KR20210010420A - Composition for preventing, ameliorating or treating fatty liver disease comprising Pediococcus inopinatus as effective component - Google Patents

Abstract

The present invention relates to a composition for preventing, mitigating, or treating fatty liver disease containing Pediococcus inopinatus as an effective ingredient. More specifically, according to the present invention, Pediococcus inopinatus not only has no cytotoxicity, but also effectively reduces accumulation of triglycerides in a hepatocarcinoma cell line (HepG_2), lowers the levels of blood ALT and TG content increased by alcohol, and provides effects of lowering, in animal models in which fatty liver is induced through high-fat diet, reducing the weight of the liver and an expression level of fat metabolism-related genes in the liver. Accordingly, the composition of the present invention can be usefully used as a health functional food, medicine, and a feed additive or veterinary composition for preventing, mitigating, and treating the fatty liver disease. The component comprises at least one, as an active ingredient, selected from a group consisting of a Pediococcus inopinatus WiKim0108 strain (Accession No.: KACC 81091BP), the culture of the strain, and a concentrate, a dried product, and an extract of the culture.

Description

Composition for preventing, ameliorating or treating fatty liver disease comprising Pediococcus inopinatus as effective component}

The present invention relates to a composition for preventing, improving or treating fatty liver disease comprising Pediococcus inofinatus as an active ingredient.

The mortality rate of liver disease in Korea is very high at 23.5 per 100,000 people, and is the number one cause of death in their 40s (41.1 people/10,000 people), the second cause of death in their 50s (72.4 people/10 million), and the third cause of death in their 30s. Liver disease, accounting for (10/10,000), is a major cause of death in the middle-aged Korean population.

Among the liver diseases, fatty liver refers to a phenomenon in which triglycerides, which are not present in normal cells, are abnormally excessively deposited in liver cells. About 5% of normal liver is composed of adipose tissue, and triglycerides, fatty acids, phospholipids, cholesterol and cholesterol esters are the main components of fat, but once fatty liver occurs, most of the components are replaced by triglycerides and the amount of triglycerides is reduced. Fatty liver is diagnosed if it is more than 5% of the weight of the liver. When the fatty liver deteriorates and the fat mass in the hepatocyte becomes large, the important components of the cells including the nucleus are pushed to one side and the function of the hepatocyte decreases, and the expanded hepatocytes due to the accumulated fat in the cells press the microvessels and lymph glands between the hepatocytes. Thus, the circulation of blood and lymph fluid in the liver is impaired. In this case, the liver cells cannot properly receive oxygen and nutrients, and liver function is degraded.

Non-alcoholic fatty liver disease (NAFLD) is defined as a case where fatty acids accumulate more than 5% in the parenchymal cells of the liver in the form of triglycerides, not liver damage caused by alcohol. Pathologically, it is classified into simple steatosis and steatohepatitis with inflammation. If left untreated for a long time, it can lead to serious liver diseases such as hepatitis, liver fibrosis, and cirrhosis. In Korea, the incidence of non-alcoholic liver disease is also increasing due to lifestyle changes.

In addition, alcoholic fatty liver occurs due to excessive drinking. Although there is a difference according to genetic characteristics and sex for each individual, liver disease such as alcoholic fatty liver is likely to occur when ingesting more than 80 g of alcohol per day. Women are more likely to develop alcoholic liver disease even in smaller doses. In general, it can be calculated that about 10g of alcohol is contained in 1 soju, 1 beer, 1 Western liquor, and 1 hop of makgeolli. Most of the symptoms are asymptomatic in patients with alcoholic fatty liver, which is the mildest form, but mild tenderness in the right upper abdomen can be complained of when mild hepatic hypertrophy (the liver is larger than normal).

