WO2020226467A1 - Enterococcus lactis wikim0107 strain having effects of regulating immune functions and alleviating inflammatory bowel disease and use thereof - Google Patents

Abstract

The present invention relates to lactic acid bacteria Enterococcus lactis having effects of regulating immune functions and alleviating an inflammatory bowel disease and use thereof. The Enterococcus lactis has no cytotoxicity, has an immune-boosting effect and NO generation and ROS generation increasing effects, increases the expression of inflammatory cytokines and the number of immune cells, and has a bowel disease alleviating effect in an inflammatory bowel disease animal model, and therefore, the Enterococcus lactis can be very helpfully used in a health functional food, medicinal product, feed composition, or veterinary composition for regulating immune functions and preventing, alleviating, or treating an inflammatory bowel disease.

Description

Enterococcus lactis WIKIm0107 strain having the effect of regulating immune function and improving inflammatory bowel disease and its use

The present invention relates to a strain of Enterococcus lactis WiKim0107 having an effect of regulating immune function and improving inflammatory bowel disease and its use.

Skate, belonging to the stingray family, is a cartilage, benthic fish, and is distributed in the coast of Heuksan Island and the west coast of Korea, and is caught in Mokpo and Yeongwang, but due to the recent decline, it is also imported from Chile, Uruguay, and Argentina. Mainly in Jeollanam-do, fermented skates are used habitually as a traditional food, and the population who enjoys its unique aroma and taste is increasing. Hongtak, which is eaten with fermented skateboard with makgeolli, is the most famous, and in the southwestern coast of Jeollanam-do, fermented skateboard is hardly missed for feast food.

Skatefish contains a lot of nitrogen compounds, urea and trimethylamine oxide, in the body to control the osmotic pressure in the deep sea, and these components are converted into ammonia and trimethylamine by fermentation, which irritates the nose. A stinging taste is produced. Skate is a high-protein, low-fat fish with 19% protein and 0.9% fat. Like sharks and rays, it contains a lot of betaine and taurine, and has a high content of calcium and urea. The reported functional effects of skate include cell membrane stabilization, cholesterol-regulating effects to prevent vascular disease and heart failure, taurine, which is important for growth and development, linolenic acid and arachidonic acid, which help reduce serum cholesterol and improve brain growth and cognitive function. It contains essential fatty acids, and contains a large amount of EPA and DHA, so it is effective in preventing coronary artery disease, thrombosis, brain development, and visual function. Collagen and cartilage present in the skin of skates contain chondroitin, a mucopolysaccharide protein, and are known as healthy and tonic foods. Recently, studies on physiological activity and component analysis of skates have been made, but studies on microorganisms present in skates are lacking. Skate is a traditional fermented food in the Jeonnam region of Korea, and there will be a variety of fermented microorganisms that are different from existing cabbage kimchi.

On the other hand, inflammatory bowel disease (IBD) is a disease including ulcerative colitis, Crohn's disease or Behcet's disease, and it is known that the activation of inflammatory cells is an important etiology, and the activation of inflammatory cells with anti-inflammatory agents, immunosuppressants, etc. Treatment through inhibition is necessary. That is, even in the case of bowel disease, the treatment method must be different depending on the specific etiology, and in particular, in the case of inflammatory bowel disease, the provision of a method for suppressing and alleviating inflammation-related symptoms is the most important factor in the treatment of the disease. Persistent or inadequate activation of the intestinal immune system plays an important role in the pathophysiology of chronic mucosal inflammation, especially infiltrating neutrophils, macrophages, lymphocytes and mast cells, resulting in mucosal destruction and ulcers.

In inflammatory bowel disease, various inflammatory cytokines are secreted from the mucous membrane of the intestine. TNF-α is highly expressed in the colon lumen and colon epithelial cells of patients with ulcerative colitis, and according to recent studies, TNF-α is known to play an important role in the pathogenesis of ulcerative colitis. Infliximab, an anti-TNF-α antibody, is known to be effective not only for the treatment of boils, but also for the treatment of previously untreated Crohn's disease, but these treatments are expensive, and some patients suffer from fluid reactions or infectious complications. The same side effects are caused. Currently, as a treatment for inflammatory bowel disease, 5-aminosalicylic acid (5-ASA) drugs that block the production of prostaglandins, such as sulfasalazine, are used, or immunosuppressants such as steroids are used. . Sulfasalazine is likely to cause side effects or adverse effects such as fullness, headache, rash, liver disease, leukopenia, agranulocytosis, and male infertility.

In addition, it is unclear whether sulfasalazine has a sufficient inhibitory effect on recurrence in patients with incisions in the intestine or patients with remission. Steroid-type immunosuppressants are adrenal cortical steroids, which are recognized for their short-term effects, but cannot improve long-term prognosis, and induced infectious diseases, secondary adrenal insufficiency, peptic ulcer, diabetes, mental disorders, steroidal nephropathy, etc. In terms of the same side effects, there is a limitation that should be used only in acute cases. There is still no reliable treatment for inflammatory bowel disease, and the development of effective treatments for these diseases is required.

