COMPOSITION COMPRISING LACTOCOCCUS CHUNGANGENSIS FOR PREVENTION OR TREATMENT OF FATTY LIVER OR METABOLIC SYNDROME

Abstract

The present invention relates to a Lactococcus chungangensis strain having preventive and reductive effects on fatty liver or metabolic syndrome. More particularly, the present invention relates to a composition at least one selected from the group consisting of the strain, a culture containing the strain, and a fermented material of the strain as an active ingredient for preventing, alleviating, and treating fatty liver or metabolic syndrome.

Claims

1. A pharmaceutical composition for the prevention or treatment of fatty liver or metabolic syndrome comprising at least one selected from the group consisting of a Lactococcus chungangensis strain, a culture containing of the strain and a fermented material of the strain as an active ingredient.

2. The pharmaceutical composition according to claim 1, wherein the Lactococcus chungangensis has an accession number of KCTC 12684BP.

3. The pharmaceutical composition according to claim 1, wherein the metabolic syndrome is any one selected from the group consisting of obesity, dyslipidemia, hyperlipidemia, diabetes and insulin resistance syndrome.

4. A food composition for preventing or improving fatty liver or metabolic syndrome comprising at least one selected from the group consisting of a Lactococcus chungangensis strain, a culture containing of the strain, and a fermented material of the strain as an active ingredient.

5. The food composition according to claim 4, wherein the food is fermented food or cheese.

6. Use of a composition comprising at least one selected from the group consisting of a Lactococcus chungangensis strain, a culture containing of the strain, and a fermented material of the strain as an active ingredient for preparing an agent for preventing or treating fatty liver or metabolic syndrome.

7. The use according to claim 6, wherein the Lactococcus chungangensis has an accession number of KCTC 12684BP.

8. The use according to claim 6, wherein the metabolic syndrome is any one selected from the group consisting of obesity, dyslipidemia, hyperlipidemia, diabetes and insulin resistance syndrome.

9. A method for treating fatty liver or metabolic syndrome comprising administering an effective amount of a composition comprising at least one selected from the group consisting of a Lactococcus chungangensis strain, a culture containing of the strain, and a ferment material of the strain as an active ingredient to a subject in need thereof.

10. The method according to claim 9, wherein the Lactococcus chungangensis has an accession number of KCTC 12684BP.

11. The method according to claim 9, wherein the metabolic syndrome is any one selected from the group consisting of obesity, dyslipidemia, hyperlipidemia, diabetes and insulin resistance syndrome.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0071] FIGS. 1a and 1b show the effect of oral administration of CAU 28 dry matter and CAU 28 cream cheese on BMI and Lee obesity index, and AUC calculated from BMI (FIG. 1a) and Lee obesity index (FIG. 1b) is displayed. Data are presented as mean±SEM (n=xx). Differences between means compared to positive controls were assessed using ANOVA. * P<0.05, ** P<0.005, *** P<0.0005, **** P<0.0001.

[0072] FIGS. 2a to 2e show the effect of oral administration of CAU 28 dry matter and CAU 28 cream cheese on organ and adipose tissue weight, and liver/weight ratio (FIG. 2a), lung/weight ratio (FIG. 2b), height/weight ratio (FIG. 2c), white fat/weight ratio (FIG. 2d) and brown fat/weight ratio (FIG. 2e) are shown (%). Data is denoted by xx (n=xx). Differences between means compared to positive controls were assessed using ANOVA. *P<0.05, **P<0.005, ***P<0.001, ****P<0.0001.

[0073] FIGS. 3a to 3d show the effect of oral administration of CAU 28 dry matter and CAU 28 cream cheese on the liver and adipose tissue, and histological analysis of liver and adipose tissue (FIG. 3a), the sizes of brown adipose tissue (FIG. 3b), abdominal adipose tissue (FIG. 3c) and subcutaneous adipose tissue (FIG. 3d) are indicated. Red arrows and squares indicate regions of interest. Differences between means compared to positive controls were assessed using ANOVA. *P<0.05, ***P<0.0005, ****P<0.0001.

[0074] FIGS. 4a and 4b show the effect of oral administration of CAU 28 dry matter and CAU 28 cream cheese on OGTT,

[0075] FIG. 4a shows the blood glucose concentration measured by OGTT at each time point after glucose ingestion, FIG. 4b shows the AUC calculated from OGTT data for 0-120 min after glucose uptake. Differences between means compared to positive controls were assessed using ANOVA. *P<0.05, **P<0.005, ***P<0.0005, ****P<0.0001.

[0076] FIGS. 5a and 5b show the effect of oral administration of L. chungangensis CAU 28 and CAU 28 cream cheese on ITT,

[0077] FIG. 5a is the blood glucose concentration measured by ITT at each time point after insulin injection, FIG. 5b shows AUC calculated from ITT data for 0-120 min after insulin injection. Differences between means compared to positive controls were assessed using ANOVA. *P<0.05, **P<0.005, ***P<0.0005.

