阅读说明：本技术 包含嗜柠檬酸明串珠菌wikim0104的用于预防、改善或治疗肥胖或脂肪肝疾病的组合物 (Composition for preventing, improving or treating obesity or fatty liver disease comprising leuconostoc citreum WIKIM0104 ) 是由 崔鹤钟 林秀吉 于 2020-02-27 设计创作，主要内容包括：本发明涉及从泡菜中分离的新型嗜柠檬酸明串珠菌WiKim0104(Leuconostoc citreum WiKim0104)以及包含其的组合物或其用途。本发明提供通过给药从泡菜中分离的新型嗜柠檬酸明串珠菌WiKim0104或包含其的组合物来预防、改善及治疗个体的炎症、肥胖或脂肪肝疾病的方法。本发明的嗜柠檬酸明串珠菌WiKim0104通过抑制肝细胞内的脂肪积聚并减少脂肪肝相关基因的表达来表现出脂肪肝生成抑制效果,因此能够以多种方式用于预防、改善及治疗炎症、肥胖或脂肪肝疾病。(The present invention relates to novel Leuconostoc citreum WiKim0104(Leuconostoc citreum WiKim0104) isolated from kimchi and a composition comprising the same or use thereof. The present invention provides a method for preventing, improving and treating inflammation, obesity or fatty liver disease in an individual by administering novel leuconostoc citreum WiKim0104 isolated from kimchi or a composition comprising the same. The leuconostoc citreum WiKim0104 of the present invention exhibits a fatty liver production inhibitory effect by inhibiting fat accumulation in hepatocytes and reducing the expression of fatty liver-related genes, and thus can be used in various ways to prevent, improve and treat inflammation, obesity or fatty liver disease.)

1. Leuconostoc citreoviride WiKim0104 with a preservation number of KCCM 12420P.

2. A composition for preventing or ameliorating inflammation, obesity or fatty liver disease, comprising as an active ingredient Leuconostoc citreoviride WiKim0104 deposited with accession No. KCCM12420P, a culture thereof, a homogenate thereof or an extract thereof.

3. The composition according to claim 2, wherein the fatty liver disease includes simple fatty liver, non-alcoholic steatohepatitis, hepatic fibrosis and cirrhosis.

4. The composition for preventing or ameliorating inflammation, obesity or fatty liver disease according to claim 2, wherein the composition is a food composition, a lactic acid bacteria starter composition for food fermentation, a feed or feed additive composition or a pharmaceutical composition.

5. The composition for preventing or improving inflammation, obesity or fatty liver disease according to claim 4, wherein the food is a health functional food, a drink, a bar-shaped food or fermented milk.

6. Use of Leuconostoc citreum WiKim0104 deposited as KCCM12420P, its culture, its homogenate, or its extract for preventing, ameliorating, or treating inflammation, obesity, or fatty liver disease.

7. The use according to claim 6, wherein the fatty liver disease includes one or more selected from the group consisting of simple fatty liver, non-alcoholic steatohepatitis, hepatic fibrosis and cirrhosis.

8. Use according to claim 6, as a food composition, a lactic acid bacteria starter composition for food fermentation, a feed or feed additive composition or a pharmaceutical composition.

9. The use according to claim 8, wherein the food composition is a health functional food, a drink, a bar-shaped food or fermented milk.

10. A method for preventing, ameliorating or treating inflammation, obesity or fatty liver disease comprising the step of administering to a subject in need thereof a composition comprising leuconostoc citreum WiKim0104 deposited under accession number KCCM12420P, a culture thereof, a homogenate thereof or an extract thereof.

11. The method for preventing, ameliorating or treating inflammatory diseases, obesity or fatty liver diseases according to claim 10, wherein the subject is an animal including a human.

12. The method of claim 10, wherein the fatty liver disease comprises one selected from the group consisting of simple fatty liver, non-alcoholic steatohepatitis, liver fibrosis and cirrhosis.

Technical Field

The present application claims priority from korean patent application No. 10-2019-0024254, which was filed on 28.2.2019, the entire contents of which are incorporated herein by reference. The present invention relates to a novel strain isolated from kimchi and a composition for preventing, improving or treating inflammation, obesity or fatty liver disease comprising the same.

