Composition for preventing, treating or improving metabolic diseases containing Lactobacillus plantarum NCHBL-004 strain or its culture broth

Abstract

The present invention relates to a composition for preventing, treating or improving metabolic diseases, comprising Lactobacillus plantarum NCHBL-004 strain or its culture solution. Since the composition suppresses adipocyte differentiation, suppresses weight gain, and also exhibits a blood glucose-lowering effect, it can be effectively used for the prevention, treatment or improvement of metabolic diseases.

Description

Technical Field

[0001] This invention was made under the support of the Ministry of Education, with the project specific number 1320213639 and the detailed project number 2021 - 3639. The specialized research management institution for this project is the Korea Foundation, the research project name is "Support Project for the Practical Application of Korean Creative Assets", the research topic name is "Animal Efficacy Evaluation of the Body Fat Inhibition and Blood Glucose Lowering Effects of Honey - derived Lactic Acid Bacteria", the competent institution is the Industry - University Cooperation Group of Chonnam National University, and the research period is from October 1, 2021 to January 31, 2022.

[0002] This patent application claims priority to Korean Patent Application No. 10 - 2022 - 0064168, filed with the Korean Intellectual Property Office on May 25, 2022, and Korean Patent Application No. 10 - 2022 - 0128076, filed with the Korean Intellectual Property Office on October 6, 2022, and the disclosure matters of the patent application are incorporated herein by reference.

[0003] The present invention relates to a composition for preventing, treating, or improving metabolic diseases, comprising Lactobacillus plantarum NCHBL - 004 strain or its culture solution. More specifically, it relates to a technology for using the culture solution or viable bacteria of Lactobacillus plantarum NCHBL - 004 strain for the prevention, treatment, or improvement of metabolic diseases such as obesity, diabetes, and non - alcoholic fatty liver disease (NAFLD).

Background Art

[0004] In recent years, along with the Westernization of the diet, the incidence of adult diseases such as obesity and diabetes due to genetic and environmental factors has been continuously increasing. According to the Korean Obesity Society, the prevalence rate of severe obesity in Korea has increased from 3.5% in 2009 to 6.01% in 2018, an increase of approximately 72% over 10 years. If this upward trend continues, it is expected that one in ten Koreans will be severely obese by 2030.

[0005] The associated social costs cannot be ignored either. The cost of obesity treatment in South Korea in 2008 alone was 1.7923 trillion won, and the fact is that this cost has been increasing every year. Amid research on substances that can prevent and treat this, in recent years, attempts have been made to develop various health functional foods. Obesity is not simply a state of being overweight but is clearly classified as a disease that can induce various diseases and even threaten life. It can lead not only to concomitant diseases such as osteoarthritis and sleep apnea due to overweight but also to metabolic-related complications such as non-alcoholic fatty liver, diabetes, hypertension, hyperlipidemia, and serious diseases such as severe cardiovascular diseases and cancer.

[0006] Non-alcoholic fatty liver disease (NAFLD) accounts for 70 - 90% of the causes of chronic hepatitis. An inflammatory reaction occurs in the liver due to an increase in oxidative stress or an increase in insulin resistance, and ultimately, it may deteriorate into severe fatty liver or non-alcoholic steatohepatitis (NASH). When it progresses to steatohepatitis, the risk of developing cirrhosis, liver failure, and hepatocellular carcinoma increases, and it may also lead to other chronic diseases such as cardiovascular diseases and result in death. It is important to cut off such a connection.

[0007] Pharmaceutical companies in South Korea or overseas have started developing NASH treatment agents, but there is still no product that has received approval from the licensing authorities. Currently used obesity treatment agents mainly act directly on the brain to induce a sense of fullness and suppress appetite. Obesity treatment agents such as orlistat reduce fat absorption and induce weight loss. Obesity treatment agents that induce a sense of fullness are designated and managed as psychotropic drugs due to dependence and tolerance. However, they may also increase the concentration of such neurotransmitters at the nerve junctions of norepinephrine or serotonin to induce a sense of fullness, or they may be induced by stimulating serotonin receptors or adrenergic receptors.