[0078] FIGS. 6a to 6f show the effect of oral administration of CAU 28 dry matter and CAU 28 cream cheese on adipokine and serum cytokine levels, and are the results of measuring each protein level using ELISA: FIG. 6a is leptin, FIG. 6b is adiponectin, FIG. 6c is TNF-α, FIG. 6d is IL-β, FIG. 6e is IL-6, FIG. 6f is the serum level of IFN-γ. Differences between means compared to positive controls were assessed using ANOVA. ***P<0.0005; ****P<0.0001.

[0079] FIGS. 7a and 7b show the effect of oral administration of CAU 28 dry matter and CAU 28 cream cheese on T cell activation, T cell activation was assessed using flow cytometry. The percentages of helper T cells (CD3.sup.+ CD4.sup.+ CD8.sup.−) (FIG. 7a) and cytotoxic T cells (CD3.sup.+ CD4.sup.− CD8.sup.+) (FIG. 7b) are indicated. Differences between means compared to positive controls were assessed using ANOVA. *P<0.05, **P<0.005.

[0080] FIGS. 8a to 8d show the effect of oral administration of CAU 28 dry matter and CAU 28 cream cheese on blood lipids, serum TC (FIG. 8a), HDL/TC (FIG. 8b), LDL/TC (FIG. 8b) and TG (FIG. 8d) values are shown. Differences between means compared to positive controls were assessed using ANOVA. **P<0.005, ***P<0.0005, ****P<0.0001.

[0081] FIGS. 9a and 9b show the effect of oral administration of CAU 28 dry matter and CAU 28 cream cheese on liver disease biomarkers, serum AST (FIG. 9a) and ALT (FIG. 9b) levels are indicated. Differences between means compared to positive controls were assessed using ANOVA. **P<0.005, ***P<0.0005, ****P<0.0001.

[0082] FIGS. 10a and 10b show the effect of oral administration of CAU 28 dry matter and CAU 28 cream cheese on excretory SOFA, excretion levels of acetic acid (FIG. 10a) and propionic acid (FIG. 10b) were measured. Differences between means compared to positive controls were assessed using ANOVA. *P<0.05, ***P<0.0005, ****P<0.0001.

[0083] FIGS. 11a and 11b are the results of observing the degree of fat accumulation (FIG. 11a) and adipocytes stained under a microscope (FIG. 11b) when 3T3-L1 adipocyte differentiation was induced and treated with CAU 28 lysate at the same time. *P<0.05, ***P<0.0005, ****P<0.0001.

[0084] FIG. 12 is the result of evaluating the mRNA expression level of fat accumulation-related factors by real-time PCR when 3T3-L1 adipocyte differentiation is induced and CAU 28 lysate is treated at the same time, and adipocyte differentiation is finished. *P <0.05, ***P<0.0005, ****P<0.0001.

[0085] FIGS. 13a and 13b are the results of measuring the degree of accumulation of triglyceride in adipocytes (FIG. 13a) and the amount of IL-6 secreted from adipocytes (FIG. 13b) when 3T3-L1 adipocyte differentiation was induced and CAU 28 lysate was treated at the same time. *P<0.05, ***P<0.0005, ****P<0.0001.

MODE FOR CARRYING OUT INVENTION

[0086] Hereinafter, the present invention will be described in detail.

[0087] However, the following examples only illustrate the present invention, and the content of the present invention is not limited to the following examples.

[0088] Experiment Method

[0089] 1. Culture and Freeze-Drying of Lactococcus chungangensis CAU 28T Lactococcus chungangensis CAU 28T, a kind of lactic acid bacteria, was used after being deposited in the Korean Collection for Type Cultures (KCTC;Taejon, Korea, accession number KCTC 12684BP), after inoculation in TSB (Bacto Tryptone 17 g, Bacto soytone 3 g, Glucose 2.5, Sodium chloride 5 g, Dipotassium hydrogen phosphate 2.5 g, D.W. 1000 ml) and incubated at 30° C., and was freeze-dried and made into a powder, it was dissolved in distilled water before use in the experiment.

[0090] In addition, after making a lysate of CAU28T using sterilized beads, was stored at −80° C. until used in the experiment, and was used after thawing in the experiment.

[0091] 2. Production of Cream Cheese

[0092] Pasteurized milk (Pasteur Milk Co., Ltd., Seoul, Korea) was heat treated at 68° C. for 30 minutes and then cooled. After the addition of 5% (v/v) Lactococcus chungangensis CAU 28T starter, it was cultured at 30° C. During this process, the milk acidified and a curd was formed. After heating at 70° C. and stirring for 5 minutes, whey was separated through filter paper. 0.5% salt was added to the separated curd. The finally produced cream cheese sample is freeze-dried and was stored in a dark room at 4° C. until the next experiment.

[0093] 3. Experimental Animals and Breeding Conditions

[0094] The mouse model used was a 6-week-old C57BL/6J mouse (n=50) model (Samtako, Osan, Korea), and C57BL/6J mice were acclimatized for 1 week prior to the experiment, and they were bred in 5 groups of 10 each. The temperature of the animal breeding room was 22° C., and the lighting was controlled for 12 hours, and these conditions were maintained for the duration of the experiment.