On the other hand, Korean Patent Publication No. 2019-0005125 discloses an anti-influenza virus composition containing Enterococcus canintestini strain, and Korean Patent No. 1909936 discloses an anti-influenza virus composition containing fermented lactic acid bacteria of natural food ingredients. Although a functional food composition for enhancing immune activity and a method for producing the same have been disclosed, there has not been disclosed a strain of Enterococcus lactis WiKim0107 having an effect of regulating immune function and improving inflammatory bowel disease of the present invention and its use.

The present invention is derived from the above requirements, the present invention provides an Enterococcus lactis WiKim0107 strain having an effect of modulating immune function and improving inflammatory bowel disease, and the Enterococcus lactis WiKim0107 strain is cytotoxic There is no immunity, it has the effect of promoting NO production and ROS production, enhancing the expression of inflammatory cytokines and the number of immune cells, and confirming that there is an effect of improving bowel disease in an animal model of inflammatory bowel disease. The invention was completed.

In order to achieve the above object, the present invention provides an Enterococcus lactis WiKim0107 strain (Accession No.: KACC 81090BP) having an effect of regulating immune function and preventing, improving or treating inflammatory bowel disease.

In addition, the present invention is an immune function comprising at least one selected from the Enterococcus lactis WiKim0107 strain, the culture of the strain, the concentrate of the culture, the dried product of the culture, and the extract of the culture as an active ingredient It provides a health functional food composition for controlling and preventing or improving inflammatory bowel disease.

In addition, the present invention is an inflammatory bowel comprising as an active ingredient at least one selected from the Enterococcus lactis WiKim0107 strain, the culture of the strain, the concentrate of the culture, the dried product of the culture, and the extract of the culture. It provides a pharmaceutical composition for preventing or treating diseases.

In addition, the present invention is an immune function comprising at least one selected from the Enterococcus lactis WiKim0107 strain, the culture of the strain, the concentrate of the culture, the dried product of the culture, and the extract of the culture as an active ingredient It provides a feed composition for controlling and preventing or improving inflammatory bowel disease.

In addition, the present invention is an immune function comprising at least one selected from the Enterococcus lactis WiKim0107 strain, the culture of the strain, the concentrate of the culture, the dried product of the culture, and the extract of the culture as an active ingredient It provides a veterinary composition for controlling and preventing or treating inflammatory bowel disease.

The present invention relates to lactic acid bacteria Enterococcus lactis having an effect of regulating immune function and improving inflammatory bowel disease and its use, and Enterococcus lactis WiKim0107 of the present invention has no cytotoxicity, has an immune enhancing effect, and NO It has the effect of promoting production and ROS production, increasing the expression of inflammatory cytokines and the number of immune cells, and has the effect of improving bowel disease in an animal model of inflammatory bowel disease, so it is used for controlling immune function and preventing, improving or treating inflammatory bowel disease. It can be very useful in health functional food, pharmaceutical, feed composition or veterinary composition.

1 is a result of confirming the anti-inflammatory activity of six lactic acid bacteria isolated from skate, (A) is the NO production rate (%), (B) is the amount of TNF-α production, (C) is the amount of IL-1β production and (D) Is the result of confirming the amount of IL-6 produced.

2 is a result of confirming the survival rate of Raw264.7 cells of the lactic acid bacteria WiKim0107 isolated from skates.

3 is a result of confirming the cell proliferation of macrophages Raw264.7 treated with Enterococcus lactis (WiKim0107) of the present invention. Con is a cell that has not been treated with anything, PBS is a cell treated with PBS solution to confirm that there is no difference due to solvent, LPS is a cell treated with lipopolysaccharide, WiKim0107 is 10 ⁴ ~ 10 ⁷ CFU/ It was treated with ml of Enterococcus lactis. * Indicates that there is a statistically significant difference compared to Con, which is p<0.05.

FIG. 4 is an analysis result of NO (Nitric oxide) production (A), iNOS expression (B) and ROS (Reactive oxygen species) production (C) according to Enterococcus lactis (WiKim0107) treatment of the present invention. * LPS treatment compared to Con; And the group treated with Enterococcus lactis (WiKim0107) of the present invention; p<0.05, indicating that NO production or iNOS expression was increased.

Figure 5 is interleukin (IL)-1β (A), IL-6 (B), tumor necrosis factor alpha (TNF-α) (C) and cyclooxygenase 2 (in accordance with the treatment of Enterococcus lactis (WiKim0107) of the present invention) It is the result of gene expression analysis of COX-2)(D). * LPS treatment compared to Con; And the group treated with Enterococcus lactis (WiKim0107) of the present invention; p<0.05, indicating that the gene expression was increased.

6 is a result of confirming the weight change (A), the distribution of immune cells (B), and the gene expression level of inflammatory cytokines (C) by administering Enterococcus lactis (WiKim0107) of the present invention to an animal model. * Indicates that the gene expression was increased in the group treated with Enterococcus lactis (WiKim0107) of the present invention compared to Con, p<0.05.