[0008] Such drugs have side effects such as fatigue, depression, auditory hallucinations, and sleep disorders when taken for more than three months, so attention should be paid to the risk of drug poisoning. Therefore, in the treatment of metabolic syndrome caused by obesity, a treatment strategy with a new mechanism of action of a material that has a high anti-obesity effect and an effect of improving metabolic diseases and few side effects is emerging.

[0009] On the other hand, microorganisms of the genus Lactobacillus are lactic acid bacteria that carry out homofermentation or heterofermentation, are often seen in the fermentation process of dairy products and vegetables, and most are classified as beneficial bacteria. In recent years, many studies have reported that by administering Lactobacillus bacteria or Bifidobacterium bacteria, which are beneficial bacteria in the intestine, that is, probiotics, effects such as weight loss, improvement of fatty liver and inflammation of adipose tissue, and alleviation of intestinal microbial imbalance can be obtained in an obese state induced by a high-fat diet.

Summary of the Invention

Problems to be Solved by the Invention

[0010] Therefore, the present inventors confirmed that Lactobacillus plantarum ( Lactobacillus plantarum ) derived from bees has excellent effects of inhibiting adipocyte differentiation and improving obesity.

[0011] Therefore, an object of the present invention is to provide a pharmaceutical composition for preventing or treating metabolic diseases, comprising Lactobacillus plantarum NCHBL-004 strain or its culture solution.

[0012] Another object of the present invention is to provide a health functional food composition for improving metabolic diseases, comprising Lactobacillus plantarum NCHBL-004 strain or its culture solution.

[0013] Still another object of the present invention relates to the use of Lactobacillus plantarum NCHBL-004 strain or its culture solution for preventing, treating or improving metabolic diseases.

Means for Solving the Problems

[0014] The present invention relates to a composition for preventing, treating, or improving metabolic diseases, comprising Lactobacillus plantarum NCHBL-004 strain or its culture solution. The composition according to the present invention suppresses adipocyte differentiation, suppresses weight gain, and also shows a blood glucose-lowering effect.

[0015] The inventors of the present invention confirmed that the live bacteria or culture solution of Lactobacillus plantarum derived from bees suppresses adipocyte differentiation from preadipocytes and shows an effect of suppressing weight gain and a blood glucose-lowering effect in an obesity-induced mouse model induced by a high-fat diet.

[0016] Hereinafter, the present invention will be described in more detail.

[0017] One aspect of the present invention is a pharmaceutical composition for preventing or treating metabolic diseases, comprising Lactobacillus plantarum NCHBL-004 strain or its culture solution.

[0018] In the present invention, the strain may be the one deposited under the accession number KCTC14909BP.

[0019] In the present invention, the strain may be live bacteria, dead bacteria, or a mixture thereof. For example, it may be live bacteria, but is not limited thereto.

[0020] In the present invention, the composition may contain the strain at a concentration of 5×10 5 ~5×10 11 CFU / ml, preferably 5×10 5 ~5×10 9 CFU / ml, 5×10 5 ~5×10 7 CFU / ml, or 5×10 7 ~5×10 11 CFU / ml. For example, it may contain the strain at a concentration of 5×10 7 ~5×10 9 CFU / ml, but is not limited thereto.

[0021] In the present invention, the culture solution may be a culture supernatant obtained by removing the strain after culturing the strain, a concentrate thereof, a fraction thereof, or a lyophilized product thereof. For example, it may be a culture supernatant, but is not limited thereto.

[0022] In the present invention, the metabolic disease may be one or more selected from the group consisting of obesity, diabetes, non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), hypertension, hyperlipidemia, cardiovascular disease, and hyperinsulinemia.

[0023] The pharmaceutical composition of the present invention may be used as a pharmaceutical composition containing a pharmaceutically effective amount of Lactobacillus plantarum NCHBL-004 strain or its culture solution and / or a pharmaceutically acceptable carrier.

