Novel lactiplantibacillus plantarum DSW3805 strain and use thereof

Abstract

The present invention relates to a composition for preventing, alleviating, or treating inflammatory bowel disease or irritable bowel syndrome, the composition comprising a Lactiplantibacillus plantarum DSW3805 strain (KCCM 13544P) or a culture medium thereof as an active ingredient.

Description

Novel Lactibactibacillus plantarum strain DSW3805 and uses thereof

[0001] The present invention relates to a novel Lactibacillus plantarum DSW3805 strain and its use.

[0002] Inflammatory bowel disease, characterized by chronic inflammation of the large intestine, is a disease marked by an inflammatory response in the colon, while irritable bowel syndrome, characterized by recurrent abdominal pain without an organic cause, is thought to have gastrointestinal infections or microinflammation as one of its major causes. Existing treatments for inflammatory bowel disease and irritable bowel syndrome are limited in their use because they not only have minimal therapeutic effects but also cause serious side effects such as headaches, rashes, liver disease, leukopenia, and male infertility. Therefore, there is a very high clinical demand for the development of new treatments that can be used effectively and safely for both diseases.

[0003] Accordingly, research on the discovery of strains effective for inflammatory bowel disease or irritable bowel syndrome has been steadily conducted, but in particular, no Lactiplantibacillus plantarum strain has been identified to date that is stable and safe for the human body as a probiotic and effective for the disease.

[0004] The present invention aims to provide a Lactiplantibacillus plantarum DSW3805 strain (KCCM 13544P) having pathogen adhesion inhibitory activity and immune modulatory ability in intestinal tissue.

[0005] However, the technical problems that the present invention aims to solve are not limited to those mentioned above, and other unmentioned problems will be clearly understood by those skilled in the art from the description below.

[0006] The present invention provides a Lactiplantibacillus plantarum DSW3805 strain (KCCM 13544P) having pathogen adhesion inhibitory activity and immune modulatory ability in intestinal tissue.

[0007] The above DSW3805 strain may include a DNA base sequence encoding the 16S rRNA of SEQ ID NO. 1.

[0008] The above pathogens may include one or more selected from the group consisting of Listeria monocytogenes, Staphylococcus aureus, Escherichia coli, and Salmonella enteritidis.

[0009] The above immunomodulatory ability may be i) a reduction in nitric oxide (NO) production; ii) a reduction in the expression of pro-inflammatory cytokines, including one or more selected from the group consisting of TNF-α, IL-1β, COX-2, IL-6, and IL-8; or iii) through the activation of the NF-κB or MAPK pathway.

[0010] The above-mentioned DSW3805 strain can increase the expression of intestinal motility-related genes in intestinal tissue, including one or more selected from the group consisting of ZO-1, Claudin-1, Occludin, and MUC2.

[0011] In one embodiment of the present invention, a pharmaceutical composition for the prevention or treatment of inflammatory bowel disease or irritable bowel syndrome is provided, comprising the Lactiplantibacillus plantarum DSW3805 strain (KCCM 13544P) or a culture medium thereof as an active ingredient.

[0012] The above pharmaceutical composition may have: i) an effect of reducing intestinal mucosal thickness and intestinal damage increased due to inflammation in the large intestine; ii) an effect of reducing the expression of pro-inflammatory cytokines including one or more selected from the group consisting of IL-6, TNF-α, IFN-γ, and IL-1β increased due to inflammation in the large intestine; iii) an effect of reducing IgA or IgG concentration increased due to inflammation in the large intestine; or iv) an effect of reducing the expression of inflammation-related factors including one or more selected from the group consisting of LTB4, PGE2, and NF-kB increased due to inflammation in the large intestine.

[0013] The above pharmaceutical composition can have a diarrhea-relieving effect by restoring the reduced moisture content of the stool.

[0014] The above pharmaceutical composition may have: i) an effect of increasing the Firmicutes:Bacteroidetes value reduced due to inflammation in the large intestine as a result of phylum analysis of feces; ii) an effect of increasing the ratio of Bifidobacterium reduced due to inflammation in the large intestine as a result of genus analysis of feces; and iii) an effect of increasing the total ratio of Lactobacillus murinus, Lactobacillus gasseri, and Lactobacillus reuteri reduced due to inflammation in the large intestine and decreasing the ratio of Helicobacter mesocricetorum increased due to inflammation in the large intestine as a result of species analysis of feces.

[0015] The above pharmaceutical composition may have: i) an effect of increasing short-chain fatty acids including one or more selected from the group consisting of acetic acid, propionic acid, and butyric acid in feces; or ii) an effect of decreasing the activity of calprotectin and β-glucuronidase in feces.

[0016] The dose of the above DSW3805 strain is 1×10 5 CFU / day to 1×10 12 It can be CFU / day.

[0017] In another embodiment of the present invention, a health functional food composition for preventing or improving inflammatory bowel disease or irritable bowel syndrome is provided, comprising the Lactiplantibacillus plantarum DSW3805 strain (KCCM 13544P) or a culture solution thereof as an active ingredient.

[0018] The Lactiplantibacillus plantarum DSW3805 strain (KCCM 13544P) according to the present invention is a novel strain having stable and safe probiotic properties, characterized by having pathogen adhesion inhibitory activity and immune modulatory ability in intestinal tissue.

[0019] The above-mentioned DSW3805 strain or culture medium thereof has the effect of reducing intestinal mucosal thickness and intestinal damage caused by inflammation in the large intestine in an animal model of colitis, and possesses anti-inflammatory and diarrhea-relieving effects, and can be usefully utilized as a composition for the prevention, improvement, or treatment of inflammatory bowel disease or irritable bowel syndrome. In particular, the above-mentioned DSW3805 strain or culture medium thereof can alter the intestinal microbial community in feces and can have the effect of increasing short-chain fatty acids.

[0020] Figure 1 shows the intestinal cell adhesion ability of L. plantarum DSW3805 confirmed through scanning electron microscopy observation.

[0021] Figure 2 shows the amount of NO produced by L. plantarumDSW3805 in SNP-induced intestinal cells.

[0022] Figures 3a to 3e show the expression of pro-inflammatory cytokines (TNF-α, IL-1β, COX-2, IL-6, and IL-8) of L. plantarum DSW3805 in LPS-induced intestinal cells.

[0023] Figures 4a to 4g show the activating effects of L. plantarumDSW3805 on the NF-κB and MAPK pathways (NF-κB pathway, p-p65 / p65, IκBα / GAPDH, MAPK pathway, p-p38 / p38, p-ERK 1 / 2 / ERK 1 / 2 and p-JNK / JNK) in LPS-induced intestinal cells.

[0024] Figures 5a to 5d show the expression of intestinal motility-related genes (ZO-1, Claudin-1, Occludin, and MUC2) of L. plantarumDSW3805 in LPS-induced intestinal cells.

[0025] Figure 6 shows the hemolytic activity of L. plantarum DSW3805.

[0026] Figure 7 shows the bile acid deconjugation of L. plantarumDSW3805.

[0027] Figure 8 shows the change in the disease activity index (DAI) according to the intake of L. plantarumDSW3805.

[0028] Figure 9 shows the change in intestinal length following the intake of L. plantarumDSW3805.

[0029] Figure 10 shows the histological changes in the intestine following the intake of L. plantarumDSW3805.

[0030] Figures 11a to 11e show changes in the intestinal microbial phylum community following the ingestion of L. plantarumDSW3805.

[0031] Figure 12 shows the results of culturing beneficial and harmful bacteria following the intake of L. plantarum DSW3805.

[0032] The inventors completed the present invention by isolating and identifying the Lactiplantibacillus plantarum DSW3805 strain (KCCM 13544P), which has a unique metabolic ability among Lactiplantibacillus plantarum strains having probiotic characteristics, confirming pathogen adhesion inhibitory activity and immune modulatory ability in intestinal tissue in vitro, and specifically demonstrating that it is effective in preventing, improving, or treating inflammatory bowel disease or irritable bowel syndrome in an animal model of colitis in vivo.

[0033]

[0034] The present invention will be described in detail below.

[0035]

[0036] The present invention provides a Lactiplantibacillus plantarum DSW3805 strain (KCCM 13544P) having pathogen adhesion inhibitory activity and immune modulatory ability in intestinal tissue.

[0037]

[0038] The above-mentioned DSW3805 strain can be viewed as a subspecies of Lactiplantibacillus plantarum. The above-mentioned DSW3805 strain was isolated from traditional fermented foods such as kimchi, and possesses stable and safe probiotic characteristics due to its excellent acid resistance, bile resistance, and intestinal adhesion ability.

[0039] As a result of analyzing the 16S rRNA to identify the isolated strain, it was confirmed to contain a DNA base sequence encoding the 16S rRNA of SEQ ID NO. 1, and was deposited with the Korean Culture Collection of Microorganisms on January 22, 2025, and assigned accession number KCCM 13544P.