7 is a change in body weight (A), intestinal disease activity index (DAI) (B) and length change of colon tissue after administration of Enterococcus lactis (WiKim0107) of the present invention to an animal model in which inflammatory bowel disease is induced This is the result of checking (C).

8 is a change in the number of immune cells (T cells, B cells, NK cells, macrophages, dendritic cells) after administration of Enterococcus lactis (WiKim0107) of the present invention to an animal model in which inflammatory bowel disease is induced This is the result of checking.

The present invention relates to an Enterococcus lactis WiKim0107 strain (Accession No.: KACC 81090BP) having an effect of regulating immune function and preventing, improving or treating inflammatory bowel disease. The Enterococcus lactis WiKim0107 strain is preferably isolated from skateboard or skate kimchi, but is not limited thereto.

In addition, the present invention is an immune function comprising at least one selected from the Enterococcus lactis WiKim0107 strain, the culture of the strain, the concentrate of the culture, the dried product of the culture, and the extract of the culture as an active ingredient It relates to a health functional food composition for controlling and preventing or improving inflammatory bowel disease.

The active ingredient is preferably prepared in any one formulation selected from powder, granule, pill, tablet, capsule, candy, syrup, and beverage, but is not limited thereto.

When using the above-mentioned strain of the present invention or a culture solution thereof as a health functional food composition, the strain or a culture solution thereof may be added as it is, or may be used with other foods or food ingredients, and may be appropriately used according to a conventional method. have. The mixing amount of the active ingredient may be appropriately determined according to the purpose of use (prevention, health or therapeutic treatment).

The health functional food composition of the present invention may further include ingredients that are commonly added at the time of food production and are food wise acceptable. For example, when prepared as a beverage, it may further include one or more components from citric acid, liquid fructose, sugar, glucose, acetic acid, malic acid, fruit juice, etc. in addition to the strain of the present invention.

The amount that can be included as an active ingredient of the health functional food composition according to the present invention is appropriately selected according to the age, sex, weight, condition, and symptoms of a person who wants a health functional food for controlling immune function and preventing or improving inflammatory bowel disease. It may be, and preferably contained in about 0.01 to 10.0g per adult basis, and by ingesting foods having such a content, it is possible to obtain an effect of controlling immune function and preventing or improving inflammatory bowel disease.

In addition, the present invention is an inflammatory bowel comprising as an active ingredient at least one selected from the Enterococcus lactis WiKim0107 strain, the culture of the strain, the concentrate of the culture, the dried product of the culture, and the extract of the culture. It relates to a pharmaceutical composition for preventing or treating diseases.

The inflammatory bowel disease is preferably ulcerative colitis, Crohn's disease, or Behcet's disease, but is not limited thereto.

The term'prevention' in the present invention refers to any action that suppresses or delays the onset of inflammatory bowel disease by administration of the pharmaceutical composition according to the present invention, and'treatment' refers to inflammatory bowel by administration of the pharmaceutical composition. It refers to any action in which the symptoms of the suspected and onset individual are improved or beneficially altered. The pharmaceutical composition of the present invention may further include suitable carriers, excipients, and diluents commonly used in the preparation of pharmaceutical compositions. The pharmaceutical compositions according to the present invention are formulated in the form of oral dosage forms such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, etc., external preparations, suppositories, and sterile injectable solutions according to a conventional method. Can be used. Carriers, excipients and diluents that may be included in the pharmaceutical composition according to the present invention include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum acacia, alginate, gelatin, calcium phosphate, calcium And various compounds or mixtures including silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and mineral oil. In the case of formulation, it is prepared using diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrants, and surfactants that are usually used. Solid preparations for oral administration include tablets, pills, powders, granules, capsules, and the like, and such solid preparations include at least one excipient, such as starch, calcium carbonate, in the strain or the endoplasmic reticulum derived from the strain. , Sucrose (sucrose) or lactose (lactose), gelatin, etc. are mixed and prepared. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used. Liquid preparations for oral use include suspensions, liquid solutions, emulsions, syrups, etc. In addition to water and liquid paraffin, which are commonly used simple diluents, various excipients such as wetting agents, sweetening agents, fragrances, and preservatives may be included. . Preparations for parenteral administration include sterile aqueous solutions, non-aqueous solutions, suspensions, emulsions, lyophilized preparations, and suppositories. As the non-aqueous solvent and suspending agent, propylene glycol, polyethylene glycol, vegetable oil such as olive oil, and injectable ester such as ethyl oleate may be used. As a base for suppositories, witepsol, macrogol, tween 61, cacao butter, laurin paper, glycerogelatin, and the like may be used.