[0024] As used herein, the term "pharmaceutically effective amount" means an amount sufficient to achieve the efficacy or activity of the above-described Lactobacillus sakei CVL-001 strain culture solution.

[0025] The pharmaceutically acceptable carrier contained in the pharmaceutical composition of the present invention is generally used in formulation, and includes, but is not limited to, lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia gum, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methylcellulose, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate, and mineral oil. The pharmaceutical composition of the present invention may further contain lubricants, wetting agents, sweeteners, flavoring agents, emulsifiers, suspending agents, preservatives, etc. in addition to the above components.

[0026] The pharmaceutical composition according to the present invention may be administered to mammals including humans by various routes. The administration method may be any commonly used method, for example, it may be administered by routes such as oral, skin, intravenous, intramuscular, subcutaneous, etc., and preferably may be administered orally.

[0027] The appropriate dosage of the pharmaceutical composition of the present invention varies depending on factors such as the formulation method, administration method, patient's age, weight, gender, pathological condition, diet, administration time, administration route, excretion rate, and reactivity, and a skilled ordinary physician can easily determine and prescribe a dosage effective for the desired treatment or prevention.

[0028] The pharmaceutical composition of the present invention is manufactured in the form of a unit dose by formulating it using a pharmaceutically acceptable carrier and / or excipient by a method that can be easily implemented by those having ordinary knowledge in the technical field to which the present invention pertains, or may be manufactured by being placed in a multi-dose container. At this time, the dosage form may be in the form of a solution, suspension, or emulsion in an oil or aqueous medium, or may be in the form of an extract, powder, granule, tablet, capsule, or gel (for example, hydrogel), and may further contain a dispersant or stabilizer.

[0029] Another aspect of the present invention is a health functional food composition for improving metabolic diseases containing Lactobacillus plantarum NCHBL-004 strain or its culture solution.

[0030] In the present invention, the strain may be the one deposited as KCTC14909BP.

[0031] In the present invention, the strain may be viable bacteria, dead bacteria, or a mixture thereof, for example, viable bacteria, but is not limited thereto.

[0032] In the present invention, the composition may contain the strain at a concentration of 5×10 5 ~5×10 11 CFU / ml, and preferably 5×10 5 ~5×10 9CFU / ml, 5×10 5 ~5×10 7 CFU / ml, or 5×10 7 ~5×10 11 CFU / ml, and may be included at a concentration of, for example, 5×10 7 ~5×10 9 CFU / ml, but is not limited thereto.

[0033] In the present invention, the culture solution may be a culture supernatant obtained by removing the strain after culturing the strain, a concentrate thereof, a fraction thereof, or a freeze-dried product thereof. For example, it may be a culture supernatant, but is not limited thereto.

[0034] In the present invention, the metabolic disease may be one or more selected from the group consisting of obesity, diabetes, non-alcoholic fatty liver, non-alcoholic steatohepatitis, hypertension, hyperlipidemia, cardiovascular disease, and hyperinsulinemia.

[0035] When the health functional food composition of the present invention is used as a food additive, the health functional food composition may be added as it is, or used together with other foods or food ingredients, and may be appropriately used by a normal method. Generally, when manufacturing foods or beverages, the food composition of the present invention may be added in an amount of 15% by weight or less, preferably 10% by weight or less, based on the raw materials.

[0036] There is no particular limitation on the type of the food. Examples of foods to which the substance can be added include meats, sausages, bread, chocolates, candies, snacks, confectioneries, pizzas, ramen, other noodles, gums, dairy products including ice creams, various soups, drinking water, tea, drink agents, alcoholic beverages, and vitamin complexes, and include any foods in the ordinary sense.