Background

Fatty liver (fatty liver) is a disease in which neutral fat accumulates in liver cells due to failure of normal fat metabolism due to excessive intake of fat and accumulation of fat in the liver, increase in synthesis, decrease in metabolism, and the like, and is classified as fatty liver when fat accounts for 5% or more of the total weight of the liver.

In addition, recently, fatty liver patients have been rapidly increased due to western-style high-fat and high-calorie dietary life and insufficient exercise due to the development of civilization, and the onset age has been widely distributed from the teens to over sixty years. The continuous accumulation of fat in the liver is accompanied by inflammation and progresses to fatty liver hepatitis, and necrosis of liver tissue due to chronic inflammation and cirrhosis of liver due to fibrosis of the liver occur. If the symptoms continue to worsen, liver cancer may develop. Therefore, fatty liver is the largest cause of hepatitis, liver cirrhosis, and liver cancer.

Currently, as a drug used in the market, there is rochelli (xenoica) which uses orlistat (orlistat) as a main material and shows an effect of improving fatty liver by treating obesity^TM(Roche Co., Ltd.) Using a West clothNuometin (reduce) with tramin (sibutramine) as main material^TMAlthough the compound has side effects such as diarrhea, abdominal pain, and insomnia (Abbott corporation), fibrate drugs such as clofibrate (clofibrate) are clinically used as a therapeutic agent for fatty liver, but side effects such as hepatic dysfunction are exhibited.

On the other hand, although patent document 1 below describes that a plurality of lactic acid bacteria exhibit an effect of improving fatty liver, there is still a real need to develop a therapeutic agent for fatty liver which has an effect of fundamentally preventing or improving fatty liver and does not exhibit the above-mentioned side effects.

On the other hand, patent document 1 discloses fatty liver improving effects of various lactic acid bacteria such as bifidobacterium and lactobacillus, but a therapeutic agent having an effect of effecting radical treatment of fatty liver and not having the above-mentioned side effects has not been developed yet. Therefore, there is a real need to develop a therapeutic agent that can radically treat fatty liver and has no side effects.

Patent document 1: korean patent application No. 10-1937365 (08 Yue in 2018)

Disclosure of Invention

Technical problem

The present invention provides a novel Leuconostoc citreum lactic acid bacterium having an excellent preventive or ameliorating effect on fatty liver diseases.

Technical scheme

Accordingly, the present inventors have made an effort to find a lactic acid bacterium strain having an effect of preventing or improving fatty liver diseases from conventional fermented foods, and as a result, isolated and identified Leuconostoc citreum WiKim0104(Leuconostoc citreum WiKim0104) as a novel lactic acid bacterium strain of genus weisse, thereby completing the present invention.

An embodiment of the present invention provides a composition for preventing or improving inflammation, obesity or fatty liver disease, comprising leuconostoc citreum WiKim0104, a culture thereof, a homogenate thereof or an extract thereof as an active ingredient.

The fatty liver disease may be one or more selected from the group consisting of simple fatty liver, non-alcoholic steatohepatitis, hepatic fibrosis and liver cirrhosis.

The composition may be a food composition, a lactic acid bacteria starter composition for food fermentation, a feed or feed additive composition or a pharmaceutical composition.

The food composition may include health functional food, drink, bar-shaped food or fermented milk.

Still another embodiment of the present invention provides use of leuconostoc citreum WiKim0104, a culture thereof, a morselized product thereof, or an extract thereof for preventing, ameliorating, or treating inflammation, obesity, or fatty liver disease.

The fatty liver disease may include one or more selected from the group consisting of simple fatty liver, non-alcoholic steatohepatitis, hepatic fibrosis and liver cirrhosis.

The present invention can be used as a food composition, a lactic acid bacteria starter composition for food fermentation, a feed or feed additive composition or a pharmaceutical composition.

An example of the present invention provides a method for preventing, improving or treating inflammation, obesity or fatty liver disease, comprising the step of administering to an individual in need thereof a composition comprising leuconostoc citrophile WiKim0104, a culture thereof, a homogenate thereof or an extract thereof. The subject may be an animal including a human.

The present invention provides a method for preventing, ameliorating or treating inflammation, obesity or fatty liver disease including one selected from the group consisting of simple fatty liver, non-alcoholic steatohepatitis, liver fibrosis and cirrhosis.

The invention provides leuconostoc citreum citrate WiKim 0104.