On the other hand, as a technology related to Pediococcus inofinatus strain, a probiotic composition including Pediococcus pentosaceus KID7 strain having a cholesterol-lowering effect is disclosed in Korean Patent No. 1673450, and Korean Patent No. 1611829 discloses obesity and obesity. Although the Pediococcus pentosaceus CBT SL4 strain and a composition containing the same are disclosed for the prevention or treatment of metabolic diseases, the prevention, improvement or prevention of fatty liver disease comprising Pediococcus inofinatus of the present invention as an active ingredient No therapeutic composition has been disclosed.

The present invention is derived from the above requirements, the present invention provides a composition for the prevention, improvement or treatment of fatty liver disease comprising Pediococcus inofinatus as an active ingredient, the Pediococcus inofinatus Liver cancer cell line (HepG ₂ ) has no cytotoxicity, effectively reduces the accumulation of triglycerides, reduces the content of ALT and TG in the blood in an animal model in which alcoholic fatty liver is induced, and animals in which fatty liver is induced through a high fat diet The present invention was completed by confirming that the model has the effect of lowering the ALT level, reducing the liver weight, and reducing the expression level of a fat metabolism-related gene in the liver.

In order to achieve the above object, the present invention Pediococcus inopinatus ( Pediococcus inopinatus) WiKim0108 strain (Accession No: KACC 81091BP), 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 health functional food composition for preventing or improving fatty liver disease comprising at least one selected from among the active ingredients.

In addition, the present invention Pediococcus inopinatus ( Pediococcus inopinatus) WiKim0108 strain (accession number: KACC 81091BP), at least one selected from the culture of the strain, the concentrate of the culture, a dried product of the culture and an extract of the culture It provides a pharmaceutical composition for preventing or treating fatty liver disease comprising as an active ingredient.

In addition, the present invention Pediococcus inopinatus ( Pediococcus inopinatus) WiKim0108 strain (accession number: KACC 81091BP), at least one selected from the culture of the strain, the concentrate of the culture, a dried product of the culture and an extract of the culture It provides a feed additive for preventing or improving fatty liver disease comprising as an active ingredient.

In addition, the present invention Pediococcus inopinatus ( Pediococcus inopinatus) WiKim0108 strain (accession number: KACC 81091BP), at least one selected from the culture of the strain, the concentrate of the culture, a dried product of the culture and an extract of the culture It provides a veterinary composition for preventing or treating fatty liver disease in animals other than humans comprising as an active ingredient.

The present invention relates to a composition for preventing, improving or treating fatty liver disease comprising Pediococcus inofinatus as an active ingredient, wherein Pediococcus inofinatus has no cytotoxicity in liver cancer cell line (HepG ₂ ), and is neutral It has the effect of effectively reducing the accumulation of fat, lowering the blood ALT and TG content increased by alcohol, and lowering ALT levels in animal models in which fatty liver was induced through a high fat diet, reducing liver weight, and reducing fat in the liver. It has the effect of reducing the expression level of metabolic-related genes.

