COMPOSITION FOR PREVENTING, ALLEVIATING, OR TREATING OBESITY OR FATTY LIVER DISEASE, COMPRISING WEISSELLA HELLENICA WIKIM0103

Abstract

The present invention relates to novel Weissella hellenica WiKim0103 isolated from Kimchi and a composition comprising the same or its use. The present invention provides a method for prevention, improvement and treatment of inflammation, obesity or fatty liver disease of a subject by administering the novel Weissella hellenica WiKim0103 isolated from Kimchi or a composition comprising thereof. The Weissella hellenica WiKim0103 according to the present invention shows an inhibitory effect for fatty liver production by inhibiting fat accumulation in liver cells and reducing expression of fatty liver-related genes, and thus it may be variously utilized for uses in prevention, improvement and treatment of inflammation, obesity or fatty liver disease.

Claims

1-12. (canceled)

13. A method of preventing, improving or treating obesity or a fatty liver disease by administering a composition comprising Weissella hellenica WiKim0103 deposited under accession number KCCM12419P, or a culture, a lysate or an extract thereof as an active ingredient to a subject in need thereof.

14. The method of claim 13, wherein the Weissella hellenica WiKim0103 deposited under accession number KCCM12419P has a 16S rDNA of SEQ ID NO: 1.

15. The method of claim 13, wherein the fatty liver disease comprises any one or more selected from the group consisting of simple fatty liver, non-alcoholic steatohepatitis, liver fibrosis and liver cirrhosis.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0054] FIG. 1 is a photograph of observing the inhibitory effect of fat accumulation in HepG2 cells according to treatment of the WiKim0103 strain according to the present invention with naked eyes. L-LAB1 represents W. hellenica WiKim01013 and L-LAB2 represents W. koreensis.

[0055] FIG. 2 is a graph of measuring the HepG2 cell-related gene expression level according to treatment of the WiKim0103 strain according to the present invention. L-LAB1 represents W. hellenica WiKim01013 and L-LAB2 represents W. koreensis. *P<0.05, compare to the CON; #P<0.05, compare to the PA; PA, palmitic acid

[0056] FIG. 3 is a graph of measuring the HT-29 cell-related gene expression level according to treatment of the WiKim0103 strain according to the present invention. L-LAB1 represents W. hellenica WiKim01013 and L-LAB2 represents W. koreensis. *P<0.05, compare to the CON; #P<0.05, compare to the PA; PA, palmitic acid

[0057] FIG. 4a to FIG. 4c are graphs showing the body weight, blood liver function values (GPT, GOT) of the fatty liver mouse model according to the intake of the WiKim0103 strain according to the present invention. L-LAB1 represents W. hellenica WiKim01013 and L-LAB2 represents W. koreensis. *P<0.05, compare to the ND; #P<0.05, compare to the HFD.

[0058] FIG. 5a to FIG. 5c are graphs of measuring the liver tissue-related gene expression level of the fatty liver mouse model according to the intake of the WiKim0103 strain according to the present invention. L-LAB1 represents W. hellenica WiKim01013 and L-LAB2 represents W. koreensis. *P<0.05, compare to the ND; #P<0.05, compare to the HFD.

[0059] FIG. 6 shows SEQ ID NO: 1 of the present application.

MODE FOR INVENTION

[0060] Hereinafter, the present invention will be described in detail by examples. The following examples are intended to illustrate the present invention only, but the scope of the present invention is not limited by the following examples.

Example 1: Identification of Weissella hellenica WiKim0103

[0061] A bacterial single colony obtained by applying an undiluted solution of Kimchi extract on an MRS medium was collected with a loop and cultured in an MRS broth. DNA extraction was performed by using QIAamp DNA Mini Kit (QIAgen, Germany). The extracted DNA was confirmed by using 1% agarose gel, and to amplify a 16S rDNA gene, Polymerase Chain Reaction (PCR) was processed by using extracted genomic DNA as a template, and 30 cycles were performed under PCR conditions of denaturation at 95° C. for 1 minute, annealing at 45° C. for 1 minute and extension at 72° C. for 1 minute and 30 seconds. For the obtained PCR products, the sequence was analyzed by requesting to Macrogen (Seoul, Korea). Identification of bacteria was performed by inductive analysis of Basic Local Alignment Search Tool (BLAST) search engine of National Center for Biotechnology Information (NCBI, www.ncbi.nlm.nih.gov) for 16S rDNA sequence.