The leuconostoc citreum provided by the invention WiKim0104 is a novel strain of leuconostoc citreum derived from pickled vegetables. Although the Leuconostoc citreoviride WiKim0104 of the present invention is isolated and identified from kimchi, the route of acquisition is not limited thereto.

The results of base sequence analysis of 16S ribosomal DNA (16S rDNA) for identifying and classifying microorganisms on the lactic acid bacteria strains isolated by the examples of the present invention showed that they have the nucleic acid sequence of SEQ ID NO: 1.

Therefore, the microorganism of the present invention having the 16S ribosomal DNA base sequence of SEQ ID NO. 1 was named Leuconostoc citreum WiKim0104 and deposited in the Korean culture Collection on 12 and 14 months in 2018 (accession No. KCCM 12420P).

The leuconostoc citreum WiKim0104 is a gram-positive bacterium, is a facultative anaerobe (viable anaerobe) capable of growing under both aerobic and anaerobic conditions, does not form spores, has no motility, and has bacillus-shaped cells.

In the following examples, it was confirmed that Leuconostoc citreum WiKim0104 of the present invention inhibits the expression of Fatty Acid Synthase (FAS), stearoyl-CoA desaturase (SCD) (lipogenic-related gene), cyclooxygenase-2 (COX-2, cyclooxygenase-2), and nuclear factor-kappa (NF-kappa) (inflammation-related gene) in hepatocytes (HepG2 cells). From the above, it was confirmed that the Leuconostoc citreum WiKim0104 strain can prevent or improve fatty liver by inhibiting fatty liver production by HepG2cells by about 30% to 35%. Therefore, the leuconostoc citreum WiKim0104 of the present invention can be used in various ways to prevent, treat or ameliorate fatty liver disease in humans or animals.

Further, it was confirmed by the following examples that the composition comprising leuconostoc citreum WiKim0104 as an active ingredient inhibits accumulation of fat and inflammation in hepatocytes (HepG2cells), has the effect of reducing body weight, improves the liver function value in blood, and reduces the expression of fat metabolism, inflammation, and liver fibrosis-related factor genes in liver tissues. Therefore, the composition can be used as an active ingredient of a composition for preventing, treating or ameliorating inflammation, obesity or one or more fatty liver diseases selected from the group consisting of simple fatty liver, non-alcoholic steatohepatitis, hepatic fibrosis and cirrhosis.

The present invention provides a composition for preventing, ameliorating or treating inflammation, obesity or fatty liver disease, comprising leuconostoc citreum WiKim0104, a culture thereof, a homogenate thereof or an extract thereof as an active ingredient.

The leuconostoc citreum WiKim0104 contained in the composition of the present invention may be present in the form of live cells or dead cells, and may be present in the form of dried or freeze-dried cells. Forms of lactic acid bacteria suitable for inclusion in various compositions and methods of formulating the same are well known in the art to which the present invention pertains. For example, leuconostoc citreum WiKim0104 can be formulated into a culture obtained by culturing the strain in a known liquid medium or solid medium, a fermented product obtained by co-culturing the strain with additional components, an extract of the strain extracted with an organic solvent, a lysate (or a disrupted product) obtained by dissolving the cell membrane of the strain or by disruption or homogenization, or the like, but the present invention is not limited thereto.

In one embodiment, the composition may be a composition comprising the Leuconostoc citreum WiKim0104 strain existing in a viable or dead form.

In the present invention, the fatty liver disease may include simple fatty liver, non-alcoholic steatohepatitis, hepatic fibrosis and cirrhosis.

Also, the fatty liver disease may include all alcoholic fatty liver and non-alcoholic fatty liver, for example, non-alcoholic fatty liver induced by high fat diet. The non-alcoholic fatty liver disease (NAFLD) may include all primary and secondary non-alcoholic fatty liver diseases, for example, primary hyperlipidemia, diabetes, or non-alcoholic fatty liver disease caused by obesity.

In one embodiment, the above composition may be a pharmaceutical composition for preventing or treating inflammation, obesity or fatty liver disease comprising a culture, a morcellate, a ferment or an extract of leuconostoc citreum WiKim0104 strain.

In the case where the composition of the present invention is used as a pharmaceutical composition, the pharmaceutical composition of the present invention can be prepared using pharmaceutically suitable and physiologically acceptable adjuvants such as excipients, disintegrants, sweeteners, binders, coating agents, swelling agents, lubricants, flavoring agents, and the like, in addition to the above-mentioned leuconostoc citreum WiKim0104 strain.