[0095] 4. Administration and Experimental Group

[0096] The experimental group was divided into 5 groups (n=10 for each group): (1) negative control, normal diet (NFD) and oral administration of PBS; (2) positive control, high-fat diet (HFD) and oral administration of PBS; (3) simvastatin group, high-fat diet (HFD) and oral administration of simvastatin; (4) L. chungangensis CAU 28 (CAU 28) group, high-fat diet (HFD) and oral administration of lyophilized L. chungangensis CAU 28; (5) CAU 28 cream cheese group, high-fat diet (HFD) and oral administration of cream cheese made with L. chungangensis CAU 28.

[0097] During the experimental period, the experimental group had free access to diet and drinking water. Cream cheese made from freeze-dried L. chungangensis CAU 28 [1×10.sup.8 CFU/mouse] and L. chungangensis CAU 28 (1.4 g/kg/mouse) was suspended in 200 μl of sterile water and administered by oral gavage. The same volume of PBS was administered to the negative control group and the positive control group. Simvastatin (10 mg/kg) was dissolved in 200 μl of sterile water and administered orally. All mice were gavaged once a week for 12 weeks.

[0098] The weight and length of the animals were measured once a week. Fecal samples were taken and an oral glucose tolerance test (OGTT) and an insulin tolerance test (ITT) were performed a few days before the end of the study period. At the end of the 12-week treatment period, mice were sacrificed by administration of isoflurane. For further analysis, blood samples and visceral and adipose tissue were immediately harvested. All procedures and procedures were approved by Food and Drug Administration (FDA) guidelines. Animals used in this study were managed according to the principles and guidelines of the Chung-Ang University Animal Care and Use Committee (IACUC) of the Laboratory Animal Research Institute (IACUC no. 2017-00044).

[0099] 5. Weight, BMI, Body Fat and Organ Weighing

[0100] The body weight and body length of each mouse were measured once a week.

[0101] BMI [weight (kg)/height.sup.2 (m.sup.2)] was measured at 12 weeks, Lee obesity index (weight×0.33/naso-anal length) was calculated at week 1 and week 12 (Novelli et al., 2007).

[0102] Liver, lung, kidney, spleen and adipose tissue (abdominal, subcutaneous and scapular fat) were dissected; Liver, lung, kidney and adipose tissue were weighed. Organs and tissues were rinsed with saline for further analysis.

[0103] 6. Glucose Tolerance Test (OGTT)

[0104] After 12 weeks of breeding, a glucose tolerance test was performed. After fasting for 16 hours, glucose (2.5 mg/g body weight, Sigma-Aldrich, St. Louis, Mo., USA) was administered by oral gavage. Blood samples were taken from the tail vein at 0, 10, 20, 30, 60, 90 and 120 minutes after glucose administration. Blood glucose levels were measured using an Accu-Check Advantage blood glucose monitor (LifeScan, Johnson & Johnson, Chesterbrook, Pa.), the area under the curve (AUC) was calculated according to the manufacturers instructions.

[0105] 7. Insulin Resistance Test (ITT)

[0106] Blood glucose changes after insulin injection were assessed by ITT. Mice were fasted for 4 h prior to intraperitoneal injection of insulin solution (0.75 UI/kg; Sigma-Aldrich, St. Louis, Mo., USA). Blood samples were taken from the tail vein at 0, 10, 20, 30, 60, 90 and 120 minutes after insulin injection. Blood glucose levels were determined by an Accu-Check Advantage glucose monitor and AUC was calculated according to the manufacturers instructions.

[0107] 8. Biochemical and Histological Analysis

[0108] 8-1. Metabolic Parameters

[0109] Blood samples were collected after 12 weeks of treatment from two mice from each experimental group in centrifuge tubes containing EDTA. Blood was drawn by puncturing the orbit through the orbit and within the orbit. Samples were centrifuged at 1200× for 15 minutes and incubated at 4° C. for 10 minutes. Serum aspartate aminotransferase (AST), alanine aminotransferase (ALT), TC, high-density lipoprotein (HDL), low-density lipoprotein (LDL) and TG were analyzed in the serum supernatant using an appropriate assay kit (Green Cross Biopharmaceutical, Yongin, Korea). The same samples were also used for hormone measurements.

[0110] 8-2. Hormone Analysis

[0111] The concentration of adiponectin in serum was measured by enzyme-linked immunosorbent assay (ELISA) using an ADP ELISA kit (CUSABIO, Houston, Tex., USA) according to the method described in the kit manual. Serum leptin concentrations were measured using a Linco Human Leptin ELISA kit (Linco Research, St. Charles, Mo., USA) according to the manufacturer's instructions. Sample absorbance was measured at 450 nm using an Infinite M200 NanoQuant plate reader (Tecan, Switzerland).