[0040]

[0041] First, the above-mentioned DSW3805 strain is characterized by having pathogen adhesion inhibitory activity in intestinal tissue, and the pathogen may include one or more selected from the group consisting of Listeria monocytogenes, Staphylococcus aureus, Escherichia coli, and Salmonella enteritidis.

[0042] In addition, the above-mentioned DSW3805 strain is characterized by having immunomodulatory ability in intestinal tissue, wherein the immunomodulatory ability may be i) a reduction in nitric oxide (NO) production; ii) a reduction in the expression of pro-inflammatory cytokines including one or more selected from the group consisting of TNF-α, IL-1β, COX-2, IL-6, and IL-8; or iii) through the activation of the NF-κB or MAPK pathway.

[0043] In addition, the above-mentioned DSW3805 strain can increase the expression of intestinal motility-related genes in intestinal tissue, including one or more selected from the group consisting of ZO-1, Claudin-1, Occludin, and MUC2.

[0044]

[0045] The present invention provides a pharmaceutical composition for the prevention or treatment of inflammatory bowel disease or irritable bowel syndrome comprising the Lactiplantibacillus plantarum DSW3805 strain (KCCM 13544P) or a culture medium thereof as an active ingredient.

[0046] In addition, a health functional food composition for preventing or improving inflammatory bowel disease or irritable bowel syndrome is provided, comprising the Lactiplantibacillus plantarum DSW3805 strain (KCCM 13544P) or a culture solution thereof as an active ingredient.

[0047]

[0048] In this specification, "Inflammatory bowel disease (IBD)" refers to a disease that causes chronic inflammation in the gastrointestinal tract accompanied by symptoms such as abdominal pain, fever, diarrhea, and bleeding. The inflammatory bowel disease is classified into two forms: ulcerative colitis and Crohn's disease. Ulcerative colitis is a type of diffuse nonspecific inflammation of unknown etiology in which the colon is mainly affected by the colon mucosa and frequently forms ulcers or erosions, accompanied by various systemic symptoms including bloody diarrhea. Crohn's disease is a granulomatous inflammatory lesion of unknown etiology in which ulcers, fibrosis, strictures, and lesions progress discontinuously from the mucosa to the entire layer of the intestinal tract from the mouth to the anus, accompanied by systemic symptoms such as abdominal pain, chronic diarrhea, fever, and malnutrition.

[0049] In this specification, "Irritable bowel syndrome (IBS)" refers to a chronic functional gastrointestinal disorder characterized by changes in the frequency or form of bowel movements and recurrent abdominal pain related to bowel movements. Chronic or recurrent unpleasant digestive symptoms occur without an organic cause. Specifically, there are changes in bowel habits such as abdominal pain, bloating, diarrhea, or constipation after meals or mild stress, and discomfort may be felt due to a sensation of incomplete bowel emptying even after defecation. Irritable bowel syndrome is one of the most common digestive diseases, affecting approximately 7 to 15% of the total population.

[0050]

[0051] As the above strain has been described above, a redundant explanation will be omitted.

[0052] The above DSW3805 strain may be prepared as live cells or dead cells. In addition, the above culture medium may be a culture filtrate, that is, a supernatant from which the cells have been removed.

[0053] The dose of the above DSW3805 strain is 1×10 5 CFU / day to 1×10 12 It can be CFU / day, and 1×10 8 CFU / day to 1×10 10 It is desirable that it be CFU / day, but is not limited thereto.

[0054]

[0055] The above pharmaceutical composition may have: i) an effect of reducing intestinal mucosal thickness and intestinal damage increased due to inflammation in the large intestine; ii) an effect of reducing the expression of pro-inflammatory cytokines including one or more selected from the group consisting of IL-6, TNF-α, IFN-γ, and IL-1β increased due to inflammation in the large intestine; iii) an effect of reducing IgA or IgG concentration increased due to inflammation in the large intestine; or iv) an effect of reducing the expression of inflammation-related factors including one or more selected from the group consisting of LTB4, PGE2, and NF-kB increased due to inflammation in the large intestine.

[0056] In addition, the above pharmaceutical composition may have a diarrhea-relieving effect by restoring the reduced moisture content of the stool.

[0057] The above pharmaceutical composition may have: i) an effect of increasing the Firmicutes:Bacteroidetes value reduced due to inflammation in the large intestine as a result of phylum analysis of feces; ii) an effect of increasing the ratio of Bifidobacterium reduced due to inflammation in the large intestine as a result of genus analysis of feces; and iii) an effect of increasing the total ratio of Lactobacillus murinus, Lactobacillus gasseri, and Lactobacillus reuteri reduced due to inflammation in the large intestine and decreasing the ratio of Helicobacter mesocricetorum increased due to inflammation in the large intestine as a result of species analysis of feces.

[0058] The above pharmaceutical composition may have: i) an effect of increasing short-chain fatty acids including one or more selected from the group consisting of acetic acid, propionic acid, and butyric acid in feces; or ii) an effect of decreasing the activity of calprotectin and β-glucuronidase in feces.

[0059]

[0060] As described above, the Lactiplantibacillus plantarum DSW3805 strain (KCCM 13544P) according to the present invention is a novel strain having stable and safe probiotic properties, characterized by having pathogen adhesion inhibitory activity and immune modulatory ability in intestinal tissue.

[0061] The above-mentioned DSW3805 strain or culture medium thereof has the effect of reducing intestinal mucosal thickness and intestinal damage caused by inflammation in the large intestine in an animal model of colitis, and possesses anti-inflammatory and diarrhea-relieving effects, and can be usefully utilized as a composition for the prevention, improvement, or treatment of inflammatory bowel disease or irritable bowel syndrome. In particular, the above-mentioned DSW3805 strain or culture medium thereof can alter the intestinal microbial community in feces and can have the effect of increasing short-chain fatty acids.

[0062]

[0063] The pharmaceutical composition according to the present invention may be formulated and used in the form of oral formulations such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, and aerosols, external formulations, suppositories, and sterile injectable solutions according to conventional methods, and may include a suitable carrier, excipient, or diluent that is conventionally used in the manufacture of pharmaceutical compositions for formulation.

[0064] The above-mentioned carrier, excipient, or diluent may include various compounds or mixtures including lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and mineral oil.

[0065] When formulating, it can be manufactured using diluents or excipients such as commonly used fillers, weights, binders, wetting agents, disintegrants, and surfactants.

[0066] A solid dosage form for oral administration can be prepared by mixing at least one excipient, such as starch, calcium bonate, sucrose or lactose, gelatin, etc. with the above-mentioned DSW3805 strain. In addition to simple excipients, lubricants such as magnesium stearate and talc may also be used.

[0067] Liquid formulations for oral administration include suspensions, liquid formulations, emulsions, syrups, etc., and may contain various excipients, such as humectants, sweeteners, flavorings, and preservatives, in addition to commonly used simple diluents like water and liquid paraffin.

[0068] Preparations for parenteral administration include sterile aqueous solutions, water-insoluble preparations, suspensions, emulsions, lyophilized preparations, and suppositories. As water-insoluble solvents and suspensions, propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate may be used. As bases for suppositories, witepsol, macrogol, Tween 61, cocoa paste, laurin paste, glycerol gelatin, etc. may be used.

[0069] The preferred dosage of the pharmaceutical composition according to the present invention varies depending on the patient's condition, body weight, severity of the disease, drug form, route of administration, and duration, but can be appropriately selected by those skilled in the art. However, for a desirable effect, it may be administered at a dose of 0.0001 to 2,000 mg / kg per day, preferably 0.001 to 2,000 mg / kg. The administration may be performed once a day or divided into several doses. However, the scope of the present invention is not limited by the above dosage.

[0070] The pharmaceutical composition according to the present invention can be administered to mammals such as rats, mice, livestock, and humans by various routes. All modes of administration may be administered, for example, orally, rectally or intravenously, intramuscularly, subcutaneously, intrathecally, or intracerebroventricularly.

[0071]

[0072] In the health functional food composition according to the present invention, when the DSW3805 strain is used as an additive to the health functional food, it may be added as is or used together with other foods or food ingredients, and may be used appropriately according to conventional methods. The mixing amount of the active ingredient may be appropriately determined according to each purpose of use, such as prevention, health, or treatment.

[0073] The formulation of health functional foods can be in the form of powders, granules, pills, tablets, or capsules, as well as in the form of general foods or beverages.

[0074] There are no specific restrictions on the types of food mentioned above, and examples of food to which the substance may be added include meat, sausage, bread, chocolate, candy, snacks, confectionery, pizza, ramen, other noodles, chewing gum, dairy products including ice cream, various soups, beverages, tea, drinks, alcoholic beverages, and vitamin complexes, and may include all food in the conventional sense.