1 is a result of confirming the cell survival rate (%) after treatment with the WiKim0108 strain on a liver cancer cell line (HepG ₂ ). N is a normal cell without any treatment.
2 is a result of confirming lipid accumulation after treatment with a 1.0 mM free fatty acid (FFA) and WiKim0108 strain in which oleate and palmidate were mixed at a concentration ratio of 2:1 in a liver cancer cell line (HepG ₂ ). . ### is a statistically significant increase in fat accumulation in the group (FFA) treated with oleate and palmitate mixture (2:1) free fatty acid compared to the normal group (N), p<0.001, *, * *, *** is oleate and palmitate mixture (2: 1) compared to the group treated with free fatty acids (FFA), fat accumulation in the group treated with the Pediococcus inofinatus strain of the present invention (WiKim0108) was statistically Was significantly decreased, * is p<0.05, ** is p<0.01, and *** is p<0.001.
3 is a result of treatment of the strain WiKim0108 in an animal model inducing alcoholic fatty liver and confirming the change in blood ALT content (A) and TG content (B). ## indicates that the blood ALT and TG content in the vehicle group was statistically significantly increased compared to the normal group, p<0.01, and * is the Pediococcus inofinatus strain of the present invention compared to the vehicle group, or Milk C, a positive control The blood ALT and TG contents in the knee treatment group were statistically significantly reduced, p<0.05.
4 is a result of confirming the change in body weight after administration of Pediococcus inofinatus strain to an animal model.
5 is a result of confirming the content in blood of ALT and AST, which are indicators of liver function. ## indicates that the content of ALT and AST in the blood of the high-fat diet group (Veh) increased statistically significantly compared to the normal group (N), p<0.01. * Indicates that the ALT content in the blood of the group administered with Pediococcus inofinatus strain (WiKim0108) was statistically significantly reduced compared to the high fat diet group (Veh), p<0.05.
6 is a result of confirming liver weight (A) and morphological change (B) after administration of Pediococcus inofinatus strain to an experimental animal. ## indicates that the liver weight of the high-fat diet group (Veh) increased statistically significantly compared to the normal group (N), p<0.01. *, ** indicates that the liver weight of the positive control group and the Pediococcus inofinatus strain (WiKim0108) administration group was statistically significantly reduced compared to the high fat diet group (Veh), * indicates p<0.05, ** indicates p< Is 0.01.
7 is a result of confirming the relative mRNA expression level of CIDEC (cell death-inducing DFFA-like effector C) related to lipid droplets in liver tissue. ### indicates that the CIDEC gene expression level of the high fat diet group (Veh) was statistically significantly increased compared to the normal group (N), p<0.001. **, *** indicates that the CIDEC gene expression levels of the positive control group and the WiKim0108 administration group were statistically significantly decreased compared to the high fat diet group (Veh), ** indicates p<0.01, and *** indicates p<0.001.

The present invention is effective in one or more selected from Pediococcus inopinatus WiKim0108 strain (Accession No.: KACC 81091BP), culture of the strain, concentrate of the culture, dried product of the culture, and extract of the culture It relates to a health functional food composition for preventing or improving fatty liver disease comprising as an ingredient.

The Pediococcus Inofinatus WiKim0108 strain is preferably isolated from skateboard or skate kimchi, but is not limited thereto.

The extraction solvent of the extract of the Pediococcus inofinatus WiKim0108 strain culture is preferably water, a lower alcohol of C ₁ to C ₄ , or a mixture thereof, but is not limited thereto.

The fatty liver disease is non-alcoholic or alcoholic simple fatty liver; Non-alcoholic or alcoholic steatohepatitis; And non-alcoholic or alcoholic cirrhosis, but is not limited thereto.

The active ingredient is characterized by reducing the content of triglycerides in liver tissue, but is not limited thereto.

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 may be appropriately selected according to the age, sex, weight, condition, and symptoms of the disease of a person who wants a functional food for preventing or improving fatty liver disease It is recommended to contain about 0.01 to 10.0g per day for adults, and by ingesting foods having such a content, the effect of preventing or improving fatty liver disease can be obtained.

In addition, the present invention Pediococcus inopinatus ( Pediococcus inopinatus) WiKim0108 strain (accession number: KACC 81091BP), at least one selected from the culture of the strain, the concentrate of the culture, a dried product of the culture and an extract of the culture It relates to a pharmaceutical composition for preventing or treating fatty liver disease comprising as an active ingredient.

The pharmaceutical composition may further include at least one selected from 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 can 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, Various compounds or mixtures including calcium 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 fatty liver disease.

In the method for preventing or treating fatty liver 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 WiKim0108 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 Pediococcus inopinatus ( Pediococcus inopinatus) WiKim0108 strain (accession number: KACC 81091BP), at least one selected from the culture of the strain, the concentrate of the culture, a dried product of the culture and an extract of the culture It relates to a feed additive for preventing or improving fatty liver disease comprising as an active ingredient.

The feed additive of the present invention corresponds to an auxiliary feed in the feed management method. 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 Pediococcus inopinatus ( Pediococcus inopinatus) WiKim0108 strain (accession number: KACC 81091BP), at least one selected from the culture of the strain, the concentrate of the culture, a dried product of the culture and an extract of the culture It relates to a veterinary composition for preventing or treating fatty liver disease in animals other than humans comprising as an active ingredient.