[0112] 8-3. Cytokine Analysis

[0113] Blood samples collected after 12 weeks of treatment were used for cytokine analysis [tumor necrosis factor (TNF-α), interferon (IFN-γ), interleukin (IL) 1β and IL-6]. Blood samples were coagulated at 4° C. for 1 hour and then centrifuged at 5000× for 1 hour. The supernatant (serum) was stored at −80° C. until analysis. Cytokine concentrations were measured using an appropriate ELISA kit (R&D Systems, Minneapolis, Minn., USA) according to the manufacturer's instructions. Sample absorbance was measured at 450 nm using an Infinite M200 NanoQuant plate reader (Tecan).

[0114] 8-4. Flow Cytometry

[0115] Spleens were harvested from sacrificed mice and crushed on a cell strainer (SPL Life Sciences, Pocheon, Korea). Cells were counted after staining with trypan blue using a TC10 automatic cell counter (Bio-Rad, Hercules, Calif., USA). It was then diluted to 2.0×10.sup.6 cells/tube and stained with phycoerythrin (PE)-labeled anti-mouse antibody: CD4 or CD8 (BD Biosciences, San Jose, Calif., USA) on ice for 20 min. Cells were analyzed by collecting at least 10,000 events on a FACSCalibur™ flow cytometer (BD Biosciences, San Jose, Calif., USA) and BD CellQuest Pro Software (version 6.0).

[0116] 8-5. Histological Analysis

[0117] Liver, abdominal, subcutaneous and scapular fats of sacrificed mice were obtained for biopsy. The intestines and adipose tissue were thoroughly washed with saline. Samples were aliquoted and fixed in 10% (v/v) neutral-buffered formalin at 0° C. for 24 h. It was then embedded in paraffin and incised at 4-5 μm to perform hematoxylin & eosin staining. The size of adipocytes in adipose tissue was measured using an optical microscope (Leica, Wetzlar, Germany).

[0118] 8-6. SCFA Analysis

[0119] Mouse stool samples were collected several days prior to sacrifice. The content of SOFA (intestinal metabolite) was determined using high performance liquid chromatography (HPLC) (Dionex Ultimate 3000, Thermo Fisher Scientific, Sunnyvale, Calif., USA). Samples were prepared by homogenization and centrifugation at 12,000×g at 4° C. for 20 minutes. SCFAs (acetic acid and propionic acid) were separated on an Aminex 87H column (300×10 mm, Bio-Rad). As the mobile phase (Fluke; Sigma-Aldrich), isocratic elution with 0.01 N sulfuric acid was used, and the flow rate was 0.5 ml/min. SCFAs were identified at a wavelength of 210 nm using a RefractoMax521 Refractive Index Detector (ERC, RefractoMAX520, Kawaguchi, Japan).

[0120] 9. Evaluation of Anti-Obesity Effect of CAU28 Strain on 3T3-L1

[0121] 9-1. Cultivation of 3T3-L1 Cell Line

[0122] The 3T3-L1 cells used in the experiment were prepared by adding FCS (Fetal Calf Serum) and Streptomycin & Penicillin antibiotics to DMEM (Dulbecco's Modified Eagle Medium) medium, and incubated at 37° C. and 5% CO.sub.2 conditions. In this experiment, after seeding the 3T3-L1 cell line with FBS instead of FCS in a 12-well plate, and then cells were incubated until full in each well.

[0123] 9-2. Differentiation Induction and Experimental Group

[0124] In each group, the negative control group in which 3T3-L1 cells were cultured by treating only the medium, the positive control group in which the medium containing differentiation components (Insulin, 3-Isobutyl-1-methylxanthine, Dexamethasone) was treated, and CAU 28 in the differentiation medium lysate-treated conditions were set as the experimental group. 4 days after differentiation induction, the positive control group was treated with a medium containing only Insulin. In the experimental group, lactic acid bacteria were continuously treated in the same medium condition to induce differentiation for 9 days, at this time, from the 7th day of induction of differentiation, the medium without differentiation components was treated for 2 days. The concentration of lysate was 1×10.sup.10 CFU/ml, and 1% was treated when the cells were treated. After a total of 9 days of differentiation, the degree of adipogenesis was confirmed from the cells and the supernatant.

[0125] 9-3. Oil Red O Staining from 3T3-L1

[0126] After the differentiation of 3T3-L1 cells was washed, the cells were fixed with formalin solution. After staining the adipocytes using Oil red 0 solution and observing them under a microscope, and after destaining with isopropanol, the degree of fat accumulation was confirmed through absorbance measurement.

[0127] 9-4. Real-Time PCR from 3T3-L1

[0128] After the differentiation of 3T3-L1 cells was washed, RNA extraction was performed using Trizol. After RNA extraction, cDNA was synthesized. Expression levels were confirmed using Real-Time PCR using primers for fat accumulation-related factors.

[0129] 9-5. Triglyceride Determination from 3T3-L1

[0130] After the differentiation of 3T3-L1 cells was removed using a scraper, the pellet obtained through centrifugation was mixed well with the solution used for triglyceride measurement, and then the cells were destroyed using sterilized beads. After centrifugation again, the supernatant was separately obtained and triglyceride was measured.