[0075] Generally, when manufacturing food or beverages, the above-mentioned DSW3805 strain may be added in an amount of 15 parts by weight or less, preferably 10 parts by weight or less, per 100 parts by weight of raw material. However, in the case of long-term consumption for the purpose of health and hygiene or health control, the above amount may be less than the above range.

[0076] The beverage among the health functional foods according to the present invention may contain various flavoring agents or natural carbohydrates as additional ingredients, as in conventional beverages. The natural carbohydrates described above may be monosaccharides such as glucose and fructose, disaccharides such as maltose and sucrose, polysaccharides such as dextrin and cyclodextrin, and sugar alcohols such as xylitol, sorbitol, and erythritol. As a sweetener, natural sweeteners such as taumatin and stevia extract, or synthetic sweeteners such as saccharin and aspartame may be used. The proportion of the natural carbohydrates may be about 0.01 to 0.04 g, preferably about 0.02 to 0.03 g, per 100 mL of the beverage according to the present invention.

[0077] In addition to the above, the health functional food composition according to the present invention may contain various nutritional agents, vitamins, electrolytes, flavoring agents, coloring agents, pectic acid and its salts, alginic acid and its salts, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohol, and carbonating agents used in carbonated beverages. Furthermore, the health functional food composition according to the present invention may contain fruit pulp for the production of natural fruit juices, fruit juice beverages, and vegetable beverages. These ingredients may be used independently or in combination. The proportion of these additives is not limited, but is generally selected in the range of 0.01 to 0.1 parts by weight per 100 parts by weight of the health functional food composition according to the present invention.

[0078]

[0079] Alternatively, the present invention provides a use for a pharmaceutical composition / health functional food for the prevention, treatment, or improvement of inflammatory bowel disease or irritable bowel syndrome comprising the Lactiplantibacillus plantarum DSW3805 strain (KCCM 13544P) or a culture medium thereof as an active ingredient.

[0080] Alternatively, the present invention provides a method for preventing, treating, or improving inflammatory bowel disease or irritable bowel syndrome, comprising the step of administering to an individual a composition containing the Lactiplantibacillus plantarum DSW3805 strain (KCCM 13544P) or a culture medium thereof as an active ingredient. In this case, "individual" refers to a subject requiring treatment for a disease, and more specifically, refers to mammals such as humans or non-human primates, mice, rats, dogs, cats, horses, and cattle.

[0081]

[0082] Preferred embodiments are presented below to aid in understanding the present invention. However, the following embodiments are provided merely to facilitate a better understanding of the invention, and the scope of the invention is not limited by the following embodiments.

[0083]

[0084] <Example>

[0085] Example 1: Isolation and identification of the Lactiplantibacillus plantarum DSW3805 strain from kimchi

[0086] To isolate novel strains, various homemade kimchi brine samples, including Napa cabbage kimchi, young radish kimchi, and cubed radish kimchi, were collected and used as samples. Novel strains were isolated by observing colony morphology after streaking the kimchi brine onto MRS agar medium using a 10-fold dilution method and incubating the cultures. To identify the isolated strains, 16S rRNA sequencing was commissioned to Bionics (Seoul, Korea). Specifically, 16S rRNA sequencing was performed, and PCR was conducted on the 16S rRNA gene using 27F and 1492R primers. Based on the results of the nucleotide sequence analysis, homology was compared with other standard strains registered in the Genebank database using the BLAST program.

[0087] As a result, the strain isolated from kimchi (source: homemade young radish kimchi from Wonju, Gangwon-do) was found to have homology with strains reported as Lactiplantibacillus plantarum, so it was named Lactiplantibacillus plantarum DSW3805 strain and deposited with the Korean Culture Collection Center (KCCM) on January 22, 2025 (KCCM 13544P). Meanwhile, commercial Lacticaseibacillus rhamnosusGG (LGG) was used as a control.

[0088]

[0089] Example 2: Characteristics of Lactiplantibacillus plantarum DSW3805 strain

[0090] (1) Acid resistance, bile resistance, intestinal adhesion

[0091] To evaluate acid resistance, artificial gastric fluid (pH 2.5, 0.3% pepsin) was prepared, and the bacterial strain was incubated for 3 hours, after which the number of viable bacteria was checked. To evaluate bile resistance, 0.3% ox gel was added to the culture medium, and the bacterial strain was incubated for 24 hours, after which the number of viable bacteria was measured. In addition, to evaluate intestinal adhesion ability, 1×10 5 After culturing the HT-29 (human colon adenocarcinoma cell line) cell line inoculated into cells / well for 24 hours, the strain (1×10⁶) 7 CFU / well) was added and cultured for an additional 2 hours. Non-attached bacteria were removed by washing three times with phosphate-buffered saline, and after detaching the cell line using 1% Triton X-100 solution, the number of bacteria attached to the cell line was measured using the plate plating method.

[0092] Analysis of viable cell count (Log CFU / mL) LGGDSW3805 Initial cell count resistant to artificial stomach acid 9.59±0.01 b 9.38±0.02a pH 2.5, 0.3% pepsin, 2 hours 9.55±0.07 a 9.52±0.20 a Survival rate (%) 99.46±0.70 a 101.50±2.15 a Initial cell count for resistance to artificial bile acid 9.59±0.01 b 9.38±0.02 a 0.3% Oxgel, 24 hours 8.71±0.02 a 9.95±0.03 c Survival rate (%) 90.86±0.22 a 106.00±0.00 b Initial cell number of adhesion cells for HT-29 cells 8.27±0.13 a 8.11±0.07 a Number of adherent cells 6.66±0.01 ab 6.31±0.04 a Adhesion rate (%) 2.56±0.73 ab 1.62±0.30 a

[0093] As shown in Table 1, the results of the acid resistance and bile resistance evaluations showed that L. plantarum DSW3805 (101.50% acid resistance, 106.00% bile resistance) was superior to LGG (99.46% acid resistance, 90.86% bile resistance). The results of the intestinal adhesion evaluation confirmed that L. plantarum DSW3805 (1.62%) was at an equivalent level to LGG (2.56%).

[0094]

[0095] (2) Scanning microscope observation of the strain

[0096] After HT-29 cells formed a monolayer, bacteria 10 9 After inoculating with CFU, the samples were incubated at 37°C for 2 hours. For scanning electron microscopy (SEM) observation, the samples were fixed with glutaraldehyde and washed with water. Dehydration was performed by stepwise treatment with ethanol. Hexamethyldisilazane was applied to form a thin coating on the surface of the samples to prevent damage and to allow for better visibility of detailed structures during SEM observation.

[0097] As shown in Figure 1, the adhesion ability to intestinal cells was confirmed through scanning electron microscopy observation, and it was confirmed that L. plantarumDSW3805 has a much superior adhesion ability compared to LGG.

[0098]

[0099] (3) Inhibitory activity of pathogen adhesion to intestinal cells

[0100] 1×10 5 10 HT-29 cells per well 6 Foodborne pathogens and mixed strains (a mixture of foodborne pathogens and bacterial strains) were inoculated at a rate of approximately cells / well and cultured at 37°C for 2 hours. After culture, the cells were washed three times with PBS to remove bacteria that were not attached to the HT-29 cells, and the attached cells were detached using 1% Triton X-100 solution. The bacterial count was then measured using the selective medium corresponding to each pathogen. The pathogens used were mixed strains: Staphylococcus aureus ATCC 25923+Staphylococcus aureus KCCM 40510; Listeria monocytogenes ATCC 15313+Listeria monocytogenes Scott A NADC 2045 4b;Escherichia coli O157; and Salmonella enteritidis ATCC 13076+Salmonella enteritidis KCCM 12021, which were cultured in TSB and used according to the bacterial count.

[0101] Strain S. aureus (Log CFU / mL) Inhibition Rate (%) L. monocytogenes (Log CFU / mL) Inhibition Rate (%) Control Group 7.24±0.30 b -6.14±0.00 c -LGG5.58±0.01 bc 95.89±0.13 cd 5.45±0.03 a 79.90±1.17 b DSW38055.67±0.05 b 95.10±0.58 cd5.67±0.05 b 66.51±3.38 a Inhibition rate (%) of strain E. coli (Log CFU / mL) Inhibition rate (%) of S. enteritidis (Log CFU / mL) Control group 6.18±0.01 d -5.27±0.21 b -LGG5.48±0.00 ab 80.31±0.00 d 4.71±0.55 ab 88.71±12.70 b DSW38055.79±0.06 c 59.51±5.58 bc 4.26±0.20 a 90.32±3.95 b

[0102] As shown in Table 2, L. plantarumDSW3805 is confirmed to have pathogen adhesion inhibitory activity against intestinal cells that is equivalent to or greater than that of LGG.

