levels of Human β-defensin 1 (HBD-1) and HBD-2 induced by the Weissella Confusa WIKIM51 strain (Wilac D001). As shown here, the mRNA expression level of HBD-1 increased in a concentration-dependent manner at all concentrations, and it was confirmed that the mRNA expression level of HBD-2 also showed an increasing trend.

[0122] 2. Animal Experiment Results - Evaluation of Intestinal Health Improvement of Wilac D001 in a Constipation-Induced Animal Model

[0123] 1) Mouse model of constipation induction using loperamide

[0124] 6-week-old male ICR mice were tested against a normal control (NC), a loperamide-induced control (LC), a positive control (PC; 100 mg / kg bw bisacodyl), and Wilac D001 low concentration (1 × 10⁶ 9 CFU / mL) administration group (Wilac D001-Low dose, WD-L), high concentration (1 × 10⁻⁶ 10A constipation animal model was established by dividing the subjects into a total of five groups, including a CFU / mL administration group (Wilac D001-High dose, WD-H), orally administering Lopmeramide to the groups excluding the NC group and sterile physiological saline to the NC group.

[0125] 2) Body weight, organ (liver, spleen) weight, and dietary intake

[0126] According to the above experimental method, Wilac D001 was orally administered to mice constipated with Loperamide for 4 weeks to confirm its efficacy in improving intestinal health. The results are shown in Table 4 below. When comparing the NC group, which was not constipated, with the LC group, which was constipated, no significant changes were observed in initial body weight, final body weight after the experiment, weight gain, or food intake. In addition, when the liver and spleen were removed after rearing and the relative weight changes relative to body weight were compared, no significant changes were observed, confirming the safety of Wilac D001.

[0127]

[0128] 3) Measurement of Gut pH in Cecum According to the above experimental method, the pH of the cecum was measured after the experiment to confirm the effect of Wilac D001 on improving the intestinal environment.

[0129] Figure 3 shows the change in stool pH caused by the Weissella Confusa WIKIM51 (Wilac D001) strain in loperamide-induced mice.

[0130] It was confirmed that the intestinal environment was improved in all Wilac D001 administration groups, with a significant decrease in intestinal pH compared to the LC group.

[0131] 4) Measurement of fecal parameters

[0132] According to the above experimental method, to determine the effect of Wilac D001 on improving bowel movements, stool samples were collected at 4-hour intervals on the 9th day of constipation induction and the weight of the stool samples was measured.

[0133] Figure 4 shows the changes in fecal parameters induced by the Weissella Confusa WIKIM51 (Wilac D001) strain in loperamide-induced mice. Here, it was confirmed that intestinal peristalsis was promoted, as the volume of feces increased in a concentration-dependent manner over a certain period in the group administered Wilac D001 (A). In addition, the moisture content was calculated by measuring the dry weight of the collected feces, and the moisture content of the feces significantly increased in the group administered Wilac D001 (B). After the experiment ended, the number of residual feces in the colon was counted, and it was confirmed that the number of residual feces was lower in the group administered Wilac D001 compared to the LC group (C).

[0134]

[0135] 5) Gastrointestinal Transit Time

[0136] According to the above experimental method, the time until the first red stool was expelled was measured after administering Carmine Red.

[0137] Figure 5 shows the change in colon transit time induced by the Weissella Confusa WIKIM51 (Wilac D001) strain in loperamide-induced mice. Here, red feces were expelled latest in the LC group administered loperamide, and it was confirmed that fecal excretion time was reduced in the WD-L and WD-H groups fed Wilac D001.

[0138]

[0139] 6) Small Intestinal Transit Rate (SIT, %)

[0140] Before the end of the experiment, loperamide and carmine red were orally administered at 30-minute intervals, and 30 minutes later, the small intestine was excised to measure the length of the small intestine and the distance traveled by carmine red to measure intestinal motility.

[0141] Figure 6 shows the gastrointestinal transit rate by the Weissella Confusa WIKIM51 (Wilac D001) strain in loperamide-induced mice. Here, it was confirmed that the Carmine Red transit distance in the group administered Wilac D001 was greater in terms of intestinal motility involved in small intestinal peristalsis.

[0142] 7) Histological analysis

[0143] According to the above experimental method, hematoxylin & eosin (H&E) staining was performed to evaluate the histopathological characteristics of the small intestine.

[0144] Figure 7 shows the results of histopathological analysis of the small intestine by the Weissella Confusa WIKIM51 (Wilac D001) strain in loperamide-induced mice. Here, villi atrophy, irregular arrangement, and thinned muscle layer thickness were observed in the LC group compared to the NC group. On the other hand, it was confirmed that these changes were significantly restored in the small intestine tissue of the Wilac D001 group.

[0145] In addition, colon tissue was sampled and H&E staining was performed.

[0146] Figure 8 shows the results of the pathological analysis of the colon by the Weissella Confusa WIKIM51 (Wilac D001) strain in loperamide-induced mice. As a result, colon damage, reduced muscle layer thickness, and short protrusions were observed in the LC group, and tissue damage was restored in the group administered Wilac D001, consistent with the results for the small intestine (A and B). Additionally, when acidic mucin was stained with alcian blue (AB), the mucin was stained more intensely in the Wilac D001-administered group compared to the LC group (A).

[0147] Consequently, in the group fed Wilac D001, loperamide-induced damage to large and small intestinal tissues was restored, and the increased production of acidic mucus suggests that Wilac D001 improved bowel movements in constipated mice.