[0062] The strain isolated by the example of the present invention was shown to have the nucleic acid sequence of SEQ ID NO: 1, as the result of 16S rDNA sequence analysis for identification of microorganisms.

[0063] Accordingly, the microorganism of the present invention was named Weissella hellenica WiKim0103, and deposited to Korean Culture Center of Microorganisms on Dec. 14, 2018 (KCCM12419P).

Example 2: Confirmation of Inhibitory Efficacy of Fat Accumulation and Inflammation in Hepatocytes (HepG2 Cells) of Weissella hellenica WiKim0103

[0064] An in vitro experimental method was devised using a transwell to mimic a human body gut-liver axis.

[0065] The Weissella hellenica WiKim0103 lactic acid bacterium was cultured in an MRS medium at 30° C. for 24 hours. At the end of the culture, to recover microbial cells, it was prepared by performing centrifugation at 6,000 rpm for 5 minutes and rinsing with PBS to remove all remaining medium components.

[0066] To culture enterocytes (HT-29 cells) and hepatocytes (HepG2 cells), RPMI-1640 medium in which penicillin/streptomycin and 10% fetal bovine serum were added was used, and co-culture was prepared by using a 6-well plate transwell. Before co-culture, enterocytes were aliquoted in a transwell membrane, and hepatocytes were aliquoted in the 6-well plate, and they were prepared by culturing under the conditions of 37° C., 5% CO.sub.2, respectively.

[0067] When the enterocytes (HT-29 cells) and hepatocytes (HepG2 cells) grew about 80% in a culture dish, the culture solution was replaced with an FBS free medium and they were cultured for 16 hours. After culturing, prepared lactic acid bacteria 1×10.sup.7 CFU were treated to the enterocytes, and co-culture of lactic acid bacteria and enterocytes and hepatocytes was performed by assembling a transwell, and fat production in hepatocytes was induced by treating 0.1 mM palmitic acid for 48 hours. After culturing with 0.1 mM palmitic acid for 48 hours, the enterocytes and hepatocytes were recovered, respectively. Fat production in the hepatocytes was confirmed by oil red-O staining, and expression of fat metabolism and inflammation-related genes, and the permeability of the enterocytes was confirmed by expression of tight junction-related genes in the enterocytes.

[0068] 1) Confirmation of Fat Accumulation by Oil Red-O Stain

[0069] The culture solution was removed and they were washed with PBS, and then fixed with 10% formaldehyde for 10 minutes. 10% formaldehyde was removed and saturated formaldehyde was added again to fix cells over 1 hour. Then, 60% isopropanol was added and removed immediately, and then washed with distilled water, and fat globules were stained with oil red-O solution for 30 minutes and then washed with distilled water. The fat accumulated by oil red-O solution represents red (FIG. 1).

[0070] As a result, it could be confirmed that fat accumulation was reduced in the hepatocytes (HepG2 cells) in which 0.1 mM palmitic acid and Weissella hellenica WiKim0103 were co-cultured than the hepatocytes (control) in which 0.1 mM palmitic acid was treated.

[0071] 2) Confirmation of Hepatocytes (HepG2 Cells) Expression Level of Fat Differentiation and Inflammation-Related Genes

[0072] To find out the expression level of fat metabolism and inflammation-related genes in hepatocytes according to the treatment of the Weissella hellenica WiKim0103 strain, quantitative real time polymerase chain reaction (qPCR) was conducted.

[0073] As a result, as shown in FIG. 2, it could be confirmed that the gene expression of fat metabolism-related factors, SREBP-1c (Sterol regulatory element-binding protein-1c), fatty acid synthase (FAS), and unsaturated fatty acid biosynthesis enzyme (Stearoyl-CoA desaturase, SCD), and inflammation-related factors, COX-2 (Cyclooxygenase-2), TNF-α (Tumor necrosis factor-alpha), and NF-κB (Nuclear factor-kappa B) was significantly reduced, in the hepatocytes (HepG2 cells) in which 0.1 mM palmitic acid and Weissella hellenica WiKim0103 were co-cultured than the hepatocytes (control) in which 0.1 mM palmitic acid was treated.