[0103]

[0104] (4) Verification of intestinal immune regulatory ability

[0105] To investigate the anti-inflammatory effects of live bacterial strains in living tissues, the reduction in nitric oxide (NO) production was measured using HT-29 cells. HT-29 cells (2×10⁶) were placed in a 96-well plate. 6 100 μL was added to each well and cultured in a CO2 incubator at 37°C in a 5% CO2 environment until a monolayer was formed. After treating with 50 μL of the strain sample for 2 hours, 50 μL of SNP was added to induce inflammation and cultured for 24 hours. 100 μL of the supernatant and 100 μL of Griess reagent were added and reacted at room temperature for 10 minutes. The absorbance was then measured at 540 nm using a plate reader, and the amount of NO produced was determined by comparing it with a standard curve for nitrite content.

[0106] As shown in Figure 2, it was confirmed that L. plantarum DSW3805 (41.9 μM NO production) has superior NO inhibitory activity compared to LGG (43.8 μM NO production) at 6 log CFU / well, and at 7 log CFU / well, it was confirmed that L. plantarum DSW3805 (29.7 μM NO production) has superior NO inhibitory activity compared to LGG (32.4 μM NO production).

[0107] After a monolayer was formed in the HT-29 cells, they were divided into LPS-treated and untreated groups, and the bacterial strain was added according to the concentration. The cell lines were washed twice, and pure RNA was obtained using an RNA isolation kit for use in the experiment. The RNA was converted to cDNA using RT-PCR reagents and PCR was performed. Primers for TNF-α, IL-1β, COX-2, IL-6, and IL-8 were used to confirm the expression of pro-inflammatory cytokines.

[0108] In the real-time qPCR analysis, similar to RT-PCR, the synthesized cDNA was used as the template strand and mixed with SYBR Green master mix (2×), 400 μM primer, and RNase-free water to a total volume of 10 μL. The process was executed at 95°C for 2 minutes for 40 cycles, followed by cycles at 95°C for 5 seconds and 65°C for 30 seconds, and finally extended under conditions of 60°C for 20 seconds. β-actin was used as the housekeeping gene and served as the standard. Amplification results were obtained using the Delta delta Cq (ΔΔCq) assay and relative quantification was performed. The purity of the amplification product was verified by examining the melting curve of the qPCR reaction and presented as a result of relative quantification.

[0109] As shown in FIGS. 3a to 3e, when TNF-α expression was checked at 7 log CFU / well, it was confirmed that L. plantarum DSW3805 (0.19) was reduced compared to PC (1.00) and LGG (0.49) treated with LPS. When IL-1β expression was checked, it was confirmed that L. plantarum DSW3805 (0.18) was reduced compared to PC (1.00) and LGG (0.61) treated with LPS. When COX-2 expression was checked, it was confirmed that L. plantarum DSW3805 (0.47) was significantly reduced compared to PC (1.00) and LGG (0.30) treated with LPS. When IL-6 expression was checked, L. It was confirmed that plantarumDSW3805 (0.20) decreased. As a result of checking IL-8 expression, it was confirmed that L. plantarumDSW3805 (0.23) decreased compared to PC (1.00) and LGG (0.67) treated with LPS.

[0110]

[0111] (5) Verification of mechanism of action using Western blot

[0112] HT-29 intestinal cells were seeded into 6-well plates (5 × 10⁴ cells / well) and cultured at 37°C in a 5% CO₂ environment until a monolayer was formed. These samples were pre-treated for 2 hours, followed by treatment with 50 μg / mL of lipopolysaccharide (LPS) and incubation for a set period. The cultured cells were lysed with Pro-Prep lysis buffer, centrifuged, and only the supernatant was collected. 20–30 μg of protein, quantified using a DC protein analysis kit, was separated by electrophoresis on a 10% sodium dodecyl sulfate-polyacrylamide gel and transferred to a polyvinyl difluoride membrane. After incubation with 5% skim milk for 1 hour, an appropriate antibody was added. After washing three times with TBS-T solution, the samples were reacted with an enhanced chemiluminescence detection kit, and the protein bands were analyzed by exposure to X-ray film in a dark room. The protein bands were quantified using ImageJ software.

[0113] As shown in Figures 4a to 4c, the effect on NF-κB activation was confirmed by its effect on p65 and IκBα. In the regulation of the inflammatory response, it was confirmed that the phosphorylation of p65 was lower in LGG compared to L. plantarumDSW3805, and that the expression of IκBα was most increased in L. plantarumDSW3805 compared to LGG.

[0114] As shown in Figures 4d to 4g, the effect on MAPK activation was confirmed through its effects on p38, ERK 1 / 2, and JNK. Phosphorylation of these factors was confirmed to inhibit phosphorylation by L. plantarum DSW3805. This is confirmed to have an effect on MAPK activation.

[0115] In other words, L. plantarumDSW3805 is confirmed to have anti-inflammatory effects through the NF-κB and MAPK signaling pathways.

[0116]

[0117] (6) Gene expression related to bowel movement

[0118] After HT-29 intestinal cells were formed into a monolayer, they were divided into LPS-treated and untreated groups, and the bacterial strain was added according to the concentration. The control group consisted of cell lines that were not treated with the strain. After washing the cell lines twice, pure RNA was obtained using an RNA isolation kit and used in the experiment. The RNA was converted to cDNA using RT-PCR reagents and PCR was performed. Gene expression was confirmed using primers of ZO-1, Claudin-1, Occludin, and MUC2.

[0119] As shown in FIGS. 5a to 5d, ZO-1 functions to connect membrane-penetrating proteins with actin, a cytoskeletal protein, and is known as a tight junction protein. Compared to the LPS-treated group (1.00), it is found in the order of LGG (1.77) and L. plantarum DSW3805 (1.49). Claudin-1 plays an important role in maintaining the function of tight junctions. In the case of Claudin-1, compared to the LPS-treated group (1.00), it is found in the order of L. plantarum DSW3805 (1.18) and LGG (1.01). Occludin is a major junction molecule present in the tight junctions of epithelial cells, and it plays a role in maintaining the polarity of epithelial cells and blocking the movement of substances through cells. Compared to the LPS-treated group (1.00) of tight junctions, LGG (1.18) and L. MUC2 is identified in order of plantarumDSW3805 (1.16). MUC2 plays a role related to mucus secretion in the intestine. In the case of MUC2, compared to the LPS treatment group (1.00), L. plantarumDSW3805 (1.30) and LGG (1.02) are identified in order.

[0120]

[0121] (7) Antibiotic susceptibility of strains

[0122] Antibiotic susceptibility of the strains was performed in accordance with the guidelines of the Ministry of Food and Drug Safety. It was confirmed using antibiotic strips and the microdilution method.

[0123] - Antibiotic strip: Strain is 0.5–1.0 McFarland (1.5–3×10⁶ 8 The strain was prepared by diluting it to a concentration of CFU / mL. A 1.8% agar medium suitable for the strain was prepared, autoclaved, and solidified at room temperature. A sterile cotton swab was soaked in the liquid medium containing the strain and removed. The strain was dispensed onto the agar medium using soft agar and left at room temperature for 10–20 minutes. An antibiotic strip was placed on the agar medium. It was incubated for 46–48 hours under anaerobic conditions at 37°C. The lowest point of the strip where no strain grew was determined as the MIC.

[0124] - Microdilution method: After selecting a medium suitable for the strain, 100 µL was dispensed into a 96-well plate. Antibiotic solutions were added to each well containing liquid medium, doubling the concentration from the no-addition (control) group. The strain to be tested (5×10 6 CFU / mL) 10 uL (final bacterial count 5×10 4 CFU / well) was inoculated into each well. Incubated for 24 to 48 hours under anaerobic conditions at 37°C. Absorbance was measured at 600 nm to determine the MIC.

[0125] Strain Antibiotic Susceptibility (expressed as Resistance (R) or Susceptibility (S) based on the 2012 EFSA MIC threshold (mg / L)) Ampicillin (AM) Gentamicin (GM) Kanamycin (KM) Streptomycin (SM) Erythromycin (EM) Clindamycin (CM) Tetracycline (TC) Chloramphenicol (CL) LGGSSSRSSSSDSW3805SSSRSSSS

[0126] As shown in Table 3, L. plantarumDSW3805 showed resistance to streptomycin.

[0127]

[0128] (8) Hemolytic activity

[0129] - Preparation of TSA Blood Agar

[0130] ① A culture medium was prepared with the following composition, autoclaved, and then heated to 40–50°C in a warm water bath (Pancreatic digest of casein USP 15.0 g, Papaic digest of soy meal USP 5.0 g, NaCl 5.0 g, Agar 15.0 g, Distilled water 1,000 mL).

[0131] ② 5% sterile sheep blood was added.

[0132] ③ 20 mL of the blood-added medium was dispensed and solidified at room temperature.

[0133] - Strain inoculation

[0134] ① The strain was streaked onto a blood electrolytic medium.