[0037] The beverage can contain various flavoring agents or natural carbohydrates as additional ingredients. The natural carbohydrates described above can include monosaccharides such as glucose and fructose, disaccharides such as maltose and sucrose, natural sweeteners such as dextrin and cyclodextrin, and synthetic sweeteners such as saccharin and aspartame. The ratio of the natural carbohydrates may be appropriately determined by those skilled in the art.

[0038] In addition to the above, the health functional food composition of the present invention can contain various nutrients, vitamins, electrolytes, flavoring agents, coloring agents, pectic acid and its salts, alginic acid and its salts, organic acids, protective colloid thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohol, carbonating agents used in carbonated beverages, etc. In addition, the health functional food composition of the present invention can contain pulp for the production of natural fruit juices, fruit juice beverages and vegetable beverages. Such components can be used independently or in combination. The ratio of such additives may also be appropriately selected by those skilled in the art.

Advantages of the Invention

[0039] The present invention relates to a composition for preventing, treating or improving metabolic diseases containing Lactobacillus plantarum NCHBL-004 strain or its culture solution. The composition suppresses adipocyte differentiation and weight gain, and also shows a blood sugar lowering effect, so it can be effectively used for the prevention, treatment or improvement of metabolic diseases.

Brief Description of the Drawings

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BEST MODE FOR CARRYING OUT THE INVENTION

[0063] The present invention relates to a pharmaceutical composition for preventing or treating metabolic diseases, comprising a Lactobacillus plantarum NCHBL-004 strain or a culture solution thereof.

EXAMPLE

[0064] Hereinafter, the present invention will be described in more detail with reference to the following examples. However, these examples are merely illustrative of the present invention, and the scope of the present invention is not limited by these examples.

[0065] Throughout this specification, “%” used to indicate the concentration of a specific substance is, unless otherwise specified, (weight / weight)% for solid / solid, (weight / volume)% for solid / liquid, and (volume / volume)% for liquid / liquid.

[0066] Example 1: Production of culture solutions of Lactobacillus kunkeei NCHBL-003 and Lactobacillus plantarum NCHBL-004 strains isolated from honeybees

[0067] 1-1. Isolation of lactic acid bacteria

[0068] To isolate lactic acid bacteria from the intestinal microbiota of honeybees, first, the target insects were collected in the Quanju area in early June. After that, they were placed in a sterilized collection container and refrigerated separately for 10 minutes before being taken out. The gastrointestinal tract was aseptically removed using microsurgical instruments in a laminar flow bench. The intestinal microbiota of honeybees was obtained by suspending the contents of the excised gastrointestinal tract in 10 ml of sterilized 0.1% peptone physiological saline supplemented with 0.1% Tween 80 (0.9% w / v NaCl, 0.1% w / v Tween 80, 0.1% w / v peptone).

[0069] For lactic acid bacteria, 500 μl of the suspension of visceral crushed matter was successively and appropriately diluted using a liquid medium (Difco TM Lactobacilli MRS Broth, BD, USA), and then spread on a solid medium (BBL TM LBS Agar, BD, USA) using an inoculation loop. After placing a BD GasPak TM EZdp, the culture was incubated at 37 °C until bacterial colonies were confirmed. The bacterial colonies confirmed from the LBS solid medium were identified as Lactobacillus by the Gram-positive result of Gram staining and the catalase-negative reaction confirmed by mixing with 3% hydrogen peroxide solution. The isolated wild isolate Lactobacillus was stored at -20 °C in MRS medium containing 15% glycerol and used in the next experiment.

[0070] Among the isolated strains, the phylogenetic identification of Lactobacillus kunkeei NCHBL-003 and Lactobacillus plantarum NCHBL-004 was performed by analyzing the nucleotide sequence of the 16S rRNA gene and comparing it with the 16S rRNA gene of Lactobacillus strains based on the similarity of the primary and secondary structures of the 16S rRNA gene using the PHYDIT program.