[0131] 9-6. Cytokine Measurement from 3T3-L1

[0132] After 4 hours of treatment with LPS (100 ng/ml) in the positive control group and the experimental group treated with lactic acid bacteria in 3T3-L1 cells after differentiation, the supernatant was obtained and cytokines were measured.

[0133] 10. Statistical Analysis

[0134] Results are presented as mean±standard error (SEM). The biochemical parameter data were normally distributed and significant differences between groups were determined by one-way ANOVA using post-hoc analysis (Duncan's test). P<0.05 was considered to represent a statistically significant difference. All analyzes were performed using the GraphPad Prism statistical package (version 7.0, GraphPad Software, La Jolla, Calif., USA).

Example 1: Effects of CAU 28 Dry Cells and CAU 28 Cream Cheese Intake on BMI and Lee Obesity Index

[0135] There was no significant difference in the initial body weight of animals in all groups (P=0.6456; Table 1), but the AUC of BMI was clearly lower in the CAU 28, CAU 28 cream cheese, negative control and simvastatin groups compared to the positive control group (P<0.0001) (FIG. 1a).

[0136] As shown in Table 1, the Lee obesity index of all animals was compared at week 1 and week 12 in order to accurately evaluate the change in obesity of experimental animals during the entire experimental period. At week 1, there was no difference in Lee obesity index between groups (P=0.1042). However, at 12 weeks, the Lee obesity index of the positive control group was significantly higher than that of the other groups (P<0.01).

[0137] The Lee obesity index of the negative control group, CAU 28 and CAU 28 cream cheese group was significantly lower at week 12 than at week 1. The Lee obesity index of the simvastatin group was slightly higher at week 12 than at week 1 (FIG. 1b).

[0138] These results indicate that oral administration of CAU 28 dry cells and CAU 28 cream cheese suppressed obesity due to HFD, thereby reducing BMI and Lee obesity index.

TABLE-US-00001 TABLE 1 Negative Positive control control Simvastatin CAU 28 CAU 28 CC P-value Initial body 23.24 ± 0.47  23.41 ± 0.33  23.73 ± 0.57  22.75 ± 0.35  23.74 ± 0.75  0.6456 weight (g) Final body 27.45 ± 0.73  41.91 ± 2.46  36.34 ± 3.13  36.47 ± 0.90  36.62 ± 2.72  <0.0001 weight (g) AUC of BMI 2.89 ± 0.08 3.57 ± 0.10 3.00 ± 0.00 3.27 ± 0.09 3.30 ± 0.11 <0.0001 Lee obesity 3.21 ± 0.13 3.23 ± 0.07 3.13 ± 0.06 3.13 ± 0.03 3.15 ± 0.01 0.1042 index (1 w) Lee obesity 2.74 ± 0.17 3.25 ± 0.05 3.43 ± 0.30 3.09 ± 0.17 3.20 ± 0.19 <0.001 index (12 w) Lung/body 0.007 ± 0.001 0.004 ± 0.001 0.006 ± 0.002 0.005 ± 0.001 0.006 ± 0.001 0.3841 weight (%) Kidney/body 0.012 ± 0.001 0.009 ± 0.001 0.010 ± 0.002 0.008 ± 0.001 0.009 ± 0.001 <0.01 weight (%) Liver/body 0.04 ± 0.00 0.032 ± 0.01  0.02 ± 0.00 0.03 ± 0.00 0.02 ± 0.00 <0.0001 weight (%) White fat/body 2.10 ± 0.08 12.02 ± 1.44  5.01 ± 1.63 8.05 ± 0.92 8.45 ± 0.50 <0.0001 weight (%) Brown fat/body 0.01 ± 0.01 0.036 ± 0.017 0.03 ±0.01  0.03 ± 0.01 0.04 ± 0.01 <0.01 weight (%) TC (mg/dl) 30.50 ± 0.50  72.50 ± 0.50  51.50 ± 1.50  48.50 ± 4.50  52.50 ± 0.50  <0.0001 TG (mg/dl) 29.50 ± 0.50  40.00 ± 5.00  30.00 ± 5.00  31.50 ± 1.50  37.00 ± 1.00  <0.01 HDL/TC (%) 0.98 ± 0.05 0.94 ± 0.00 1.00 ± 0.02 1.00 ± 0.02 0.99 ± 0.01 0.1295 LDL/TC (%) 11.50 ± 0.02  0.16 ± 0.01 0.12 ± 0.00 0.15 ± 0.01 0.13 ± 0.00 <0.01 AST (U/l) 35.00 ± 1.00  61.50 ± 6.50  54.50 ± 2.50  47.50 ± 4.50  38.00 ± 4.00  <0.0001 ALT (U/l) 9.50 ± 0.50 17.00 ± 3.00  10.00 ± 0.00  9.00 ± 0.00 10.00 ± 0.00  <0.001

Example 2: Effects of CAU 28 Dry Cells and CAU 28 Cream Cheese Intake on Organ and Fat Percentage

[0139] Adipose tissue-to-body weight ratios for liver, lung, kidney and adipose tissue (abdominal, subcutaneous and scapular adipose tissue) were determined to investigate the effects of CAU 28 dry cells and CAU 28 cream cheese on organs and adipose tissue.