[0135] - Analysis of hemolysis results

[0136] ① Observed with transmitted light to confirm the hemolytic reaction.

[0137] ② α-hemolysis: Reduced methemoglobin in red blood cell hemoglobin to form green colonies; β-hemolysis: Destroyed red blood cells to form clear colonies; γ-hemolysis: No hemolytic phenomenon was observed.

[0138] As shown in Figure 6, γ-hemolytic activity was confirmed for LGG and L. plantarum DSW3805, confirming their safety.

[0139]

[0140] (9) Toxin production through LDH analysis

[0141] - Cell culture and preparation

[0142] ① Penicillin-streptomycin solution (100 U / mL) was added to the sterile DMEM medium to prevent contamination.

[0143] ② Activated Caco-2 or HT-29 cell line (10 4 cells) were inoculated into 100 μL of prepared DMEM cell medium.

[0144] ③ After transferring the cell medium inoculated into each well of a 96-well plate, the cells were cultured at 37°C in 5% CO2 for 24 hours.

[0145] ④ After removing the medium following culture, the cells were washed twice with PBS containing 1% BSA, and 100 μL of new cell medium was added.

[0146] - Strain inoculation

[0147] ① Test strains in each well (10 7 ~10 9 CFU / well) was inoculated and cultured for 24 hours at 37°C in 5% CO2.

[0148] ② As comparison groups, a positive control group (Lysate solution added - surfactant) and a negative control group (No lysate solution, No bacterial cell addition) were prepared.

[0149] - LDH analysis (using a kit)

[0150] ① After centrifugation for 5 minutes (250 × g), 50 μL of the supernatant was transferred to a new 96-well plate.

[0151] ② 50 μL of LDH reaction mix was added and incubated at room temperature for 30 minutes to produce NADH.

[0152] ③ The generated NADH converted INT-Formazan (colorless) to Formazan (purple).

[0153] ④ After measuring the absorbance of the changed color at 450 nm, the cytotoxicity (%) was calculated according to the formula.

[0154] Strain cytotoxicity (%) 10 7 CFU / mL10 8 CFU / mL10 9CFU / mLStaphylococcus aureusATCC 25923 (comparative strain)--15.90±0.38% c LGG4.51±0.19% ab 3.19±0.29% a 0% a DSW38053.62±0.02% a 3.24±0.01% a 0% a

[0155] As shown in Table 4, the results of the LDH assay showed that LGG and DSW3805 strains were 10 7 CFU / mL ~ 10 9 It exhibited cytotoxicity within 4% at CFU / mL. When treated with the pathogenic bacterium S. aureus ATCC 25923, 10 9 High cytotoxicity was observed at CFU / mL (15.90%). All strains were confirmed to have lower cytotoxicity compared to the control strain. In other words, the results of the cytotoxicity assay for toxin production using the LDH assay are confirmed to be safe.

[0156]

[0157] (10) D-lactate production

[0158] 1) Sample preparation

[0159] ① The supernatant was obtained after culturing the strain at 37℃ for 24 hours.

[0160] ② Standard samples were prepared based on the D-lactate provided in the kit.

[0161] 2) D-lactic acid production test (using a kit)

[0162] 1. 50 μL of the prepared sample was dispensed into each well of a 96-well plate, and then 50 μL of the reaction mix (46 μL of D-lactate assay buffer, 2 μL of D-lactate assay substrate, and 2 μL of D-lactate enzyme mix) was dispensed.

[0163] ② Incubated at room temperature for 30 minutes.

[0164] ③ After measuring the absorbance of the changed color at 450 nm, the amount of D-lactate was measured according to the formula.

[0165] 3) D-Lactate Judgment Criteria: The racemase enzyme was determined to be positive or negative based on the presence or absence of D-Lactate production.

[0166] Strain LGGDSW3805D-Lactate 1.53±0.09 g / L a (16.99±0.94mM / L)1.59±0.03g / L ab (17.65±0.34 mM / L)L-lactate 1.53±0.01 g / L a (16.99±0.11mM / L)1.62±0.00g / L b (17.99±0.00 mM / L)

[0167] While L-lactic acid can be metabolized, D-lactic acid cannot. Probiotic strains possess DL-lactate racemase, an enzyme that converts L-lactate into D-lactate; therefore, if enzyme activity is strong, D-lactate can accumulate in newborns, children, and patients with short bowel syndrome, potentially causing acidosis. Consequently, the WHO has recommended a daily intake of D-lactic acid of 100 mg / kg or less.

[0168] As shown in Table 5, it was confirmed that LGG and DSW3805 strains produce 1.53 g / L and 1.59 g / L of D-lactate, respectively, and 1.53 g / L and 1.62 g / L of L-lactate, respectively.

[0169]

[0170] (11) Bile acid separation

[0171] 1) Preparation for BSH (Bile Salt Hydrolase) Activity Test on Agar Medium

[0172] ① MRS agar medium was prepared with 0.5% taurodeoxycholic acid (TDCA; bile acid, Sigma) added.

[0173] 2) BSH (Bile Salt Hydrolase) Activity Test and Analysis on Agar Medium

[0174] ① The strain was streaked onto MRS agar medium supplemented with 0.5% TDCA and cultured under anaerobic conditions at 37°C for 2 days.

[0175] ② In the case of strains with BSH activity, opaque white colonies were formed around the colonies.

[0176] As shown in Figure 7, no aggregation was observed for bile acid deconjugation in LGG and L. plantarumDSW3805.

[0177]

[0178] Example 3: Animal model experiment of DSS (dextran sulfate sodium)-induced colitis using Lactiplantibacillus plantarum DSW3805 strain

[0179] (1) Preparation of strain sample

[0180] Strain samples were cultured in a fermentation tank according to the lactic acid bacteria production method presented by the Korea Food Research Institute, then freeze-dried with a preservative for use in animal experiments.

[0181]

[0182] (2) Preparation of DSS-induced colitis animal model

[0183] 1) Preparation of experimental animals

[0184] Six-week-old male ICR mice were acclimatized by rearing them on standard feed for one week under constant temperature and humidity conditions (23±1℃, 53±2%). Subsequently, a randomized block design based on body weight was used to divide the normal control group, the control group (colitis-induced group), and the low-concentration strain group (10 8 CFU / day), strain high concentration group (10 9 The test substance was administered orally once a day for one week, divided into CFU / day. Only water was supplied to the experimental animals starting 24 hours before DSS administration.

[0185] 2) Induction of colitis

[0186] The experimental group was given drinking water in which 5% DSS was dissolved, while the control group was given water freely for 7 days. During the 7 days of DSS supply, the test substance was administered orally once a day, and on the 7th day of DSS supply, the experimental animals were sacrificed and their colons were removed.

[0187] Group Oral Administration (Days 1-14) DSS (Days 8-14) NC Normal Control Distilled Water Distilled Water PC Control Distilled Water 5% DSS (Drinking Water) LGGLGG Live Bacteria High Concentration Group 10 9 CFU / dayDSW3805-LDSW3805 Live bacteria low concentration group 10 8 CFU / dayDSW3805-HDSW3805 Live Bacteria High Concentration Group 10 9 CFU / day

[0188] 3) Analysis of disease activity

[0189] The disease activity index (DAI) was analyzed to assess the degree of colitis induction during the DSS supply period. Daily weight loss, stool consistency, the degree of blood in the stool, and the presence or absence of anal blood were visually checked.<DAI 평가 기준> It was evaluated according to [the criteria], calculated on a 0-4 point scale, and summed.

[0190] Group NCPCLGGDSW3805-LDSW3805-H Day 7 Disease Activity Index (Score) 0.00±0.0 a 13.3±0.6 d 11.4±0.7 bc 13.0±0.5 cd 11.2±0.7 b Weight reduction 0.00±0.00 a 3.00±0.24 bc 2.67±0.29 b 3.33±0.17 c 3.11±0.20 bc Diarrhea 0.00±0.00 a 3.33±0.17 c 3.22±0.15 c 3.33±0.17 c 2.67±0.24 bBloody stool 0.00±0.00 a 3.22±0.15 b 3.11±0.11 b 3.00±0.00 b 3.00±0.00 b Rectal bleeding 0.00±0.00 a 3.78±0.15 d 2.75±0.25 b 3.33±0.17 cd 3.14±0.14 bc

[0191] As shown in Table 7, when comparing the changes in disease activity index during the DSS administration period among the DSS administration groups excluding the NC group, the DAI in the DSW3805-H group was significantly reduced compared to the PC group on the 7th day of DSW3805 administration. The DAI on the 7th day of DSS administration was compared. The DAI in the PC group was significantly increased compared to the NC group. There was no change in the DSW3805-L group due to intake, while the DAI in the DSW3805-H group was significantly reduced compared to the PC group. Although it was confirmed to be diarrhea symptoms associated with the DSS administration group, it was confirmed that diarrhea associated with irritable bowel syndrome was significantly improved in the DSW3805-H group.