[0071]

Table 1

[0072] 1-2. Production of Bacterial Culture Solution

[0073] Live bacteria of Lactobacillus kunkeei NCHBL-003 and Lactobacillus plantarum NCHBL-004, which are lactic acid bacteria isolated from the internal intestine of honeybees, were cultured in DMEM (high glucose) medium for 24 hours, and then centrifuged to separate the supernatant. After adjusting the supernatant to pH 7.4, it was sterilized using a syringe filter (0.2 μm) and stored. 8 CFU / ml) and centrifuged, and the supernatant was separated. After adjusting the supernatant to pH 7.4, it was sterilized using a syringe filter (0.2 μm) and stored.

[0074] Example 2: Experiment on the inhibitory effect on adipocyte differentiation (in vitro)

[0075] 2-1. Cytotoxicity Evaluation

[0076] Cytotoxicity can be evaluated by measuring LDH (Lactate dehydrogenase) released from dead cells. 3T3-L1 preadipocyte cells purchased from the Korean Cell Line Bank were subcultured and maintained by adding 10% BCS (Bovine calf serum) and 1% PS (penicillin) to DMEM medium. The cells were seeded at a density of 1×10 5 cells / ml, 0.2 ml each, together with the medium supplemented with 10% FBS and 1% PS in a 48-well plate. After seeding in the plate, when the density reached 100%, it was designated as Day-2, and while culturing for another 2 days, all the cells in the cultured plate were induced to be in a cell cycle arrest state at the G1 stage.

[0077] To measure spontaneous LDH, wells without any treatment were prepared as the control group. Specifically, from Day 0 of 3T3-L1 preadipocytes, MDI (IBMX, Dexamethasone, Insulin) was added to the medium supplemented with 10% FBS (Fetal bovine serum) to induce adipocyte differentiation. On Day 2, the cells were transferred to the medium containing only insulin. From Day 4, the medium was changed at two-day intervals until Day 8 during differentiation.

[0078] To measure the maximum LDH, wells treated with the culture solution were prepared as the experimental group. Although prepared in the same way as the control group, on Day 0, which was two days after the 3T3-L1 preadipocytes grew to 100% density, the culture solution (12.5%, 25%, 50%) was treated. The culture solution was diluted using DMEM medium.

[0079] After culturing the cells for 24 hours, LDH analysis (LDH assay) was performed to evaluate cytotoxicity. After preparing wells added with triton X-100 solution, LDH substrate mixture was added, and the absorbance was measured to determine the percentage of toxicity.

[0080] The LDH released from the culture solutions of Lactobacillus kunkeei NCHBL-003 and Lactobacillus plantarum NCHBL-004 (0, 12.5%, 25%, 50%) was calculated as a percentage to evaluate cytotoxicity.

[0081] As can be confirmed from Figure 1, no cytotoxicity was observed at all concentrations of the culture solution treated on 3T3-L1.

[0082] 2-2. Evaluation of the inhibition on adipocyte differentiation

[0083] 3T3-L1 was seeded at 1×10 in a 12-well plate 5Cells were inoculated at a density of cells / ml with 1 ml each of medium supplemented with 10% FBS and 1% PS. Two days after the cells grew to 100% density (Day 0), the culture medium (0, 12.5, 25, 50%) and MDI, a differentiation inducer, were added to the treatment.

[0084] On Day 2, the culture medium was replaced while adding the culture medium (0, 12.5, 25, 50%) and insulin for treatment. On Day 4, the culture medium was replaced while adding the culture medium (0, 12.5, 25, 50%) for treatment. This was repeated at two-day intervals until differentiation progressed. When differentiated adipocytes were observed, for Oil Red O staining, the culture medium was replaced with 4% formalin to fix the cells for 20 minutes, and then washed twice with distilled water.

[0085] Thereafter, adipocytes were stained for 20 minutes using a staining reagent prepared by mixing Oil Red O staining reagent and distilled water (D.W) at a ratio of 6:4. After washing twice with distilled water and observing and photographing under a microscope, 100% isopropanol was added to extract the Oil Red O staining, and the degree of differentiation was quantitatively analyzed by measuring the absorbance at 510 nm.