For administration, the pharmaceutical composition may contain one or more pharmaceutically acceptable carriers in addition to the above-described active ingredients, and preferably formulated into a pharmaceutical composition.

For example, for formulation into tablet or capsule form, the active ingredient may be combined with an oral, non-toxic and pharmaceutically acceptable inactive carrier such as ethanol, glycerol, water, etc. If desired or necessary, a binder, a lubricant, a disintegrant, a color former, or a mixture thereof may be suitably contained. Suitable binding agents include, but are not limited to, starch, gelatin, natural sugars such as glucose or beta-lactose, corn sweeteners, natural or synthetic gums such as acacia, tragacanth or sodium oleate, sodium stearate, magnesium stearate, sodium benzoate, sodium acetate, sodium chloride, and the like. The disintegrator includes, but is not limited to, starch, methyl cellulose, agar, bentonite, xanthan gum, and the like. In the composition which can be formulated into a liquid solution, the pharmaceutically acceptable carrier may be sterilized and used in a physiological saline solution, sterilized water, ringer's solution, buffered physiological saline, albumin injection solution, glucose solution, maltodextrin solution, glycerin, ethanol, or a mixture thereof suitable for living bodies, and other conventional additives such as an antioxidant, a buffer, a bacteriostatic agent, and the like may be added as needed. In addition, diluents, dispersants, surfactants, binders and lubricants may be added to prepare injection preparations such as aqueous solutions, suspensions and emulsions, pills, capsules, granules or tablets.

Further, the method disclosed in Remington's Pharmaceutical Science, Mack Publishing Company, Easton PA can be used as an appropriate method in the related art, and the preparation can be preferably made according to various diseases or according to the ingredients.

In one embodiment, the present invention provides a food composition for preventing or improving inflammation, obesity or fatty liver disease, comprising as an active ingredient leuconostoc citreum WiKim0104, a culture thereof, a homogenate thereof, a fermentation thereof or an extract thereof. The food composition may be in the form of functional health food, beverage, or bar food.

In the present invention, the food composition containing the above-mentioned strain as an active ingredient may include beverages such as fermented milk. Accordingly, the present invention provides a lactic acid bacteria starter for fermentation comprising consisting of leuconostoc citreum WiKim0104 or a culture thereof.

The food composition of the present invention can be formulated in the same manner as the above-described pharmaceutical composition to be used as a functional food, or can be added to various foods. Examples of foods to which the composition of the present invention can be added include beverages, vitamin complexes, health supplements, and the like.

The food composition of the present invention may comprise ingredients that are normally added when preparing a food product, for example, comprising proteins, carbohydrates, fats, nutrients, flavoring agents and aroma agents. Examples of the carbohydrate include conventional sugars such as monosaccharides including glucose and fructose, disaccharides including maltose, sucrose and oligosaccharides, and polysaccharides such as dextrin and cyclodextrin, and sugar alcohols such as xylitol, sorbitol and erythritol. As the flavoring agent, natural flavoring agents (thaumatin, stevia extract (e.g., rebaudioside A, glycyrrhizin, etc.)) and synthetic flavoring agents (saccharin, aspartame, etc.) can be used. For example, when the food composition of the present invention is prepared into drinks and beverages, citric acid, liquid fructose, white sugar, glucose, oxalic acid, malic acid, fruit juice, various plant extracts, and the like may be included.

The above-described composition of the present invention can be used as a feed additive or feed.

In the case of use as a feed additive, the above composition may be prepared as a highly concentrated solution of 20% to 90%, or in the form of powder or granules. The feed additive may further comprise one or more of organic acids such as citric acid, fumaric acid, adipic acid, lactic acid, and malic acid, phosphates such as sodium phosphate, potassium phosphate, acid pyrophosphate, and polyphosphate (total phosphate), and natural antioxidants such as polyphenol, catechin, alpha-tocopherol, rosemary extract, vitamin C, green tea extract, licorice extract, chitosan, tannic acid, and phytic acid. In the case of use as a feed, the above composition may be formulated in a conventional feed form, and may contain conventional feed ingredients together.