[0074] 3) Confirmation of Expression Level of Tight Junction-Related Genes in Enterocytes (HT-29 Cells)

[0075] To find out the expression level of tight junction-related genes in the enterocytes according to the treatment of the Weissella hellenica WiKim0103 strain, quantitative real time polymerase chain reaction (qPCR) was conducted.

[0076] As a result, as shown in FIG. 3, it could be confirmed that the expression of tight junction-related ZO-1 (Zonula occludens-1) and Occludin genes was significantly increased in the enterocytes in which 0.1 mM palmitic acid and Weissella hellenica WiKim0103 were co-cultured than the enterocytes (control) in which 0.1 mM palmitic acid was treated.

Example 3: Confirmation of Body Weight Reduction Efficacy and Improvement of Liver Function Values in Blood

[0077] Five-week-old male mice (C57BL/6J) were adapted by chow diet (CD; Purina, Korea) in the breeding room environment under the conditions of temperature 20±2° C., humidity 50±5% and light-dark cycle 12 hours unit for 1 week. The experimental animals were fed a normal diet (D12450B, Research Diets, New Brunswick, N.J.) or high-fat diet (D12451, Research Diets, New Brunswick, N.J.) in which 45% of total calories were fat, and were adapted to the diet for 1 week, and then the experiment was performed. The experimental groups consisted of 4 groups in total, which were the normal diet intake group (ND), high-fat diet intake group (HFD), intake group receiving daily oral administration of Weissella hellenica WiKim0103 at a concentration of 2×10.sup.9 CFU/200 μl with the high-fat diet (HFD+L-LAB1), and intake group receiving daily oral administration of Weissella koreensis at a concentration of 2×10.sup.9 CFU/200 μl with the high-fat diet ((HFD+L-LAB2), and fatty liver was induced through diet for 20 weeks. The normal diet intake group (ND) and high-fat diet intake group (HFD) were orally administered with the same amount of phosphate buffered saline (PBS) daily. In 20 weeks after performing the experiment, the body weight of each experimental group was measured (FIG. 4a). In addition, to measure the liver function values (serum GOT, serum GPT) in blood of the experimental groups, they were fasted 16 hours before the end of the experiment, and blood was collected from the abdominal vena cava, and serum was separated and used in the experiment. The GOT and GPT in blood were measured by using an automatic biochemical measuring device (FUJI DRI-CHEM 7000i, Fujifilm, Tokyo, Japan), and in FIG. 4b and FIG. 4c, GLUTAMATE-OXALOACETATE TRANSAMINASE (GOT) and GLUTAMATE-PYRUVATE TRANSAMINASE (GPT) in blood were shown as graphs.

[0078] As a result, as shown in FIG. 4a to FIG. 4c, it could be confirmed that the body weight and liver function values in blood were significantly reduced when the high-fat diet and Weissella hellenica WiKim0103 were ingested together (HFD+L-LAB1), compared to the high-fat diet intake mice (HFD).

Example 4: Confirmation of Expression Level of Fat Metabolism, Inflammation and Liver Fibrosis-Related Genes in Liver Tissue

[0079] To find out the expression level of fat metabolism, inflammation and liver fibrosis-related genes in the liver tissue according to the intake of the Weissella hellenica WiKim0103 strain, using the liver tissue extracted after the end of experiment, QPCR (quantitative real time polymerase chain reaction) was conducted.

[0080] As a result, as shown in FIG. 5a to FIG. 5c, it could be confirmed that the expression of genes of the fat metabolism-related factor, SREBP-1c (Sterol regulatory element-binding protein-1c), inflammation-related factor, NF-κB (Nuclear factor-kappa B), and liver fibrosis-related factor, Col1α1 (Collagen type I alpha 1) was reduced, according to the intake of the Weissella hellenica WiKim0103 strain.

INDUSTRIAL AVAILABILITY

[0081] The present invention provides a novel strain. In addition, the strain of the present invention may be used as a food, feed or feed additive, pharmaceutical composition for preventing, improving or treating inflammation, obesity or fatty liver disease, or the like.

[Accession Number]

[0082] Depository authority name: Korean Culture Center of Microorganisms (Overseas)
Accession number: KCCM12419P
Accession date: 2018 Dec. 14