[0140] The liver/body weight of the positive control group was significantly higher than that of the CAU 28, CAU 28 cream cheese and simvastatin groups (P<0.01) (FIG. 2a). This means that oral administration of CAU 28 freeze-dried group and CAU 28 cream cheese group prevented hepatomegaly. In addition, the effect of CAU 28 cream cheese intake group was more pronounced than that of CAU 28 freeze-dried cell intake.

[0141] There was no significant difference in lung and height/weight ratio in the positive control group, CAU 28, CAU 28 cream cheese, and simvastatin groups (P=0.3841) (FIGS. 2b and 2c). These results suggest that the administration of CAU 28 dry cells and CAU 28 cream cheese did not damage the lungs and kidneys in HFD-induced obese mice.

[0142] Body fat percentage is the ratio of the weight of fat to body weight and can be used to determine the degree of obesity in an animal. Abdominal and subcutaneous adipose tissue represent white fat. Scapular adipose tissue represents brown fat. The white fat/weight ratio of the positive control group was significantly higher than that of the negative control group (P<0.0001). This indicated that HFD increased the white fat/body weight ratio in obese mice. On the other hand, the white fat/weight ratio was clearly decreased in the CAU 28, CAU 28 cream cheese and simvastatin groups compared to the positive control group (P<0.0001) (FIG. 2d).

[0143] Also, the brown fat/weight ratio of the positive control group was not significantly different from the CAU 28, CAU 28 cream cheese and simvastatin groups (P=0.4594) (FIG. 2e). Therefore, oral administration of CAU 28 dry cells and CAU 28 cream cheese decreased the white fat/weight ratio while increasing the brown fat/body weight ratio in HFD-induced obese mice.

Example 3: Effects of CAU 28 Dry Cells and CAU 28 Cream Cheese Intake on Hepatic Steatosis and Adipocyte Size

[0144] As a result of histological analysis, it was confirmed that lipid accumulation was abnormal in the liver of the positive control group (FIG. 3a). In the positive control group, vesicle damage was observed in the liver tissue, the fat content of the liver was high, and cell contents leak, the cells swell and the cell membranes are destroyed as a large amount of fat accumulated in the blood vessels. On the other hand, as a result of oral administration of CAU 28 dry cells and CAU 28 cream cheese to HFD-induced obese mice, hepatic steatosis was less than that of the positive control group, hepatocytes had fewer fat vacuoles, morphologically, it was similar to that of the negative control group. Hepatic fat droplets in the simvastatin group were smaller than in the positive control group, but the number did not decrease significantly in the simvastatin group.

[0145] Hypertrophy of adipocytes in abdominal and subcutaneous fat was more pronounced in the positive control group and the simvastatin group than in the CAU 28 and CAU 28 cream cheese groups, but the brown adipocytes were the opposite. These results indicate that ingestion of CAU 28 dry cells and CAU 28 cream cheese suppressed the accumulation of lipids in hepatocytes and adipocytes. That is, the effects of CAU 28 dry cells and CAU 28 cream cheese were more pronounced than the effects of simvastatin and reduced the degree of liver damage in the HFD-induced obese group.

[0146] As shown in FIG. 3b, the size of the brown and white adipose tissue (abdominal and subcutaneous) of the mouse was measured. The brown adipose tissue of the positive control group was smaller than that of the CAU 28 cream cheese group (P<0.001) and the negative control group (P<0.05). However, there was no difference in size between the CAU 28 dry cells and the positive control (P=0.9167) (FIG. 3b). Abdominal and subcutaneous adipose tissue were significantly smaller in all other groups than the positive control group (P<0.001) (FIGS. 3c and 3d).

Example 4: Effects of CAU 28 Dry Cells and CAU 28 Cream Cheese Intake on OGTT

[0147] OGTT was used to determine the glycemic response to surgically administered glucose in all groups of animals after 12 weeks. In all groups, the blood glucose concentration increased immediately after glucose administration and reached the highest concentration 20-30 minutes after glucose administration. (FIG. 4a). The peak blood glucose concentration of the CAU 28 and CAU 28 cream cheese groups was lower than that of the positive control group and the simvastatin group.

[0148] The blood glucose concentration in all groups decreased immediately after reaching the peak level, but the blood glucose concentration AUC in CAU 28 cream cheese and the negative control group was significantly lower than that in the positive control group (P<0.01), there was no significant difference between the blood glucose concentrations of CAU 28, simvastatin and positive control (P=0.2082) (FIG. 4b).

[0149] These results suggest that the intake of CAU 28 dry cells and CAU 28 cream cheese reduced the blood glucose concentration in HFD-induced obese mice.