The pharmaceutical composition of the present invention may be administered in a pharmaceutically effective amount, and the term'pharmaceutically effective amount' of the present invention is sufficient to prevent or treat a disease at a reasonable benefit/risk ratio applicable to medical prevention or treatment. It means an amount, and the effective dose level is the severity of the disease, the activity of the drug, the patient's age, weight, health, sex, the patient's sensitivity to the drug, the time of administration of the composition of the present invention used, the route of administration and the rate of excretion, treatment The duration, factors including drugs used in combination or co-use with the composition of the present invention used, and other factors well known in the medical field. The pharmaceutical composition of the present invention may be administered as an individual therapeutic agent or administered in combination with other therapeutic agents, and may be administered sequentially or simultaneously with a conventional therapeutic agent. And can be administered single or multiple. Considering all of the above factors, it is important to administer an amount capable of obtaining the maximum effect in a minimum amount without side effects.

The frequency of administration of the composition of the present invention is not particularly limited thereto, but may be administered once a day or divided into several doses. The above dosage does not in any way limit the scope of the present invention.

The term "individual" of the present invention includes, without limitation, mammals including mice, livestock, humans, etc. that are likely to develop inflammatory bowel disease or have developed.

In the method for preventing or treating inflammatory bowel disease of the present invention, the route of administration of the pharmaceutical composition may be administered through any general route as long as it can reach the target tissue. The pharmaceutical composition of the present invention is not particularly limited thereto, but may be administered through a route such as oral administration or rectal administration, and in some cases, may be administered by other routes depending on the purpose. However, since the WiKim0107 strain may be denatured by gastric acid when administered orally, the oral composition should be coated with an active agent or formulated to protect it from degradation in the stomach. In addition, the composition can be administered by any device capable of moving the active substance to the target cell.

In addition, the present invention is an immune function comprising at least one selected from the Enterococcus lactis WiKim0107 strain, the culture of the strain, the concentrate of the culture, the dried product of the culture, and the extract of the culture as an active ingredient It relates to a feed composition for controlling and preventing or improving inflammatory bowel disease.

The strain is as described above, and may be added as a feed composition for the purpose of regulating immune function and preventing or improving inflammatory bowel disease. The feed composition may contain a feed additive. The feed additive of the present invention corresponds to an auxiliary feed in the feed management law.

In the present invention, the term'feed' may mean any natural or artificial diet, one meal meal, etc., or a component of the one meal meal for animals to eat, ingest, and digest. The kind of feed is not particularly limited, and feed commonly used in the art may be used. Non-limiting examples of the feed include vegetable feeds such as grains, root fruits, food processing by-products, algae, fiber, pharmaceutical by-products, oils and fats, starches, meals or grain by-products; Animal feeds such as proteins, inorganic logistics, oils and fats, minerals, oils and fats, single-cell proteins, zooplanktons or foods. These may be used alone or in combination of two or more.

In addition, the present invention is an immune function comprising at least one selected from the Enterococcus lactis WiKim0107 strain, the culture of the strain, the concentrate of the culture, the dried product of the culture, and the extract of the culture as an active ingredient It relates to a veterinary composition for controlling and preventing or treating inflammatory bowel disease.

Excipients and diluents that may be included in the veterinary composition of the present invention include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum acacia, alginate, gelatin, calcium phosphate, calcium silicate, Cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and mineral oils. The veterinary composition of the present invention may further include a filler, an anti-aggregating agent, a lubricant, a wetting agent, a spice, an emulsifying agent, a preservative, and the like. After the veterinary composition according to the present invention is administered to an animal, the active ingredient is rapidly, sustained or It can be formulated using methods well known in the art to provide delayed release, and the formulations are powders, granules, tablets, capsules, suspensions, emulsions, solutions, syrups, aerosols, soft or hard gelatin capsules, suppositories. , Sterile injectable solutions, sterile external preparations, and the like. The veterinary composition according to the present invention may vary depending on the age, sex, and weight of the animal, and may be administered once to several times a day, and the dosage may be increased or decreased depending on the route of administration, sex, weight, age, and the like. Therefore, the dosage does not limit the scope of the present invention in any way.

Hereinafter, the present invention will be described in more detail using examples. These examples are only for describing the present invention in more detail, and it is apparent to those of ordinary skill in the art that the scope of the present invention is not limited thereto.

<Materials and methods>

1. Isolation of new strains

As for the new strain used in the present invention, take skate (10g) purchased from the Skate Farming Corporation in Youngsanpo, Naju, Jeollanam-do, dilute it with sterilized physiological saline, spread on a solid medium for separating MRS lactic acid bacteria, and incubate for 48 hours at 30°C. Different microorganisms were separated and used according to the shape of. The isolated microorganisms were cultured at 30° C. for 48 hours in minimal nutrient medium (1% glucose, 0.5% yeast extract).

2. Confirmation of anti-inflammatory activity of new strains isolated from skates

(1) cell culture

The macrophage cell line Raw264.7 cells (Murine macrophage cell line) used in the present invention was pre-sale and used from the Korean Cell Line Bank (KCLB, Seoul, Korea). Cell culture medium was used by adding 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin (100U/ml) to DMEM (Dulbecco's modified Eagle's medium, Gibco, Co., USA) medium, and 37°C, 5% Incubated for 48 hours in CO ₂ conditions.