[0192] 4) Analysis of body weight, colon length, and spleen weight

[0193] Body weight and spleen weight were measured using a scale, and the length of the large intestine was measured using a ruler.

[0194] Group NCPCLGGDSW3805-LDSW3805-H Body weight (g) 33.6±1.3 b 29.9±0.9 a 32.2±0.8 ab 30.5±0.9 a 31.0±0.9 ab Large intestine length (cm) 8.1±0.3 c 5.6±0.3 a 6.0±0.2 ab 5.6±0.2 a 6.6±0.3 b Spleen weight (g) 0.08±0.01 a0.15±0.01 c 0.12±0.01 bc 0.11±0.01 b 0.13±0.01 bc

[0195] As shown in Table 8, the PC group showed a significantly lower body weight than the NC group. The DSW3805-L group showed no change due to intake, and the DSW3805-H group showed an increase in body weight compared to the PC group, but this was not statistically significant. Additionally, the PC group showed a significantly higher spleen weight compared to the NC group. The DSW3805-L group showed a significantly lower spleen weight compared to the PC group, and the DSW3805-H group showed a lower spleen weight compared to the PC group, but this was not statistically significant.

[0196] As shown in Figure 9, the PC group had a significantly reduced intestinal length compared to the NC group. The DSW3805-L group showed no significant difference in intestinal length compared to the PC group, while the DSW3805-H group had a significantly increased intestinal length compared to the PC group.

[0197]

[0198] (2) Cytokine analysis

[0199] Colon tissue was isolated, and TNF-α, IFN-γ, IL-1β, and IL-6 activities were measured using enzyme immunoassay (ELISA).

[0200] Group NCPCLGGDSW3805-LDSW3805-HIL-6(pg / mg protein)1.5±0.1 a 3.2±0.3 b 1.8±0.2 a 2.0±0.3 a 1.7±0.2 a TNF-α(pg / mL)176.3±17.2 a 313.9±30.0 c 270.3±3.5 bc 226.7±13.9 ab 225.4±11.5 ab IFN-γ(pg / mL)500.7±74.8 b 702.2±14.0c 440.2±40.5 b 311.5±27.3 a 272.9±11.2 a IL-1β(ng / mL)44.3±1.2 a 89.9±7.2 c 60.8±4.8 b 38.5±4.5 a 42.2±5.5 a

[0201] As shown in Table 9, the production of IL-6, TNF-α, IFN-γ, and IL-1β in the PC group was significantly increased compared to the NC group. In both the DSW3805-L and DSW3805-H groups, the production of IL-6, TNF-α, IFN-γ, and IL-1β was significantly decreased compared to the PC group, and there was no difference based on intake concentration.

[0202]

[0203] (3) Immunological analysis

[0204] Colon tissue was isolated, and IgG and IgA concentrations were measured using enzyme immunoassay (ELISA).

[0205] GroupNCPCLGGDSW3805-LDSW3805-HIgA(ug / mg protein)3.2±0.1 a 4.6±0.2 b 3.5±0.1 a 3.6±0.3 a 3.2±0.1 a IgG(ug / mg protein)26.2±2.8 a 44.4±1.5 b 38.2±0.9 b 27.6±2.8 a 27.5±3.6 a

[0206] As shown in Table 10, the PC group had significantly higher IgA and IgG concentrations compared to the NC group. Both the DSW3805-L and DSW3805-H groups had significantly lower IgA concentrations compared to the PC group, and there was no difference based on intake concentration.

[0207]

[0208] (4) Analysis of inflammation-related factors

[0209] Colon tissue was isolated, and leukotriene B4 (LTB4) and prostaglandin E2 (PGE2) content were measured by enzyme immunoassay (ELISA), and NF-kB expression was measured by RT-PCR and Western blot techniques.

[0210] GroupNCPCLGGDSW3805-LDSW3805-HLTB4(ng / mg protein)117.6±2.0 ab 133.3±6.6 b 129.9±4.4 ab 113.5±8.5 ab 109.7±9.0 ab PGE2(μg / mg protein)557.9±26.6 a 765.1±94.6 b 557.8±44.6 a 530.8±64.8 ab 423.8±29.2 a NF-kB (pg / mL)9.0±0.4 ab 14.5±1.5 b 5.1±1.4 a 10.2±1.0 ab 10.6±3.8 ab NF-κB (relative gene expression)1.2±0.1 a 34.7±2.7 c 7.1±0.40 b 8.5±0.8 b 9.1±0.2 b

[0211] As shown in Table 11, the PC group showed increased production of LTB4, PGE2, and NF-kB compared to the NC group, but this was not statistically significant. Both the DSW3805-L and DSW3805-H groups showed decreased production of LTB4, PGE2, and NF-kB compared to the PC group, but this was not statistically significant, and there were no differences based on intake concentration. Upon confirmation through NF-κB gene expression, NF-kB gene expression was significantly increased in the PC group compared to the NC group. Both the DSW3805-L and DSW3805-H groups showed significantly decreased NF-kB gene expression compared to the PC group, and there were no differences based on intake concentration.

[0212]

[0213] (5) Histological evaluation of the intestine

[0214] 1) Visual observation

[0215] The extent of colonic lesions, such as redness, thickening, and ulcers caused by tissue necrosis of the mucosal tissue, was observed visually according to the histological evaluation scale of the intestine. The extent of colonic lesions, including the presence or absence of redness, thickening, and ulcers, was observed according to the <visual evaluation criteria>, and the results were calculated by summing the scores evaluated on a scale of 0 to 10.

[0216] Group NCPCLGGDSW3805-LDSW3805-H Macroscopic score 0.3±0.2 a 9.3±1.5 c 6.8±1.4 bc 4.3±0.4 b 6.2±0.7 b

[0217] As shown in Table 12, the PC group showed a significant increase in visual damage scores compared to the NC group. Both the DSW3805-L and DSW3805-H groups showed a significant decrease in damage scores compared to the PC group, and there was no difference based on intake concentration.

[0218] 2) Microscopic observation

[0219] After double-staining colon tissue sections with H&E, items such as ulcers, inflammation, and depth of injury were observed microscopically according to the histological evaluation scale of the intestine. The intestinal mucosal thickness of the tissue sections was measured using a scanner program (Motic DSAssistant Software). Intestinal tissues with induced intestinal disease exhibited longer mucosal thickness than normal tissues due to typical inflammatory changes, such as the loss of intestinal crypts, thickening of the intestinal mucosa, and infiltration of inflammatory cells. After H&E staining of the tissue sections, the degree of colonic lesions, including edema, erosion, inflammation, necrosis, and ulceration of the intestinal mucosal tissue, was observed according to the <evaluation criteria through microscopic observation>, and the results were calculated by summing the scores evaluated on a scale of 0 to 6.

[0220] Group NCPCLGGDSW3805-LDSW3805-H Mucosal thickness (μm) 162.9±3.8 a 271.7±16.3 c 204.7±21.8 b 148.0±8.1 a 183.5±26.1 ab Pathological score 3.0±3.0 a 34.3±6.4 c 15.0±4.5 ab 23.0±5.6 bc 19.7±5.2 abc

[0221] As shown in Table 13 and Figure 10, the intestinal mucosal thickness of the PC group significantly increased compared to the NC group. Both the DSW3805-L and DSW3805-H groups showed a significant decrease in intestinal mucosal thickness compared to the PC group. Additionally, the damage score measured by microscopic examination significantly increased in the PC group compared to the NC group. Although both the DSW3805-L and DSW3805-H groups showed a decrease in damage score compared to the PC group, this difference was not statistically significant.

[0222]

[0223] (6) Analysis of diarrhea relief / antidiarrheal effects

[0224] After ingesting the test substance, the moisture content in the stool was measured to determine if diarrhea was alleviated. Immediately after defecation, the wet weight of the stool was measured, and then the stool was freeze-dried to measure the dry weight.

[0225] - Moisture content of feces (%) = (Wet weight of feces - Dry weight of feces) / Wet weight of feces × 100

[0226] Group NCPCLGGDSW3805-LDSW3805-H Wet stool weight (mg / day) 710.8±27.7 b 610.1±20.0 a 683.4±11.9 ab 742.3±18.4 b 728.5±40.9 b Fecal moisture content (%) 28.7±1.0 b 20.9±1.9 a 23.6±2.2 ab 28.3±2.3 b 24.1±2.4 ab

[0227] As shown in Table 14, the wet weight of the feces in the PC group was significantly lower than that of the NC group. Both the DSW3805-L and DSW3805-H groups had significantly higher wet weights of feces compared to the PC group, and there was no difference due to intake concentration.