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.

1. Liver cancer cell line (HepG ₂ ) In culture

The liver cancer cell line (HepG ₂ ) was purchased and used from ATCC (American Type Culture Collection). The liver cancer cell line (HepG ₂ ) was cultured in DMEM-F12 (Dulbecco's modified Eagle's medium) medium containing 10% FBS and 1% penicillin-striptomycin. The cells were cultured in a 5% CO ₂ incubator at 37°C, and the cultured cells were tested by dispensing a constant number of cells (1×10 ⁵ cells/500 μl well) into a 24-well plate. After dispensing the cells in a 24-well plate, the cells were completely attached to the 24-well plate to form a cell shape, and the cells were used when they reached 75% confluence.

2. Preparation of Free Fatty Acid (FFA)

(1) Preparation of 100mM palmitate (Sigma P-0500) and 100mM oleate (Sigma P-75010) stock solutions. Prepared using a 0.1M NaOH solution as a solvent at 70 ℃, filtered using a 0.2㎛ filter, and then sterilized.

(2) Preparation of 5% (W/v) BSA (Sigma A-6003) solution containing no free fatty acids. It was prepared using third distilled water as a solvent, sterilized, and stored in a refrigerator at 4°C.

(3) Preparation of 5 mM mixed fatty acid stock solution mixed so that the concentration of oleate and palmitate is 2:1. At 60° C., the concentration of each fatty acid prepared in (1) above using a 5% (w/v) BSA solution that does not contain free fatty acids dissolved in the tertiary distilled water prepared in (2) above is 10 ml of a fatty acid stock solution was prepared so that it became 5 mM.

The 100mM palmitate and 100mM oleate stock solutions prepared in (1) above were 165µl (1.65mM) of palmitate and 330µl (3.3mM) of oleate, and 9,505µl of 5% ( w/v) BSA solution was shaken while dropping and mixing (vortexing). Thereafter, it was cooled to room temperature, and then filtered through a 0.2 μm filter (the prepared mixed solution is stable even if stored at -20°C for 3 to 4 weeks).

3. Alcoholic fatty liver induction

C57BL/6J (12 weeks old, male, 27~30g, Japan SLC, Inc.) was maintained in an SPF laboratory animal facility controlled at 24°C, 50% humidity, and 12 hours light and dark cycle. As for the alcohol formula, Lieber-DeCarli Ethanol Rat Diet and Lieber-DeCarli Regular Control Rat Diet were used in the central laboratory animals, and edible alcohol was additionally added according to the final alcohol concentration (%). Eight animals were used for each group, and 4 animals were placed in one cage and liquid food was fed. The alcohol diet was adapted by increasing alcohol concentration incrementally, at 0.5 and 1% for 1 day each; 2 days each at 1.5, 2.0 and 2.5%; After increasing the alcohol concentration at 3 and 4% each for 1 day, 5% alcohol was fed for 11 days.

Thereafter, on the 12th day, after oral administration of maltose to the normal group, alcohol (20%) was orally administered to the alcohol diet group, and blood and organs were removed.

The drug was orally administered to each group once a day for 12 days from the 3% alcohol diet, the normal group was administered DW for SPF, the vehicle group was administered saline, and the Pediococcus inofinatus strain (WiKim0108) was administered. Silver Pediococcus inofinatus strain was administered in an amount of 3×10 ⁸ CFU per head, and the milk thistle administration group was administered with 50 mg/kg of milk thistle as a positive control.

4. Blood analysis

For the analysis of blood indices (ALT, TG), Fuji dry-chem 700i was used.