(2) Cell viability

Raw264.7 cells were dispensed into a 96-well plate at a concentration of 1×10 ⁵ cells/ml and incubated for 3 hours, followed by treatment with LPS (1µg/ml) and samples, and cultured for 24 hours. DMEM medium containing 10% MTS solution in the cultured 96-well plate was treated with 100 µl of each well, incubated for 3 hours, and absorbance at 490 nm using an ELISA microplate reader (Infinite 200 pro, TECAN, Austria). Was measured. Cell viability was calculated using the following formula (1).

Equation (1)

Cell viability (% control) = [(absorbance of treated sample)/(absorbance of control group)]×100

(3) Measurement of NO (Nitric oxide) production

DMEM medium containing 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin (100U/ml) was dispensed into a 96-well plate at a concentration of 1×10 ⁵ cells/ml, and the samples were treated for 1 hour. During the culture, LPS (1 ㎍ / ㎖) was treated and incubated for 24 hours in a 37 ℃, 5% CO ₂ incubator. After incubation, 100 μl of the supernatant was transferred to a new 96-well plate, and the same amount of the Grease reagent mixture (Stock-I: 0.2% naphylendia HCl, Stock-II: 2% sulfanilamide in 5% H ₃ PO ₄ ) was added. After 15 minutes, absorbance was measured at 540 nm using an ELISA microplate reader (Infinite 200 pro, TECAN, Austria). At this time, sodium nitrate was used as a standard product.

(4) Inflammatory cytokine analysis

To measure the amount of inflammatory cytokine production, Raw264.7 cells were dispensed into a 96-well plate at a concentration of 1×10 ⁵ cells/ml and incubated for 2 hours, and then each sample was treated by concentration and incubated for 1 hour. . Thereafter, LPS (1 μg/ml) was treated and incubated for 24 hours in a 37°C, 5% CO ₂ incubator. The obtained supernatant was used as a sample, and the absorbance was measured at 450 nm using a Quantikine ELISA kit (R&D system, USA), and the measured pro-inflammatory cytokines were TNF-α, interleukin-1β (IL- 1β) and interleukin-6 (IL-6) production was analyzed by the sandwich-ELISA method according to the experimental method specified in the kit.

(5) Statistical processing

The results obtained in the experiment were measured three times and expressed as mean ± standard deviation. After performing one way ANOVA using SPSS (Statistical Package for Social Science Inc., Chicago, IL, USA), Student's t The significance was verified at the p<0.05 level by analyzing with -test.

3. Identification of lactic acid bacteria

The skate microorganism Enterococcus lactis WiKim0107 with excellent anti-inflammatory activity was identified through 16S ribosomal DNA gene sequence analysis. After taking the cells of the selected microorganism and washing the cells twice in sterilized physiological saline, DNA was extracted using a DNeasy tissue kit (Qiagen, Valecia, CA, USA). Universal primer for amplification of 16S ribosomal DNA gene of extracted DNA; 27F (5'-AGAGTTTGATCATGGCTCAG-3') (SEQ ID NO: 1) and 1492R (5'-GGATACCTTGTTACGACTT-3') (SEQ ID NO: 2) primers were used. In PCR reaction, in 10 µl of Takara Perfect Premix (Takara, Japan) containing 0.4mM dNTP, 0.5 unit of Taq polymerase, and 4mM Mg ²⁺ , 1 µl of DNA template (20 µg/ml), 1.0 µM forward primer and 1.0 μM reverse primers were added at 1 μl each, and distilled water was added to the rest to obtain a total volume of 20 μl.

PCR amplification was performed with a Mastercycler gradient (Eppendorf, Hamburg, Germany). The PCR reaction was performed at 95°C for 5 minutes (initial denaturation), 94°C for 45 seconds (denaturation), 52°C for 45 seconds (annealing), 72°C for 1 minute (extension) for 30 cycles, and 72°C for 5 minutes. Final extension was performed. The amplified fragment of about 1400bp was transformed after binding to a T vector (Invitrogen, Carlsbad, CA, USA). Base sequence determination was performed using a T vector sequencing primer,

The results were identified by comparison with the ribosomal DNA gene sequencing of GenBank (NCBI, Bethesda, MD, USA) using the BLAST analysis program (http://www.ncbi.nlm.nih.gov). The determined nucleotide sequence was aligned with Clustal_W and BioEdit programs, and the generated gap was treated as a missing trait in the subsequent analysis process. The calculation of the genetic distance between nucleotide sequences and the generation of neighbor-joining, maximum-likelihood and parsimony phylogenies were performed using the Mega program, and bootstrap analysis was performed with the same specifications for each of the above three analyses to determine the support of the phylogeny. (1,000 replicates).

4. Analysis of cell proliferation ability of Enterococcus lactis

Raw264.7 cells were dispensed into a 96-well culture plate under a condition of 5×10 ³ cells/well and stabilized for 24 hours. 1 ㎍ / ㎖ of lipopolysaccharide (Lipopolysaccharide, LPS; Sigma) and 10 ⁴ ~ 10 ⁷ CFU / ㎖ of Enterococcus lactis ( Enterococcus lactis ) respectively treated and cultured for 24 hours MTS assay (Promega) Cell proliferation changes were analyzed using.