[0228] In addition, the PC group had a significantly lower fecal moisture content compared to the NC group. The DSW3805-L group had a significantly higher fecal moisture content compared to the PC group, and the DSW3805-H group also had a higher fecal moisture content compared to the PC group, but the difference was not statistically significant.

[0229]

[0230] (7) Measurement of the number of bacteria in the intestines (beneficial / harmful bacteria)

[0231] 0.1 g of fecal matter was inoculated into 900 μL of Brain Heart Infusion (BHI) broth containing 0.05% L-cysteine ​​hydrochloride hydrate. 10 3 and 10 5 The diluted solution was spread onto a 100 mm agar plate and incubated at 37°C to count the number of colonies.

[0232] As shown in Figure 12, when beneficial and harmful bacteria were plated and cultured on each selective medium, the number of Lactobacillus bacteria was found to be 6.82±0.32, 7.05±0.55, 7.46±0.27, and 7.44±0.59 log CFU / g in the NC, PC, LGG, and DSW3805-H groups, respectively. The number of Bifidobacterium bacteria was found to be 7.14±0.43, 6.71±0.53, 7.22±0.40, and 7.34±0.30 log CFU / g in the NC, PC, LGG, and DSW3805-H groups, respectively. On the other hand, the bacterial counts of Staphylococcus aureus were found to be 4.67±0.87, 5.57±1.03, 4.90±0.57, and 4.42±0.34 log CFU / g in the NC, PC, LGG, and DSW3805-H groups, respectively, with the lowest bacterial count of Staphylococcus aureus found in the DSW3805-H group.

[0233]

[0234] (8) Metagenomics Analysis

[0235] Fecal samples were collected and provided to the Korea Institute of Medical Science for metagenomic analysis.

[0236] Microbiome analysis was performed using Miseq (Illumina) instruments, and metagenome analysis was conducted on the FASTQ files obtained from Miseq analysis using the EZBioCloud (CJ Bioscience, Inc.) platform and the BaseSpace (Illumina) platform. The diversity and uniformity of microbial communities within the samples were analyzed using the Shannon diversity index and Simpson index, and the correlation between samples was confirmed using beta diversity.

[0237] Microbiome analysis for improving the gut environment was conducted on a total of 39 fecal samples from 5 groups. According to the Alpha-diversity results, the Shannon index, which is an indicator of microbial diversity in the samples, was 4.73 for the control group, while the DSS-treated groups showed values ​​similar to or lower than those of the DSS-treated group.

[0238]

[0239] Figures 11a to 11e show changes in the intestinal microbial phylum community following the ingestion of L. plantarumDSW3805.

[0240] Analysis of community changes in the gut phylum revealed that Firmicutes were the dominant species in all groups, accounting for a high proportion of 62.9% in the DSW3805-H group and the lowest proportion of 56.7% in the LGG group. The next dominant species were identified as Bacteroidetes and Proteobacteria.

[0241] In the case of Bacteroidetes, the NC group accounted for the highest proportion at 31.7%, while the DSW3805-H group accounted for the lowest proportion at 24.1%. The Firmicutes:Bacteroidetes ratio decreased in the DSS group (1.75:1) compared to the NC group (2.23:1). In contrast, the LGG and DSW3805-H groups showed ratios of 2.09:1 and 2.53:1, respectively, with the DSW3805-H group having the highest proportion. It is confirmed that the decrease in Firmicutes:Bacteroidetes observed in inflammatory bowel disease is alleviated upon probiotic treatment. Proteobacteria include various pathogenic bacteria such as E. coli, Salmonella, and Helicobacter. While the NC group was 4.1%, it increased to 8.4% in the PC group. In particular, the LGG group and the DSW3805-H group were 10.2% and 8.4%, respectively.

[0242] Group NCPCLGGDSW3805-HFirmicutes: Bacteroidetes2.23±0.48:1 b 1.75±0.47:1 a 2.09±0.50:1 ab 2.53±0.56:1 c

[0243] Analysis of community changes in the intestinal microbial class revealed that Clostridia includes genera similar to Clostridium, such as Clostridium perfringens, Clostridium difficile, Clostridium tetani, Clostridium botulinum, and Clostridium acetobutylicum. The PC group accounted for 31.2%, while the LGG group accounted for 35.6%, and the DSW3805-H group accounted for the lowest percentage at 33.6%.

[0244] Analysis of changes in the intestinal microbial family community showed that the proportion of beneficial bacteria, Bifidobacteriaceae, was 2.51% in the control group, 0.54% in DSS, 0.93% in LGG, and 2.71% in DSW3805, while the proportion of harmful bacteria, Helicobacteriaceae, was 2.49% in the NC group, 5.12% in the PC group, 5.16% in the LGG group, and 4.68% in the DSW3805-H group.

[0245] Analysis of changes in the gut microbial community revealed that Bifidobacterium was distributed as follows: NC group (2.51%), PC group (0.54%), LGG group (0.93%), and DSW3805-H group (2.71%), with the DSW3805-H group accounting for the highest proportion.

[0246] Analysis of changes in the intestinal microbial community revealed that the total proportions of Lactobacillus murinus, Lactobacillus gasseri, and Lactobacillus reuteri were NC group (15.78%), PC group (10.31%), LGG group (10.21%), and DSW3805-H group (12.12%). The proportions of Bifidobacterium pseudolongum were NC group (2.50%), PC group (0.49%), LGG group (0.91%), and DSW3805-H group (2.32%). The proportions of the harmful bacterium Helicobacter mesocricetorum were NC group (2.12%), PC group (4.67%), LGG group (4.84%), and DSW3805-H group (4.04%).

[0247]

[0248] (9) Short-chain fatty acid analysis

[0249] Fecal samples were collected and provided to the Korea Institute of Medical Science for short-chain fatty acid analysis.

[0250] Group short-chain fatty acids (μmol / g) butyric acid, acetate, propionic acid PC3 3.46±7.97 a 10.19±3.48 a 16.30±3.79 a LGG57.16±14.95 b 17.73±6.39 b 31.90±8.90 b DSW3805-H78.53±12.29 c 23.52±4.14 b 36.28±11.96 b

[0251] As shown in Table 16, the DSW3805 group was found to have higher levels of short-chain fatty acids, such as acetic acid, propionic acid, and butyric acid, compared to the LGG group.

[0252]

[0253] (10) Analysis of harmful intestinal enzyme activity

[0254] 1) Preparation of crude enzyme solution

[0255] 1 g of the contents of the cecum was suspended in 20 mL of cold sterile physiological saline and centrifuged at 500 rpm for 5 minutes. The supernatant, from which fiber and other impurities were removed, was centrifuged at 8,000 rpm for 30 minutes, and the precipitate was suspended in 20 mM phosphate buffer to be used as the crude enzyme solution.

[0256] 2) Measurement of calprotectin and β-glucuronidase activity

[0257] 0.38 mL of 0.1 M phosphate buffer and 0.02 mL of 10 mM ρ-nitrophenyl-β-D-glucuronide were added to 0.1 mL of enzyme solution and reacted at 37°C for 60 minutes. After stopping the reaction by adding 0.5 mL of 0.5 N NaOH, 1 mL of distilled water was added and centrifuged (2,000×g, 20 min), and the absorbance of the supernatant was measured at 405 nm.

[0258] Group Calprotectin (ng / g) β-Glucuronidase (U / mg) Control 0.52±0.05 ab 4.66±0.67a DSS0.53±0.15 b 5.03±0.93 ab LGG0.50±0.11 b 4.85±0.99 ab DSW3805-H0.38±0.19 a 4.58±0.88 a

[0259] As shown in Table 17, calprotectin is identified as a factor in inflammatory bowel disease, and it is known that calprotectin levels increase when inflammation occurs. When calprotectin was tested in the feces, it was confirmed to be decreased in the DSW3805 group. In addition, when the activity of β-glucuronidase, a harmful intestinal enzyme, was checked, a value of 4.58 U / mg was confirmed in the DSW3805 group, which was lower than that of LGG.

[0260]

[0261] The following describes an example of a composition comprising the strain or culture solution of the present invention as an active ingredient, but the present invention is not intended to be limited but merely to be described in detail.

[0262]

[0263] Preparation Example 1: Preparation of a powder

[0264] 20 mg of Lactiplantibacillus plantarum DSW3805 strain or culture

[0265] Lactose monohydrate 100 mg

[0266] Talc 10 mg

[0267] The above ingredients were mixed and filled into an airtight bag to produce a powder.

[0268]

[0269] Formulation Example 2: Preparation of Tablets

[0270] 10 mg of Lactiplantibacillus plantarum DSW3805 strain or culture

[0271] 100 mg of corn starch

[0272] Lactose monohydrate 100 mg

[0273] Magnesium stearate 2 mg

[0274] After mixing the above ingredients, tablets were manufactured by compressing them according to the conventional method of manufacturing tablets.