Example 1. Pediococcus inofinatus ( Pediococcus inopinatus ) Isolation and identification of WiKim0108

(1) Isolation of new strains

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

(2) Identification of lactic acid bacteria

The skate kimchi microorganism WiKim0108 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 amplifying the 16S ribosomal DNA gene of the 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, 4mM Mg ²⁺ , 1 μl of DNA template (20 μg/ml), 1.0 μM forward primer 1.0 μM reverse primer was added to each 1 μl, and distilled water was added to the remainder to give 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), 30 cycles, and 72°C for 5 minutes. The final extension was carried out for minutes. The amplified fragment of about 1400bp was transformed after binding to a T vector (Invitrogen, Carlsbad, CA, USA). Sequencing was performed using T vector sequencing primers, and the results were ribosomal of GenBank (NCBI, Bethesda, MD, USA) using BLAST analysis program (http://www.ncbi.nlm.nih.gov). It was identified by comparison with DNA gene sequencing. 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).

For the identification and classification of the lactic acid bacteria WiKim0108 strain isolated from skate kimchi, as a result of analyzing the 16S rRNA nucleotide sequence, the new strain WiKim0108 was 97% consistent with the Pediococcus inopinatus standard strain BT (SEQ ID NO: 3). Subsequently, the new strain was named Pediococcus inopinatus WiKim0108 ( Pediococcus inopinatus WiKim0108). In addition, the Pediococcus Inofinatus WiKim0108 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 81091BP.

Example 2. Confirmation of cytotoxicity of WiKim0108 strain

The cultured HepG ₂ cells were diluted by concentration for 24 hours and treated, and MTS assay (CellTiter Aqueous One Solution Cell proliferation assay kit, 3-(4,5-dimethylthiazol-2-yl)-5-(3) -carboxymethoxyphenyl)-2-(4-sulfophynyl)-2H-tetrazolium, inner salt; MTS, Promega Co.Madison, WI, USA) to determine cell viability at 490nm by microplate reader (Molecular Devices, Sunnyvale, CA, USA) It was measured as.

As a result, as disclosed in FIG. 1, the cell survival rate (%) when the WiKim0108 strain of the present invention was treated was approximately similar to that of the normal group (N). Therefore, it was found that the WiKim0108 strain of the present invention had little cytotoxicity.

Example 3. Confirmation of changes in lipid accumulation according to treatment of WiKim0108 strain

In the liver cancer cell line (HepG ₂ ), it was confirmed whether the increased lipid accumulation was reduced by the treatment of the WiKim0108 strain by treating the prepared 1 mM oleate and palmitate mixture (2:1) free fatty acid.

As a result, as disclosed in FIG. 2, it was confirmed that the lipid accumulation of the WiKim0108 strain was reduced in a concentration-dependent manner.

Example 4. Confirmation of changes in blood ALT and TG content according to treatment of WiKim0108 strain

(1) Changes in blood ALT content

As a result of confirming the change in blood ALT content in the animal model in which fatty liver was induced by alcohol intake, the blood ALT content in the vehicle group administered with alcohol compared to the normal group was significantly increased as disclosed in FIG. 3(A), Compared to the vehicle group with increased ALT content due to alcohol, the blood ALT content of the milk thistle administration group (positive control group) and the WiKim0108 administration group was significantly decreased.

(2) Changes in blood TG content

As a result of confirming the change in the blood TG content in the animal model in which fatty liver was induced by alcohol intake, the TG content in the blood in the vehicle group administered with alcohol compared to the normal group was increased, as disclosed in FIG. As a result, the blood TG content of the WiKim0108-administered group was significantly decreased compared to the vehicle group with increased TG content.

Example 5. Confirmation of weight change in animal model

Six-week-old male C57BL/6J mice were purified, followed by ingestion of a 60 kcal% fat diet (D12492, Research Diet, Inc) to induce non-alcoholic fatty liver. When the average body weight reached 28-29g, the WiKim0108 strain was administered orally for 10 weeks (1×10 ⁸ , 1×10 ⁹ CFU/mice/day), and the body weight was measured weekly, and as a positive control, milk thistle (MT, 100mg/kg/day) was administered.