5. Analysis of NO (Nitric oxide) production of Enterococcus lactis

The Raw264.7 cells in 96-well culture plate 5 × 10 ³ cells / well seeded in condition was stabilized for 24 h, LPS (1㎍ / ㎖) and 10 ⁵ ~ 10 ⁷ CFU / ㎖ of Enterobacter nose kusu lactis ( Enterococcus lactis ) was treated respectively. After culturing for 24 hours, each cell culture solution was separated, and the change in NO production was analyzed using a grease reagent system (Promega).

6. Analysis of ROS (Reactive oxygen species) production of Enterococcus lactis

Raw264.7 cells were dispensed in a 6-well culture plate under conditions of 1×10 ⁵ cells/well and stabilized for 24 hours, followed by LPS (1 μg/ml) and 10 ⁵ ~10 ⁷ CFU/ml of Enterococcus rock Teeth ( Enterococcus lactis ) were each treated and cultured for 24 hours. Each cell was stained with CellRox Deep Red Reagent (Thermo Fisher Scientific), and then the change in ROS production was analyzed using a flow cytometer.

7. Gene expression analysis using Real Time-PCR

After dispensing RAW264.7 cells in a 6-well culture plate under conditions of 1×10 ⁵ cells/well and stabilizing for 24 hours, LPS (1 μg/ml) and 10 ⁵ ~ 10 ⁷ CFU/ml Enterococcus Rock Teeth ( Enterococcus lactis ) were each treated and cultured for 24 hours. After RNA was extracted from each cell using TRIzol reagent (Invitrogen), cDNA was synthesized using TaqMan Reverse Transcription Master Mix (Applied Biosystems).

The primers for β-actin, iNOS, interleukin (IL)-1β, IL-6, TNF-α (tumor necrosis factor alpha), and COX-2 (cyclooxygenase 2) were purchased from TaqMan Gene Expression assay (Applied Biosystems). The relative expression levels were analyzed using ABI QuantStudio 6 Flex Real Time-PCR and TaqMan PCR Master Mix. The relative expression levels of each gene were quantified with β-actin and calculated by the ΔΔCt method.

8. Animal breeding conditions

A 5-week-old male mouse (C57BL/6N, Japan SLC Inc.) was purchased from the central experimental animal, adapted for one week, and used in the experiment, and the mice in a healthy state were used for the experiment by observing the general condition during the adaptation period. The breeding environment was maintained at a temperature of 23±3℃, a humidity of 50±5%, and a contrast period of 12 hours (07:00-19:00/lighting time). During the test period, the experimental animals were reared in a polycarbonate cage (200×320×145mm, Three-shine Co., Daejon Korea) in 3 animals per group, and the feed was Harlan 2018S (Harlan ^TM , USA) for mice. I was fed free. For negative water, reverse osmosis (RO) water was disinfected with ultraviolet light and fed freely.

9. Immunity-enhancing animal testing

Animal experiments were conducted in accordance with the NIH (National Institutes of Health)'s guidelines for the management and use of experimental animals, and was conducted with the approval of the Animal Experimental Ethics Committee of the Korea Institute of Oriental Medicine (approval number: 18-042). The experimental group was divided into a normal group (WT), a control group (Con), and an Enterococcus lactis administration group (WiKim0107), and the sample was administered orally using a mouse zone at a concentration of 10 ⁸ CFU/100 μl. I did. The sample administration period was administered daily for a total of 2 weeks, and the mice were sacrificed at 2 weeks to analyze the immune enhancing effect.

10. Analysis of the number of immune cells using flow cytometry

After the spleen tissue was excised from each experimental group, it was stored in DMEM medium (Dulbeco's modified eagle's media; Gibco) containing 1% Fetal bovine serum (Gibco). Each spleen tissue was extracted using DMEM medium containing 1% FBS and a 40 μm cell strainer (Falcon), and then dispensed into 5 ml FACs tubes (Falcon) under 1×10 ⁶ cell conditions. After washing the dispensed splenocytes twice with PBS (Phosphate buffer saline) containing 1% FBS, CD4-FITC, CD8-APC, CD45R/B220-FITC, NK1.1-APC, CD11c-FITC, CD11b- Each APC antibody was treated and reacted at 4° C. for 20 minutes. After washing twice with PBS containing 1% FBS, the change in the number of immune cells was analyzed using a flow cytometer.

11. Animal test inducing inflammatory bowel disease

In order to induce inflammatory bowel disease in mice, 5% (w/v) dextran sulfate sodium salt (DSS, MP Biomedicals) negative water was prepared. The experimental group was divided into a normal group (WT), a control group (Con), a positive control group (5-ASA), and an Enterococcus lactis administration group (WiKim0107), and the sample administration was conducted at a concentration of 10 ⁸ CFU/100 μl. It was orally administered using a mouse bolus. The sample administration period was 3 weeks in total and was administered daily. After 2 weeks of sample administration, 5% DSS negative water was freely fed for 6 days to the experimental group excluding the normal group (WT), and mice were sacrificed at the 3rd week of sample administration. The improvement effect was analyzed.