Example 5: Effects of CAU 28 Dry Cells and CAU 28 Cream Cheese Intake on ITT

[0150] To evaluate the insulin resistance of mice in each group, blood glucose changes were measured by short-term ITT after insulin injection. Blood glucose concentration increased immediately after insulin injection in all groups, and the peak reached 20 minutes after insulin injection (FIG. 5a). Thereafter, the blood glucose concentration immediately decreased and reached a new low level 30 minutes after the insulin injection, which was maintained until the end of the measurement (120 minutes). The blood glucose concentration in the simvastatin group was significantly higher than that in the positive control group (P<0.01). However, the blood glucose concentration of CAU 28, CAU 28 cream cheese and the negative control group was lower than that of the positive control group (FIG. 5b).

[0151] In addition, there was a significant difference only in the AUC values of the CAU 28 cream cheese group, the negative control group and the positive control group (P <0.05) (FIG. 5b).

[0152] These results indicated that ingestion of CAU 28 dry cells and CAU 28 cream cheese reduced blood glucose concentration in ITT.

Example 6: Effect of CAU 28 Dry Cells and CAU 28 Cream Cheese Intake on Blood Inflammatory Markers

[0153] To investigate the effect of CAU 28 dry cells and CAU 28 cream cheese intake on cytokine and chemokine production, cytokine and chemokine concentrations in the serum of animals in each group were investigated.

[0154] Serum leptin levels in the positive control group were significantly higher than those in the negative control group (P<0.0001), and this indicates that HFD induces leptin resistance (FIG. 6a). The serum leptin concentration of the CAU 28 and CAU 28 cream cheese groups was significantly lower than that of the positive control group (P <0.0001). There was no statistically significant difference in serum leptin concentrations between the simvastatin group and the positive control group (P=0.9971). These results indicate that oral administration of CAU 28 dry cells and CAU 28 cream cheese reduced serum leptin levels in HFD-induced obese mice.

[0155] Serum adiponectin levels were clearly higher in the negative control group than in the positive control group (FIG. 6b). It is known that increased oxidative stress decreases adiponectin secretion, and adiponectin is mainly secreted from adipocytes and is known to play a role in improving hyperlipidemia by increasing insulin sensitivity and reducing sugar production in the liver. That is, it was shown that a high-fat diet resulted in a decrease in adiponectin levels in HFD-induced obese mice, but serum adiponectin levels in the CAU 28 and CAU 28 cream cheese groups (P<0.0001) and in the simvastatin group (P<0.001) were significantly higher than in the positive control group.

[0156] Therefore, these results indicate that oral administration of CAU 28 dry cells and CAU 28 cream cheese increases serum adiponectin levels in HFD-induced obese mice.

[0157] When fat accumulates in the body, NADPH (nicotinamide adenine dinucleotide phosphate) oxidase is activated and systemic oxidative stress is increased. Increased oxidative stress is known to increase inflammatory cytokines TNF-α (tumor necrosis factor-alpha), MCP-1 (monocyte chemotactic protein-1), IL-6 (interleukin-6), and the like. Serum concentrations of TNF-α, IL-β, IFN-γ, and IL-6 were significantly higher in the positive control group than in the negative control group, CAU 28, and CAU 28 cream cheese group (P<0.0001) (FIG. 6c-f). TNF-α, IL-β, IFN-γ, and IL-6 are inflammatory cytokines. Therefore, these data suggest that oral administration of CAU 28 dry cells and CAU 28 cream cheese can reduce or inhibit oxidative stress by reducing the level of pro-inflammatory cytokines in HFD-induced obese mice.

Example 7: Effects of CAU 28 Dry Cells and CAU 28 Cream Cheese Intake on Lymphocytes

[0158] To investigate the effect of HFD on the immune system of HFD-induced obese mice, the number of T lymphocytes in the spleen was measured (FIGS. 7a and 7b).

[0159] The number of CD4.sup.+ T cells was significantly lower in the positive control group than in the negative control group (P<0.05) (FIG. 7a). This indicates that HFD reduced CD4.sup.+ T cell counts in obese mice. As a result of the measurement, the number of CD4.sup.+ T cells in the CAU 28 and CAU 28 cream cheese groups was higher than that of the positive control group.

[0160] The number of CD8.sup.+ T cells in the negative control group and the CAU 28 cream cheese group was higher than that in the positive control group, but the CD8.sup.+ T cell number in the CAU 28 group was lower than that in the positive control group. (FIG. 7b).

[0161] Therefore, oral administration of CAU 28 dry cells and CAU 28 cream cheese was shown to enhance the immune response by increasing the number of CD4.sup.+ helper T cells.

Example 8: Effects of CAU 28 Dry Cells and CAU 28 Cream Cheese Intake on Blood Composition

[0162] 8-1. Measurement of TC, HDL/TC, LDL/TC and TG Levels

[0163] After 12 weeks, the serum lipid levels of each group were examined (FIGS. 8a to 8d). As a result, the TC, TG, and LDL/TC values of the negative control group, CAU 28 and CAU 28 cream cheese groups were significantly lower than those of the positive control group (P<0.05) (FIGS. 8a, 8c and 8d). The HDL/TC ratio of the negative control group was higher than the HDL/TC ratio of the positive control group (P=0.2721) (FIG. 8b).