The feed additive and feed may further comprise: cereals, such as crushed or broken wheat, oat, barley, corn and rice; vegetable protein feeds such as feeds containing rape, soybean and sunflower as main ingredients; animal protein feeds such as blood meal, meat meal, bone meal and fish meal; sugar and dairy products include, for example, dry ingredients composed of various milk powders and whey powders, and may further include nutritional supplements, digestion and absorption promoters, growth promoters, and the like.

The feed additives described above may be administered to the animal alone or in combination with other feed additives in an edible carrier. Also, the above feed additives can be easily administered to animals as topcoats, or by mixing them directly with animal feeds, or in separate oral dosage forms separate from the feeds. In the case where the above feed additive is administered separately from animal feed, it may be prepared in a form of immediate release or sustained release in combination with a pharmaceutically acceptable edible carrier well known in the art to which the present invention pertains. Such edible carriers may be solid or liquid, such as corn starch, lactose, sucrose, soy flakes, peanut oil, olive oil, sesame oil, and propylene glycol. In the case of solid carriers, the feed additive may be in the form of tablets, capsules, powders, lozenges or sugar-containing tablets or top-dressing in microdispersed form. In the case of a liquid carrier, the feed additive may be in the form of a gelatin soft capsule, syrup or suspension, emulsion or solution.

The feed additive and feed may contain an auxiliary agent such as a preservative, a stabilizer, a wetting agent or emulsifier, a solubilizer, and the like. The feed additive can be added into animal feed by soaking, spraying or mixing.

The feed or feed additive of the present invention can be applied to the diet of various animals including mammals, poultry and fishes.

In the present invention, animals include mammals, and mammals include not only pigs, cattle, sheep, goats, experimental rodents but also pets (e.g., dogs and cats) other than experimental rodents, and the poultry may be chickens, turkeys, ducks, geese, pheasants, quail, and the fish may be trout, but are not limited thereto.

The compositions of the present invention may comprise about 10 on a single dose basis⁶cfu/ml to 10¹²The Leuconostoc citreoviride WiKim0104 strain of cfu/ml, for example, may contain 10⁷cfu/ml to 10¹¹cfu/ml、10⁸cfu/ml to 10¹⁰cfu/ml. In the case of administration of the strain, preferably, administration is in a viable state, and may be either killed prior to ingestion or in a attenuated (attenuation) state. When the culture medium is prepared using a culture supernatant or the like, a sterilization process by a heat treatment process may be further performed. The amount of strain required for minimal efficacy and the extent of daily intake may vary depending on the physical or health status of the ingesting person, but may typically be about 10⁶cfu/ml to 10¹²cfu/ml, for example, may be 10⁷cfu/ml to 10¹¹cfu/ml、10⁸cfu/ml to 10¹⁰cfu/ml。

Another embodiment of the present invention may provide the use of leuconostoc citreum WiKim0104 for preventing, treating or ameliorating inflammation, obesity or fatty liver disease in a human or an animal.

Another embodiment of the present invention provides a use of leuconostoc citreum WiKim0104, a culture thereof, a disrupted product thereof, or an extract thereof for preventing, ameliorating, or treating inflammation, obesity, or fatty liver disease.

Yet another embodiment of the present invention provides leuconostoc citreum WiKim0104, a culture thereof, a homogenate thereof, or an extract thereof for use in the prevention, amelioration, or treatment of obesity or fatty liver disease.

An example of the present invention provides a method for preventing, improving or treating inflammation, obesity or fatty liver disease, comprising the step of administering to an individual in need thereof a composition comprising leuconostoc citrophile WiKim0104, a culture thereof, a homogenate thereof or an extract thereof. The above-mentioned subject is an animal including a human, and the above-mentioned animal may be included in the scope of the present invention as long as it is the above-mentioned animal which takes in feed. The fatty liver disease may include one or more selected from the group consisting of simple fatty liver, non-alcoholic steatohepatitis, hepatic fibrosis and liver cirrhosis.

Advantages and features of the present invention and methods of accomplishing the same may be understood more clearly by reference to the detailed description of embodiments that follow. However, the present invention is not limited to the embodiments disclosed below, but can be implemented in various different embodiments, which are provided only for the purpose of making the disclosure of the present invention complete and fully informing the scope of the present invention to those skilled in the art to which the present invention pertains, and the present invention is defined only by the scope of the claims.