12. Analysis of representative disease indicators of inflammatory bowel disease

After free feeding of 5% DSS negative water, the body weight of each experimental group was measured daily using a Sartorius Secura 2102-1S balance, and the change in DAI (Disease activity index) was recorded. DAI (a disease activity index indicating the degree of colitis) was given a score based on Table 1, and then expressed as a total. After the mice were sacrificed, colon tissues of each experimental group were removed and the length of the colon was measured and analyzed.

DAI (Disease activity index) score

Score	Weight loss	Stool consistency	Visible blood in feces
0	No weight loss	Normal	No bleeding
One	1~5%
2	6-10%	Loose	Slight bleeding
3	11-15%
4	15% or more	Diarrhea	Gross bleeding

Example 1. Selection of lactic acid bacteria with excellent anti-inflammatory activity

As a result of measuring the anti-inflammatory activity of the lactic acid bacteria culture medium for 100 species of Lactobacillus , Leuconostoc , Pediococcus , and Weissella isolated from skate and skate kimchi, 10 strains with excellent anti-inflammatory activity were first selected. In order to confirm the anti-inflammatory activity of the selected 10 lactic acid bacteria, the NO concentration in the Raw264.7 cell culture was measured. As a result of checking the inhibition rate of NO (nitric oxide) production in Raw264.7 cells using LPS (1㎍/㎖), it was confirmed that the NO production of the Wikim0107 strain was at a level of 50.5% compared to the LPS treatment group (FIG. 1A).

In addition, TNF-α, IL-1β and IL-6, which are cytokines produced by Raw264.7 cells by LPS stimulation, were analyzed. In order to investigate the factors involved in the inhibition of NO production, the production of TNF-α, an inflammatory cytokine, which is a tumor necrosis factor, was measured as 1363.2 pg/ml (Fig. 1B), and IL-1β production was measured as 45.7 pg/ml. Compared with the treated group (68 pg/ml), it was significantly suppressed (Fig. 1C). IL-6 production was also measured as 761.1pg/ml, and showed a tendency to decrease significantly compared to the group treated with only LPS (1211.1pg/ml) (Fig. 1D). These results confirmed that skate-derived lactic acid bacteria have a great effect on anti-inflammatory action by increasing the ability to inhibit the production of intrinsic inflammatory substances.

By performing a second repeated experiment in the same way, the WiKim0107 strain was finally selected, and as a result of confirming the survival rate of Raw264.7 cells by MTS assay, it showed a high survival rate of 130% or more compared to the control, and the cells according to the number of viable cells Since there was no decrease in survival rate, it was confirmed that there was no significant toxic reaction to Raw264.7 cells (FIG. 2).

Thereafter, a total nucleotide sequence of 1394 bp (SEQ ID NO: 3) was determined with the 16S rRNA of the strain WiKim0107 having excellent anti-inflammatory activity isolated from the skate.

For the identification and classification of the lactic acid bacteria, as a result of analyzing the 16S rRNA nucleotide sequence, it was confirmed that the new strain WiKim0107 isolated from skates was 97% identical to the Enterococcus lactis standard strain BT, and Enterococcus lactis WiKim0107 ( Enterococcus lactis WiKim0107 ). In addition, the Enterococcus lactis WiKim0107 was deposited with the Agricultural Genetic Resource Center of the National Academy of Agricultural Sciences on March 8, 2019, and was given the deposit number KACC 81090BP.

Example 2. Enterococcus lactis ( Enterococcus lactis ) Cytotoxicity and immune enhancing efficacy

In order to verify the cytotoxicity and immune enhancing effect of Enterococcus lactis , changes in cell proliferation were observed in RAW264.7 cells. Compared to the control group, cell proliferation was increased in a concentration-dependent manner in the experimental group treated with Enterococcus lactis , and a low increase in cell proliferation was confirmed compared to the experimental group in which the inflammatory response was induced by treatment with LPS. Enterobacter nose kusu lactis cytotoxicity of Enterobacter nose kusu lactis (Enterococcus lactis) through the cell proliferation of RAW264.7 cells not inhibited by the treatment of (Enterococcus lactis) is no, the cell proliferation is higher than the control group and the LPS It was confirmed that there is an immunity enhancing effect through being lower than that of the treated inflammation group (FIG. 3).

Example 3. NO and ROS generation amount change confirmation

As a result of confirming the change in NO production, it was confirmed that in the experimental group treated with Enterococcus lactis , the amount of NO produced increased compared to the control group, and the gene expression of iNOS, which was related to NO production, was also increased.

In addition, as a result of confirming the change in the amount of ROS production, the amount of ROS production was increased in the experimental group treated with Enterococcus lactis compared to the control group. It was confirmed that the amount of NO and ROS production increased than that of the control group and lower than that of the inflammatory group treated with LPS (FIG. 4).