[0164] Administration of CAU 28 cream cheese significantly reduced TC (P<0.05) and decreased TG level (P=0.05) compared to the positive control group. In addition, administration of CAU 28 dry cells and simvastatin significantly reduced TC and TG levels in HFD-induced obese mice (P=0.6401) (FIGS. 8a and 8d).

[0165] The LDL/TC ratio of the simvastatin, CAU 28 and CAU 28 cream cheese groups was lower than that of the positive control group (FIG. 8c), but the HDL/TC ratio of the positive control group was higher than the HDL/TC ratio of the other experimental groups, but there was no statistical significance (P=0.1295, Table 1) (FIG. 8b).

[0166] These results indicate that CAU 28 dry cells and CAU 28 cream cheese intake reduced TC and/or TG serum levels in HFD-induced obese mice.

[0167] 8-2. Measurement of AST and ALT Levels

[0168] When the structure and function of the cell membrane are destroyed, ALT (Alanine aminotransferase), an enzyme widely present in the cytoplasm of the liver, is released into the blood, so blood ALT levels are frequently used as indicators of liver damage. Serum concentrations of AST and ALT, biomarkers of liver disease, were significantly higher in the positive control group than in the CAU 28 and CAU 28 cream cheese groups (P<0.01) (FIGS. 9a and 9b).

[0169] The serum concentration of the positive control group was significantly higher than that of the simvastatin group (P<0.001), but the serum AST concentration of the positive control group and the simvastatin group was not statistically different (P=0.1849) (FIG. 9b).

[0170] These results showed that CAU 28 dry cells and CAU 28 cream cheese intake prevented liver damage in HFD-induced obese mice.

Example 9: Effect of CAU 28 Dry Cells and CAU 28 Cream Cheese Intake on Fecal SCFA

[0171] Acetic acid and propionic acid are absorbed into the blood and enter the metabolic pathway through the liver. It is speculated that SOFA, mainly propionic acid, improves glucose resistance and inhibits cholesterol synthesis in the liver, and this is probably due to suppressing the increase in serum free fatty acid concentration and improving insulin sensitivity. It was found that the SOFA also affects the expression of PPAR, and the synergistic action of PPAR was found to increase the production of ApoA1, a major component of HDL.

[0172] Therefore, the levels of acetic acid and propionic acid, which are short chain fatty acids (SCFAs), were measured in mouse feces after 12 weeks.

[0173] SOFA levels were significantly higher in the negative control group than in the positive control group (P<0.001) (FIGS. 10a and 10b). This indicated that HFD induced a decrease in acetic acid and propionic acid levels in the gut of HFD-induced obese mice.

[0174] In addition, the concentrations of acetic acid and propionic acid in the stool were significantly higher in the CAU 28 cream cheese group than in the positive control group (P<0.05). Both acetic acid and propionic acid were higher in the CAU 28 group than in the positive control group (P=0.0749).

[0175] These results confirmed that not only had the effect of improving the intestinal flora, but also improved insulin resistance and glucose resistance, and had the effect of inhibiting cholesterol synthesis in the liver as oral administration of CAU 28 dry cells and CAU 28 cream cheese increased the intestinal levels of acetic acid and propionic acid in HFD-induced obese mice.

Example 10: Anti-Obesity Effect and Inhibition of Fat Differentiation of CAU28 Strain on 3T3-L1

[0176] When the CAU28 strain for 3T3-L1 was treated, the degree of fat accumulation was quantified, and adipocytes stained with a microscope were confirmed.

[0177] As a result, the degree of fat accumulation was reduced in the experimental group treated with lactic acid bacteria. There was also a statistically significant difference when compared with the positive control group treated with the differentiation component (FIG. 11a). As a result of confirming the stained adipocytes under a microscope, the degree of adipocyte staining was significantly reduced compared to that of the positive control group (FIG. 11b).

[0178] Next, when 3T3-L1 was treated with CAU28, mRNA expression levels of fat accumulation-related factors, triglyceride accumulation, and IL-6 secretion were checked.

[0179] As a result, the experimental group treated with CAU28 showed a decrease in the expression level of fat accumulation-related factors compared to the positive control group, and there was a statistically significant difference (FIG. 12). In addition, the amount of triglyceride accumulated in adipocytes was decreased (FIG. 13a), and it was confirmed that the secretion amount of IL-6 was also significantly decreased (FIG. 13b).

INDUSTRIAL APPLICABILITY

[0180] Lactococcus chungangensis strain, as a therapeutic composition comprising at least one selected from the group consisting of a culture of the strain and a fermented product of the strain as an active ingredient to prevent and treat fatty liver or metabolic syndrome, has excellent industrial applicability as it can be usefully used for the prevention or development of therapeutic agents for fatty liver or metabolic syndrome because it reduces blood glucose levels, has the effect of suppressing weight gain induced by a high-fat diet, has the effect of reducing the fat in the tissues produced by obesity and inhibiting liver damage.

[0181] [Accession Number]

[0182] Name of deposit institution: Korea Research Institute of Bioscience and Biotechnology

[0183] accession number: KCTC12684BP

[0184] deposit date: 20140926