ADVANTAGEOUS EFFECTS OF INVENTION

The leuconostoc citreum WiKim0104 of the present invention is lactic acid bacteria isolated from kimchi, shows an effect of suppressing fatty liver generation by suppressing intracellular fat accumulation and reducing expression of fatty liver-related genes, and can be used for preventing, improving and treating inflammation, obesity or fatty liver disease in various ways. Further, it can be effectively used as a starter for fermentation.

Drawings

FIG. 1 is a bar graph for measuring the expression level of HepG2 cell-associated genes treated with the WiKim0104 strain of the invention. L-LAB1 denotes Leuconostoc citreum WiKim0104(Leu. citreum WiKim0104), L-LAB2 denotes Leuconostoc mesenteroides (Leu. mesenteroides), and L-LAB3 and L-LAB4 denote Leuconostoc citreum strains #2 and #3(Leu. citreum strain #2 and #3), respectively, isolated in the laboratory.

Fig. 2a to 2c are bar graphs showing body weight, and numerical values of liver function in blood (glutamate pyruvate transaminase (GPT), Glutamate Oxaloacetate Transaminase (GOT)) of a mouse model of fatty liver which ingested WiKim0104 strain of the present invention. P < 0.05, compared to normal dietary intake group (compare to the ND); # P < 0.05, L-LAB1 represents Leuconostoc citreoviride WiKim0104 compared to the high fat diet intake group (compare to the HFD).

FIGS. 3a to 3c are histograms for measuring the degree of expression of liver tissue-related genes in a mouse model of fatty liver in which the WiKim0104 strain of the present invention was ingested. P < 0.05, compared to normal dietary intake group; # P < 0.05, L-LAB1 indicating Leuconostoc citrophilum WiKim0104 compared to the high fat diet intake group.

FIG. 4 shows SEQ ID NO 1 of the present application.

Detailed Description

The present invention will be described in detail below with reference to examples. The following examples merely illustrate the present invention, and the scope of the present invention is not limited to the following examples.

Examples

Example 1: identification of leuconostoc citreoides WiKim0104

The single colony obtained by smearing the stock solution of kimchi extract in MRS medium was collected using an inoculating loop and cultured in MRS broth (broth). The DNA was extracted using QIAamp DNA Mini Kit (QIAgen, Germany). The extracted DNA was confirmed using 1% agarose gel, and in order to amplify the 16S ribosomal DNA gene (16S rDNA gene), Polymerase Chain Reaction (PCR) was performed using the extracted genomic DNA as a template, under the conditions of: denaturation (denaturation) at a temperature of 95 ℃ for 1 minute, conjugation (annealing) at a temperature of 45 ℃ for 1 minute, extension (extension) at a temperature of 72 ℃ for 1 minute and 30 seconds, and the above process was cycled 30 times. The obtained polymerase chain reaction product was assigned to the company Macrogen (seoul, korea) for sequence analysis. Identification of bacteria was performed by induced analysis of 16S ribosomal DNA sequences using the Search engine of the Local Alignment algorithm based Search Tool (BLAST) of the National Center for Biotechnology Information (NCBI, www.ncbi.nlm.nih.gov).

The results of 16S ribosomal DNA base sequence analysis for identifying microorganisms performed on the strains isolated by the present examples of the invention show that they have the nucleic acid sequence of SEQ ID NO. 1.

Therefore, the microorganism of the present invention was named Leuconostoc citreum WiKim0104 and was deposited in the Korean culture Collection (accession No: KCCM12420P) at 12.12.14.2018.

Example 2: confirmation of the efficacy of Leuconostoc citreoviride WiKim0104 in the inhibition of fat accumulation and inflammation in hepatocytes (HepG2cells)

An experimental procedure was designed to mimic the human gut-liver axis (gut-liver axis) in vitro (in vitro) using an invasion cell (transwell).

Leuconostoc citreum WiKim0104 lactic acid bacteria were cultured in MRS medium at a temperature of 30 ℃ for 24 hours. At the end of the culture, the cells were collected by centrifugation at 6000rpm for 5 minutes, and the cells were washed with Phosphate Buffered Saline (PBS) to remove all the remaining medium components.