[0148] 8) NGS Fecal Gut Microbiome Analysis

[0149] One day before the end of the experiment, fecal samples were collected, and a paired-end sequencing library was constructed by amplifying and indexing the 16S rRNA V3-V4 region. Sequencing data was generated from the constructed sequencing library, and the microbial community was analyzed using Quantitative Insights Into Microbial Ecology (QIIME2) software and the v128 SILVA ribosomal RNA database, which has 97% similarity.

[0150] Figure 9 shows the results of microbial community analysis using the Weissella Confusa WIKIM51 (Wilac D001) strain in loperamide-induced mice. Consequently, low alpha diversity was observed in the LC group with induced constipation, whereas high alpha diversity was observed in the group fed Wilac D001 (A). Additionally, when the F / B ratio was measured at the phylum level, the F / B ratio increased in the LC group with induced constipation. Thus, Wilac D001 restored the intestinal microbial imbalance caused by constipation (B).

[0151] Figure 10 shows the differential abundance of loperamide-induced mouse taxa. Analysis of the gut microbial composition revealed that f_S24_7, which contributes to the stability and regulation of the gut ecosystem, was abundant in the WD-L group compared to the LC group (A). When comparing the LC group and the WD-H group, the WD-H group exhibited a diverse gut microbial community, and it was confirmed that the abundance of Akkermansia Muciniphilia increased. The abundance of Hellenica, a beneficial gut bacterium, also increased in the WD-H group (B).

[0152] When considering all these results together, treatment with Weissella Confusa WIKIM51 (Wilac D001) in a loperamide-induced constipation mouse model relieved constipation by increasing fecal water content and bowel motility, restored damage to large and small intestinal tissues, and improved the intestinal environment by raising intestinal pH. Furthermore, Wilac D001 upregulated the diversity of the intestinal microbial community and increased the abundance of beneficial intestinal bacteria such as Akkermasa and Weissella, indicating its potential for improving intestinal health. Therefore, it is believed that Weissella Confusa Wilac D001 has an effect on improving intestinal health.

[0153] Specific parts of the present invention have been described in detail above. It is evident to those skilled in the art that such specific descriptions are merely preferred embodiments and do not limit the scope of the invention. Accordingly, the actual scope of the invention is defined by the appended claims and their equivalents.

[0154]

[0155] As such, the present invention confirmed that the kimchi-derived strain Weissella Confusa WIKIM51 (Wilac D001) has an effect of improving bowel movements in a mouse model of constipation induction, and also has an effect of restoring damage to large and small intestine tissues and increasing beneficial bacteria in the intestines. Therefore, according to the present invention, a composition containing the kimchi-derived Weissella Confusa WIKIM51 (Wilac D001) strain as an active ingredient is expected to be useful as a pharmaceutical and functional food composition for improving intestinal health or preventing or treating intestinal health-related diseases.

[0156]

[0157] [Consignment Number]

[0158] Depository Name: Korean Culture Collection Center (KCCM)

[0159] Trustee Number: KCCM11914P

[0161] Date of entrustment: 20161012

[0160]

Claims

1. Weissella confusa WIKIM51 strain having efficacy in improving gut health (Deposit No.: KCCM11914P).

2. In Paragraph 1, The above Weissella Confusa WIKIM51 strain is characterized by being derived from kimchi.

3. A pharmaceutical composition for improving intestinal health characterized by comprising the Weissella Confusa WIKIM51 strain according to claim 1 or 2 as an active ingredient.

4. In Paragraph 3, A pharmaceutical composition for improving intestinal health, characterized in that the above-mentioned improvement of intestinal health is the improvement of constipation.

5. In Paragraph 3, A pharmaceutical composition for improving intestinal health, characterized in that the above-mentioned improvement of intestinal health is the recovery of damage to large and small intestine tissues.

6. In Paragraph 3, A pharmaceutical composition for improving intestinal health, characterized in that the above-mentioned improvement of intestinal health is an increase in beneficial bacteria in the intestines.

7. A functional food composition for improving gut health characterized by comprising the Weissella Confusa WIKIM51 strain according to Claim 1 or 2 as an active ingredient.

8. A feed composition for improving intestinal health characterized by containing the Weissella Confusa WIKIM51 strain according to claim 1 or 2 as an active ingredient.

9. A pharmaceutical composition for the prevention or treatment of intestinal health-related diseases, characterized by comprising the Weissella Confusa WIKIM51 strain according to Claim 1 or 2 as an active ingredient.

10. In Paragraph 9, A pharmaceutical composition for the prevention or treatment of intestinal health-related diseases, characterized by the above-mentioned irritable bowel syndrome, inflammatory bowel disease, leaky gut syndrome, intestinal bacterial imbalance, constipation, diarrhea, small intestinal bacterial overgrowth, colorectal cancer, intestinal obstruction, food allergy and intolerance, diverticulitis, and lactose intolerance.

11. A functional food composition for preventing or improving intestinal health-related diseases, characterized by comprising the Weissella Confusa WIKIM51 strain according to claim 1 or 2 as an active ingredient.

12. A feed composition for preventing or improving intestinal health-related diseases, characterized by containing the Weissella Confusa WIKIM51 strain according to claim 1 or 2 as an active ingredient.

13. A method for improving intestinal health comprising the step of administering the composition according to claim 3 to an individual other than a human.

14. A method for preventing or improving intestinal health-related diseases, comprising the step of administering a composition according to claim 9 to an individual other than a human.

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