As a result, as disclosed in FIG. 4, the weight of the high fat diet group (HFD) increased compared to the normal group, and in contrast, the weight of the WiKim0108 strain administration group and the milk thistle administration group (MT) of the present invention decreased slightly. I did.

Example 6. Checking the content of liver function indicators (ALT, AST) in blood

After Example 5, the content of ALT and AST, which are indicators of liver function, in blood (serum) collected from the animal model by cardiac puncture was measured.

As a result, as disclosed in FIG. 5, the ALT and AST content in the blood of the high fat diet group (Veh) compared to the normal group (N) was statistically significantly increased, and Pediococcus inofinatus compared to the high fat diet group (Veh) The ALT content in the blood of the strain (WiKim0108) administration group was statistically significantly reduced.

Example 7. Liver weight and liver morphological change

After Example 6, the experimental animals were sacrificed and the weight and morphological changes were confirmed after liver tissue extraction.

As a result, as disclosed in FIG. 6(A), the liver weight of the high fat diet group (HFD) increased compared to the normal group, and in contrast, the liver weight of the WiKim0108 strain administration group and the milk thistle administration group (MT) of the present invention was increased. It was confirmed that there was a statistically significant decrease. In addition, the morphological change of the liver was confirmed as disclosed in FIG. 6(B), and the liver of the high fat diet group (Veh) became enlarged compared to the normal group (N), and the liver of the WiKim0108 strain administration group of the present invention compared to the high fat diet group It was confirmed that the shape was similar to that of the normal group.

Example 8. Confirmation of expression levels of genes involved in regulation of fat metabolism and absorption of fatty acids

RNA was isolated from a sample of 30 mg of liver tissue obtained in Example 7, and cell death-inducing DFFA (CIDEC) related to lipid droplets involved in the regulation of fat metabolism through reverse transcription and Q-PCR using Taqman probe (ABI). -like effector C) relative mRNA expression levels were confirmed.

As a result, as disclosed in FIG. 7, the CIDEC gene expression level of the high fat diet group (Veh) was statistically significantly increased compared to the normal group (N), and the positive control group and Pediococcus inofina compared to the high fat diet group (Veh) The amount of CIDEC gene expression in the group administered with the tooth strain (WiKim0108) was statistically significantly reduced.

[Statistics processing]

The results obtained in Example 2 of the present invention were tested for significance between the control and the control group by performing the LSD test using the SPSS ver 20.0 program.

The results obtained in Example 3 were expressed as the mean ± standard error of the mean (SEM), for statistical analysis, ANOVA was performed in GraphPad Prism 8.1.2, and for post-mortem analysis, the significance was tested by performing Dunnett's multiple comparisons test.

The results obtained in Examples 5 to 8 of the present invention were tested for significance between the control group and the experimental group by performing the LSD test and the Student's t-test using the SPSS ver 20.0 program.