To culture intestinal cells (Η Τ -29cells) and liver cells (HepG2cells), co-culture was prepared using RPMI-1640 medium supplemented with penicillin/streptomycin (penicillin/streptomycin), 10% fetal bovine serum (total bovine serum) and 6-well (6-well) culture plates to invade the chamber. Before CO-culture, intestinal cells were seeded on an invasion cell membrane (membrane), and hepatocytes were seeded on 6-well plates, each at 37 ℃ with 5% CO₂And culturing under the conditions of (1).

When intestinal cells (HT-29cells) and hepatic cells (HepG2cells) grew to around 80% in the culture dish (dish), the culture solution was replaced with a medium containing no fetal bovine serum (FBS free) and cultured for another 16 hours. After the cultivation, 1X 10 was used⁷Intestinal cells were treated with prepared lactic acid bacteria of CFU, and the invasion chamber was assembled to co-culture lactic acid bacteria, intestinal cells, and hepatocytes, and treated with 0.1mM palm oil (palmitic acid) for 48 hours to induce lipogenesis in hepatocytes. After culturing the cells with 0.1mM palm oil for 48 hours, quantitative real-time polymerase chain reaction (qPCR) was performed to confirm the lipid metabolism and inflammation-related genes in hepatocytes treated with Leuconostoc citreophilum WiKim0104 strainThe level of expression. As a result, as shown in fig. 1, it was confirmed that the gene expression of fatty acid synthase, unsaturated fatty acid synthase (Stearoyl-CoA desaturase, SCD)), inflammation-related factor (cyclooxygenase-2 (COX-2, cycloxygenase-2)), and Nuclear factor κ B (Nuclear factor-kappa beta) was significantly reduced in hepatocytes co-cultured with 0.1mM palm oil and leuconostoc citreum WiKim0104, as compared to hepatocytes treated with 0.1mM palm oil (control group).

Example 3: confirming weight reducing effect and improving liver function value in blood

Male mice (C57BL/6J) of 5 weeks of age were acclimatized for one week by normal diet (CD, Purina, Korea) in a nursery environment at a temperature of 20. + -. 2 ℃ and a humidity of 50. + -. 5% with a light-dark cycle 5(light-dark cycle) of 12 hours. The experiment was carried out after adapting the experimental animals to a one-week diet either on a normal diet (D12450B, Research Diets, new binishi weck, new jersey, usa) or on a high fat diet with 45% of the total calories as fat (D12451, Research Diets, new binishi weck, new jersey, usa). The experimental groups consisted of normal diet intake group (ND), high fat diet intake group (HFD), high fat diet and daily oral administration of 2X 10⁹The intake group (HFD + L-LAB1) of CFU/200. mu.l concentration of Leuconostoc citrophilum WiKim0104 consisted of 3 groups, and fatty liver was induced by 20 weeks of diet supply. The normal diet intake group (ND) and the high fat diet intake group (HFD) were orally administered with an equal amount of Phosphate Buffered Saline (PBS) daily. After 20 weeks of experiment, body weight of each experimental group was measured. In order to measure the liver function values (serum glutamic-oxaloacetic transaminase (serum GOT) and serum glutamic-pyruvic transaminase (serum GPT)) in the blood of the experimental group, the feeding was stopped 16 hours before the end of the experiment, and blood was collected from the vena cava and then serum was isolated and used in the experiment. Serum glutamic-oxaloacetic transaminase and glutamic-pyruvic transaminase were measured by an automatic biochemical analyzer (FUJI DRI-CHEM 7000i, Fujifilm corporation, Tokyo, Japan), and serum glutamic-oxaloacetic transaminase (GLUTAMATE-OXALOACETATE TRANSAMINASE, GOT), glutamic-pyruvic transaminase (GLUTAMATE-PYRUVATE) and the like are shown in FIGS. 2b and 2cTRANSAMINASE, GPT) is displayed.

As a result, as shown in fig. 2a to 2c, it was confirmed that the liver function values in the body weight and blood were significantly reduced in the case of taking a high-fat diet together with leuconostoc citreum WiKim0104 (HFD + L-LAB1) compared to the mice taking a high-fat diet (HFD).

Example 4: confirmation of expression levels of genes involved in fat metabolism, inflammation and hepatic fibrosis in liver tissues

Real-time quantitative polymerase chain reaction was performed using liver tissues removed after completion of the experiment to confirm the expression levels of genes related to fat metabolism, inflammation, and hepatic fibrosis in liver tissues ingested with the Leuconostoc citreum WiKim0104 strain.