[0275]

[0276] Formulation Example 3: Preparation of capsules

[0277] 10 mg of Lactiplantibacillus plantarum DSW3805 strain or culture

[0278] Microcrystalline cellulose 3 mg

[0279] Lactose monohydrate 14.8 mg

[0280] Magnesium stearate 0.2 mg

[0281] After mixing the above ingredients, a capsule was manufactured by filling it into a gelatin capsule according to a conventional method for manufacturing capsules.

[0282]

[0283] Formulation Example 4: Preparation of an injectable

[0284] 10 mg of Lactiplantibacillus plantarum DSW3805 strain or culture

[0285] Mannitol 180 mg

[0286] 2974 mg sterile distilled water for injection

[0287] Sodium monohydrogen phosphate 26 mg

[0288] After mixing the above ingredients, the above ingredients were prepared in an amount of 2 mL per ampoule according to the conventional method of preparing injectables.

[0289]

[0290] Formulation Example 5: Preparation of a liquid formulation

[0291] 10 mg of Lactiplantibacillus plantarum DSW3805 strain or culture

[0292] 10 g isomerized sugar

[0293] 5 g mannitol

[0294] Appropriate amount of purified water

[0295] Appropriate amount of lemon flavor

[0296] The above ingredients are dissolved in purified water according to a conventional manufacturing method, an appropriate amount of lemon scent is added, then purified water is added to adjust the total volume to 100 mL, sterilized, and filled into a brown bottle to prepare a liquid preparation.

[0297]

[0298] Preparation Example 6: Preparation of a health functional food

[0299] 10 mg of Lactiplantibacillus plantarum DSW3805 strain or culture

[0300] Appropriate amount of vitamin mixture

[0301] Vitamin A Acetate 70 µg

[0302] Vitamin E 1.0 mg

[0303] Vitamin B1 0.13 mg

[0304] Vitamin B2 0.15 mg

[0305] Vitamin B6 0.5 mg

[0306] Vitamin B12 0.2 µg

[0307] Vitamin C 10 mg

[0308] Biotin 10 µg

[0309] Nicotinamide 1.7 mg

[0310] Folic acid 50 µg

[0311] Calcium pantothenate 0.5 mg

[0312] Appropriate amount of mineral mixture

[0313] Ferrous sulfate 1.75 mg

[0314] Zinc oxide 0.82 mg

[0315] Magnesium carbonate 25.3 mg

[0316] Potassium dihydrogen phosphate 15 mg

[0317] 55 mg dicalcium phosphate

[0318] Potassium citrate 90 mg

[0319] Calcium carbonate 100 mg

[0320] Magnesium chloride 24.8 mg

[0321] The composition ratio of the above vitamin and mineral mixture is composed of ingredients that are relatively suitable for health functional foods as a preferred example, but the mixing ratio may be modified at will. The above ingredients may be mixed according to a conventional method for manufacturing health functional foods, then granules may be prepared and used to manufacture health functional foods according to a conventional method.

[0322]

[0323] Preparation Example 7: Preparation of a health drink

[0324] 10 mg of Lactiplantibacillus plantarum DSW3805 strain or culture

[0325] 15 g of Vitamin C

[0326] Vitamin E (powder) 100 g

[0327] Iron lactate 19.75 g

[0328] 3.5 g zinc oxide

[0329] Nicotinamide 3.5 g

[0330] Vitamin A 0.2 g

[0331] Vitamin B1 0.25 g

[0332] Vitamin B2 0.3g

[0333] Quantification of purified water

[0334] The above ingredients are mixed according to a conventional method for manufacturing health drinks, then stirred and heated at 85°C for about 1 hour, the resulting solution is filtered, obtained in a sterilized 2 L container, sealed and sterilized, and then refrigerated and used to manufacture the health drink composition of the present invention.

[0335]

[0336] Although the above composition ratio was formulated by mixing ingredients relatively suitable for beverages as a preferred embodiment, the mixing ratio may be arbitrarily modified according to regional and ethnic preferences, such as consumer groups, countries of demand, and intended uses.

[0337]

[0338] The foregoing description of the present invention is for illustrative purposes only, and those skilled in the art will understand that other specific forms can be easily modified without altering the technical spirit or essential features of the present invention. Therefore, the embodiments described above should be understood as illustrative in all respects and not restrictive.

[0339]

[0340] [Consignment Number]

[0341] Depository Name: Korean Culture Collection of Microorganisms (Overseas)

[0342] Accession Number: KCCM 13544P

[0343] Date of Trust: January 22, 2025

[0344]

[0345]

[0346] In this regard, the depositor 'Baekhyeon-dong' in the Microorganism Deposit Certificate entered into an assignment agreement to transfer the right to obtain a patent related to the said microorganism to the applicant 'WithBio Co., Ltd.' as of February 24, 2025, after depositing the said microorganism for patenting.

Claims

1. Lactiplantibacillus plantarum DSW3805 strain (KCCM 13544P) having pathogen adhesion inhibitory activity and immunomodulatory ability in intestinal tissue.

2. In Paragraph 1, The above DSW3805 strain is characterized by including a DNA base sequence encoding the 16S rRNA of SEQ ID NO. 1, DSW3805 strain (KCCM 13544P).

3. In Paragraph 1, DSW3805 strain (KCCM 13544P), characterized in that the above pathogen comprises one or more selected from the group consisting of Listeria monocytogenes, Staphylococcus aureus, Escherichia coli, and Salmonella enteritidis.

4. In Paragraph 1, The above immune regulatory ability is i) Reduction in nitric oxide (NO) production; ii) reduction in the expression of pro-inflammatory cytokines comprising one or more selected from the group consisting of TNF-α, IL-1β, COX-2, IL-6, and IL-8; or iii) DSW3805 strain (KCCM 13544P), characterized by activation of the NF-κB or MAPK pathway.

5. In Paragraph 1, The above DSW3805 strain is characterized by increasing the expression of intestinal motility-related genes, including one or more selected from the group consisting of ZO-1, Claudin-1, Occludin, and MUC2, in intestinal tissue (KCCM 13544P).

6. A pharmaceutical composition for the prevention or treatment of inflammatory bowel disease or irritable bowel syndrome comprising the Lactiplantibacillus plantarum DSW3805 strain (KCCM 13544P) or a culture medium thereof as an active ingredient.

7. In Paragraph 6, The above pharmaceutical composition is i) Effect of reducing intestinal mucosal thickness and intestinal damage increased by inflammation in the large intestine; ii) Effect of reducing the expression of pro-inflammatory cytokines, including one or more selected from the group consisting of IL-6, TNF-α, IFN-γ, and IL-1β, which are increased due to inflammation in the large intestine; iii) an effect of reducing IgA or IgG concentrations increased due to inflammation in the large intestine; or iv) A pharmaceutical composition for the prevention or treatment of inflammatory bowel disease or irritable bowel syndrome, characterized by having an effect of reducing the expression of inflammation-related factors, comprising one or more selected from the group consisting of LTB4, PGE2, and NF-kB, which are increased due to inflammation in the large intestine.

8. In Paragraph 6, A pharmaceutical composition for the prevention or treatment of inflammatory bowel disease or irritable bowel syndrome, characterized by having a diarrhea-relieving effect by restoring the reduced water content of feces.

9. In Paragraph 6, The above pharmaceutical composition is i) Results of phylum series analysis of feces, showing an increasing effect of Firmicutes:Bacteroidetes values ​​reduced by inflammation in the colon; ii) Results of genus lineage analysis of feces, showing an effect of increasing the proportion of Bifidobacterium reduced by inflammation in the colon; iii) A pharmaceutical composition for the prevention or treatment of inflammatory bowel disease or irritable bowel syndrome, characterized by having, as a result of species lineage analysis of feces, an increase in the total proportion of Lactobacillus murinus, Lactobacillus gasseri, and Lactobacillus reuteri reduced due to inflammation in the large intestine, and a decrease in the proportion of Helicobacter mesocricetorum increased due to inflammation in the large intestine.

10. In Paragraph 6, The above pharmaceutical composition is i) an effect of increasing short-chain fatty acids comprising one or more selected from the group consisting of acetic acid, propionic acid, and butyric acid in feces; or ii) A pharmaceutical composition for the prevention or treatment of inflammatory bowel disease or irritable bowel syndrome, characterized by having an effect of reducing the activity of calprotectin and β-glucuronidase in feces.

11. In Paragraph 1, The dose of the above DSW3805 strain is 1×10 5 CFU / day to 1×10 12 A pharmaceutical composition characterized by having a CFU / day.

12. A health functional food composition for the prevention or improvement of inflammatory bowel disease or irritable bowel syndrome comprising the Lactiplantibacillus plantarum DSW3805 strain (KCCM 13544P) or a culture solution thereof as an active ingredient.

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