Institute of Agricultural Biotechnology KACC81091BP 20190308

<110> Korea Institute of Oriental Medicine Korea Food Research Institute <120> Composition for preventing, ameliorating or treating fatty liver disease comprising Pediococcus inopinatus as effective component <130> PN20184 <160> 3 <170> KopatentIn 2.0 <210> 1 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> universal primer; 27F <400> 1 agagtttgat catggctcag 20 <210> 2 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> universal primer; 1492R <400> 2 ggataccttg ttacgactt 19 <210> 3 <211> 1359 <212> DNA <213> Pediococcus inopinatus <400> 3 tggcgaacgg gtgagtaaca cgtgggtaac ctgcccagaa gtgggggata acacctggaa 60 acagatgcta ataccgcata ataaagtaaa ccgcatggtt ttcttttaaa agatggcttc 120 ggctatcact tctggatgga cccgcggcgt attagctagt tggtgagata aaggcccacc 180 aaggctgtga tacgtagccg acctgagagg gtaatcggcc acattgggac tgagacacgg 240 cccagactcc tacgggaggc agcagtaggg aatcttccac aatggacgaa agtctgatgg 300 agcaacgccg cgtgagtgat gaaggcttta gggtcgtaaa actctgttgt tggagaagaa 360 cgtgtgtgag agtaactgct catgcagtga cggtatccaa ccagaaagcc acggctaact 420 acgtgccagc agccgcggta atacgtaggt ggcaagcgtt atccggattt attgggcgta 480 aagcgagcgc aggcggtttc ttaagtctaa tgtgaaagcc ttcggcttaa ccgaagaagt 540 gcattggaaa ctgggaaact tgagtgcaga agaggacagt ggaactccat gtgtagcggt 600 gaaatgcgta gatatatgga agaacaccag tggcgaaggc ggctgtctgg tctgtaactg 660 acgctgaggc tcgaaagcat gggtagcgaa caggattaga taccctggta gtccatgccg 720 taaacgatga atgctaagtg ttggagggtt tccgcccttc agtgctgcag ctaacgcatt 780 aagcattccg cctggggagt acgaccgcaa ggttgaaact caaaagaatt gacgggggcc 840 cgcacaagcg gtggagcatg tggtttaatt cgaagctacg cgaagaacct taccaggtct 900 tgacatcttc tgctaaccta agagattagg cgttcccttc ggggacagaa tgacaggtgg 960 tgcatggttg tcgtcagctc gtgtcgtgag atgttgggtt aagtcccgca acgagcgcaa 1020 cccctattat tagttgccag cattaagttg ggcactctag tgagactgcc ggtgataaac 1080 cggaggaagg tggggacgac gtcaaatcat catgcccctt atgacctggg ctacacacgt 1140 gctacaatgg acggtacaac gagttgcgag accgcgaggt taagctaatc tcttaaaacc 1200 gttctcagtt cggactgcag gctgcaactc gcctgcacga agttggaatc gctagtaatc 1260 gcggatcagc atgccgcggt gaatacgttc ccgggccttg tacacaccgc ccgtcacacc 1320 atgagagttt gtaacaccca aagccggtgg agtaacctt 1359

Claims (8)

Pediococcus inopinatus ( Pediococcus inopinatus) WiKim0108 strain (accession number: KACC 81091BP), a culture of the strain, a concentrate of the culture, a dried product of the culture and one or more selected from the extract of the culture as an active ingredient Health functional food composition for preventing or improving fatty liver disease. The method of claim 1, wherein the extraction solvent of the extract of the Pediococcus inopinatus WiKim0108 strain culture is water, a lower alcohol of C ₁ to C ₄ or a mixture thereof. Health functional food composition for improvement. The method of claim 1, wherein the fatty liver disease is non-alcoholic or alcoholic simple fatty liver; Non-alcoholic or alcoholic steatohepatitis; And health functional food composition for preventing or improving fatty liver disease, characterized in that any one selected from non-alcoholic or alcoholic cirrhosis. The health functional food composition for preventing or improving fatty liver disease according to claim 1, wherein the active ingredient reduces the content of triglycerides in liver tissue. Pediococcus inopinatus ( Pediococcus inopinatus) WiKim0108 strain (accession number: KACC 81091BP), a culture of the strain, a concentrate of the culture, a dried product of the culture and one or more selected from the extract of the culture as an active ingredient A pharmaceutical composition for preventing or treating fatty liver disease. The pharmaceutical composition for preventing or treating fatty liver disease according to claim 5, further comprising at least one selected from pharmaceutically acceptable carriers, excipients, and diluents in addition to the active ingredient. Pediococcus inopinatus ( Pediococcus inopinatus) WiKim0108 strain (accession number: KACC 81091BP), a culture of the strain, a concentrate of the culture, a dried product of the culture and one or more selected from the extract of the culture as an active ingredient Feed additive for preventing or improving fatty liver disease. Pediococcus inopinatus ( Pediococcus inopinatus) WiKim0108 strain (accession number: KACC 81091BP), a culture of the strain, a concentrate of the culture, a dried product of the culture and one or more selected from the extract of the culture as an active ingredient A veterinary composition for preventing or treating fatty liver disease in animals other than humans.

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