As a result, it was confirmed that, as shown in FIGS. 3a to 3c, the expression of the leukocyte differentiation antigen 36(CD36, Cluster of differentiation 36), inflammation-related factor kappa B, and liver fibrosis-related factor type I Collagen alpha 1(Col1 alpha 1, Collagen type I alpha 1) genes was reduced with the ingestion of the Leuconostoc citreum WiKim0104 strain.

Industrial applicability

The present invention provides novel strains. Also, the strain of the present invention can be used as a food, feed or feed additive, pharmaceutical composition, or the like for preventing, ameliorating, or treating inflammation, obesity, or fatty liver disease.

Deposit number

The name of the depository institution: korea center for preservation of microorganisms (foreign)

The preservation number is: KCCM12420P

The preservation date is as follows: 12 months and 14 days in 2018

International recognition of the Budapest treaty on the deposit of microorganisms for patent procedures

International format

For the original deposited certificate

Issue according to rule 7.1

Preservation of a person: korea institute of food

Address: postal code 55365, Kyodo Otsu county, Kirgi, Korea, county, Yiwei farm Life road No. 245

Sequence listing

<110> Korean Food Institute (Korea Food Research Institute)

<120> composition for preventing, improving or treating obesity or fatty liver disease comprising Leuconostoc citreoviride WIKIM0104

<150> 10-2019-0024254

<151> 2019-02-28

<160> 1

<170> SIPOSequenceListing 1.0

<210> 1

<211> 1430

<212> DNA

<213> Leuconostoc citrophilum WIKIM0104(Leuconostoc citreum WiKim0104)

<400> 1

gatgaacgct ggcggcgtgc ctaatacatg caagtcgaac gcgcagcgag aggtgcttgc 60

acctttcaag cgagtggcga acgggtgagt aacacgtgga taacctgcct caaggctggg 120

gataacattt ggaaacagat gctaataccg aataaaactt agtatcgcat gatatcaagt 180

taaaaggcgc tacggcgtca cctagagatg gatccgcggt gcattagtta gttggtgggg 240

taaaggctta ccaagacgat gatgcatagc cgagttgaga gactgatcgg ccacattggg 300

actgagacac ggcccaaact cctacgggag gctgcagtag ggaatcttcc acaatgggcg 360

caagcctgat ggagcaacgc cgcgtgtgtg atgaaggctt tcgggtcgta aagcactgtt 420

gtatgggaag aaatgctaaa atagggaatg attttagttt gacggtacca taccagaaag 480

ggacggctaa atacgtgcca gcagccgcgg taatacgtat gtcccgagcg ttatccggat 540

ttattgggcg taaagcgagc gcagacggtt gattaagtct gatgtgaaag cccggagctc 600

aactccggaa tggcattgga aactggttaa cttgagtgtt gtagaggtaa gtggaactcc 660

atgtgtagcg gtggaatgcg tagatatatg gaagaacacc agtggcgaag gcggcttact 720

ggacaacaac tgacgttgag gctcgaaagt gtgggtagca aacaggatta gataccctgg 780

tagtccacac cgtaaacgat gaatactagg tgttaggagg tttccgcctc ttagtgccga 840

agctaacgca ttaagtattc cgcctgggga gtacgaccgc aaggttgaaa ctcaaaggaa 900

ttgacgggga cccgcacaag cggtggagca tgtggtttaa ttcgaagcaa cgcgaagaac 960

cttaccaggt cttgacatcc tttgaagctt ttagagatag aagtgttctc ttcggagaca 1020

aagtgacagg tggtgcatgg tcgtcgtcag ctcgtgtcgt gagatgttgg gttaagtccc 1080

gcaacgagcg caacccttat tgttagttgc cagcattcag ttgggcactc tagcgagact 1140

gccggtgaca aaccggagga aggcggggac gacgtcagat catcatgccc cttatgacct 1200

gggctacaca cgtgctacaa tggcgtatac aacgagttgc caacctgcga aggtgagcta 1260

atctcttaaa gtacgtctca gttcggactg cagtctgcaa ctcgactgca cgaagtcgga 1320

atcgctagta atcgcggatc agcacgccgc ggtgaatacg ttcccgggtc ttgtacacac 1380

cgcccgtcac accatgggag tttgtaatgc ccaaagccgg tggcctaacc 1430