Example 4. Confirmation of changes in gene expression of cytokines (IL-1β, IL-6, TNF-α, COX-2)

Changes in gene expression of cytokines (IL-1β, IL-6, TNF-α, COX-2) expressed by the activation of immune cells to confirm the immune enhancing efficacy of Enterococcus lactis Observed. As a result, gene expression of cytokines was increased in the experimental group treated with Enterococcus lactis compared to the control group, and a low gene expression increased compared to the inflammatory group treated with LPS (FIG. 5).

Example 5 Enterococcus lactis in an animal model ( Enterococcus lactis ) Of the immune system

Enterococcus lactis ( Enterococcus lactis ) was orally administered to C57BL/6N mice for 2 weeks to confirm the immune enhancing efficacy of Enterococcus lactis in an animal model, and then the mice were sacrificed. During the administration period, no change in body weight and no gross signs of abnormality were observed in the mice, and it was found that there is no toxicity of Enterococcus lactis . After separating the cells from the spleen tissue, the number of immune cells (T cells, B cells, NK cells, macrophages, dendritic cells) was confirmed using a flow cytometer. As a result, Enterococcus lactis was compared with the normal group and the control group. It was confirmed that the number of CD4 ⁺ T cells, B cells, macrophages and dendritic cells increased in the experimental group administered ( Enterococcus lactis ). In addition, as a result of analyzing gene expression changes of cytokines (IL-1β, IL-6, TNF-α, COX-2) in the spleen tissue, it was confirmed that gene expression of cytokines was increased compared to the normal group and the control group. (Fig. 6).

Example 6. Enterococcus lactis in an animal model ( Enterococcus lactis ) To improve inflammatory bowel disease

In animal models, Enterobacter nose Syracuse lactis (Enterococcus lactis) Inflammatory Bowel an enterovirus nose Syracuse lactis (Enterococcus lactis), for a total of three weeks in C57BL / 6N mice to confirm the disease improvement was orally administered dosage period second week of May % DSS negative water was fed freely for 6 days to induce inflammatory bowel disease, and then the mice were sacrificed. As a result of observing changes in mouse weight and DAI in an animal model of inflammatory bowel disease, it was confirmed that the weight loss and DAI increased in the control group, the positive control group, and the experimental group administered Enterococcus lactis compared to the normal group. I did. As a result of observing the change in the length of the colon tissue isolated from the mouse, the length of the colon of the control group and the positive control group, and the experimental group administered Enterococcus lactis decreased compared to the normal group, and was positive compared to the control group. It was confirmed that the decrease in colon length was alleviated in the control group, the experimental group administered with Enterococcus lactis (FIG. 7).

After extracting the spleen tissue from the mouse and separating the cells, the change in the number of immune cells (T cells, B cells, NK cells, macrophages, dendritic cells) was confirmed using a flow cytometer. The number of immune cells was decreased, and it was observed that the decrease in the number of immune cells was alleviated in the experimental group administered with the positive control group and Enterococcus lactis compared to the control group (FIG. 8).

[Accession number]

Name of deposit institution: Institute of Agricultural Biotechnology

Accession number: KACC81090BP

Consignment Date: 20190308

Claims (9)

1. Enterococcus lactis ( Enterococcus lactis ) WiKim0107 strain (Accession No.: KACC 81090BP) having immune function control and prevention, improvement or therapeutic activity of inflammatory bowel disease.
2. The Enterococcus lactis WiKim0107 strain according to claim 1, wherein the Enterococcus lactis WiKim0107 strain is isolated from skateboard or skate kimchi.
3. The immune function regulation and prevention of inflammatory bowel disease comprising at least one selected from the strain of claim 1 or 2, the culture of the strain, the concentrate of the culture, the dried product of the culture, and the extract of the culture as an active ingredient, or Health functional food composition for improvement.
4. The method of claim 3, wherein the active ingredient is prepared in any one formulation selected from powders, granules, pills, tablets, capsules, candy, syrup and beverages, for controlling immune function and preventing or improving inflammatory bowel disease. Health functional food composition.
5. A pharmaceutical composition for preventing or treating inflammatory bowel disease, comprising as an active ingredient at least one selected from the strain of claim 1 or 2, the culture of the strain, the concentrate of the culture, the dried product of the culture, and the extract of the culture .
6. The pharmaceutical composition for preventing or treating inflammatory bowel disease according to claim 5, wherein the inflammatory bowel disease is ulcerative colitis, Crohn's disease, or Behcet's disease.
7. The pharmaceutical composition for preventing or treating inflammatory bowel disease according to claim 5, further comprising a pharmaceutically acceptable carrier, excipient, or diluent in addition to the active ingredient.
8. The immune function control and prevention of inflammatory bowel disease comprising at least one selected from the strain of claim 1 or 2, the culture of the strain, the concentrate of the culture, the dried product of the culture, and the extract of the culture as an active ingredient, or Feed composition for improvement.
9. The immune function control and prevention of inflammatory bowel disease comprising at least one selected from the strain of claim 1 or 2, the culture of the strain, the concentrate of the culture, the dried product of the culture, and the extract of the culture as an active ingredient, or Veterinary composition for treatment.

Images