[0086] As can be confirmed from FIGS. 2A and 2B, when MDI was added to 3T3-L1 cells, the differentiation into adipocytes increased, and the number of adipocytes decreased significantly in a concentration-dependent manner by treatment with Lactobacillus kunkeei NCHBL-003 culture supernatant. However, the number of adipocytes did not decrease by treatment with Lactobacillus plantarum NCHBL-004 culture supernatant.

[0087] From this, it was confirmed that the Lactobacillus kunkeei NCHBL-003 culture supernatant inhibits the differentiation into adipocytes.

[0088] 2-3. Evaluation of the inhibition of adipocyte differentiation-related transcription factors

[0089] In the adipogenesis process occurring in adipose tissue, lipid biosynthesis, lipid droplet formation, lipid accumulation, etc. are increased by major transcription factors such as SREBP1c (sterol regulatroy element binding protein1c), PPARγ (peroxisome proliferator-activated receptor-γ), and C / EBPα (CCAAT-enhancer binding protein α). Thus, in 3T3-L1 cells differentiated into adipocytes, the protein expression levels of the aforementioned transcription factors were measured by Western blotting with or without treatment with lactic acid bacteria culture broth.

[0090] Specifically, 3T3-L1 cells inoculated at a density of 1×10 5 cells / ml in a 12-well plate were cultured until Day 8 with treatment with lactic acid bacteria culture broth as described in the aforementioned experimental method. After collecting the cells of each group with a cell scraper, proteins were separated using a protein lysis buffer containing a protease inhibitor.

[0091] The same amount of each protein was loaded onto an SDS-PAGE gel and separated by protein molecular weight size, and then transferred to a PVDF (Polyvinylidene fluoride) membrane. The transferred membrane was reacted with primary antibodies SREBP1c (#ab28481, Abcam), PPAR γ (#2443S, Cell Signaling Technology), C / EBPα (#8178T, Cell Signaling Technology), and β-actin (#sc-47778, Santa cruz Biotechnology) at 4°C for 18 hours, and then washed with TBST buffer.

[0092] Anti-rabbit IgG, anti-mouse IgG, and HRP-linked secondary antibody (#31640, Invitrogen), which are secondary antibodies, were reacted at room temperature for 2 hours and then washed with TBST. The detection reagent was treated on the membrane, and the photograph was developed to measure the expression level of the target protein.

[0093] As can be confirmed from Figure 3, when adipocyte differentiation was induced by MDI treatment, the protein expression level of related transcription factors increased, and the expression level of transcription factors decreased significantly in a concentration-dependent manner by treatment with the culture solution of Lactobacillus kunkeei NCHBL-003. However, the expression level of transcription factors did not decrease by treatment with the culture solution of Lactobacillus plantarum NCHBL-004.

[0094] From this, it was confirmed that the culture solution of Lactobacillus kunkeei NCHBL-003 suppresses adipocyte differentiation by decreasing the expression level of transcription factors.

[0095] Example 3: Preparation of viable cells of Lactobacillus kunkeei NCHBL-003 and Lactobacillus plantarum NCHBL-004

[0096] To produce viable cells for feeding mice, viable cells of Lactobacillus kunkeei NCHBL-003 and viable cells of Lactobacillus plantarum NCHBL-004 (1*10 8 CFU / ml) were cultured in a solid medium (MRS agar) at 30 °C for 24 hours. A single colony was collected and pre-cultured in a liquid medium (MRS broth). The pre-culture was carried out at 150 rpm and 30 °C for 24 hours in 5 ml of the medium, and then diluted 10-fold and main-cultured under the same conditions for 3 hours.

[0097] To adjust the number of bacteria, the concentration was adjusted with PBS using a spectrophotometer so that an O.D. value of 0.6 was obtained at an absorbance of 600 nm. Under this condition, the Lactobacillus kunkeei NCHBL-003 strain was 1.57*109 Only at CFU / ml, Lactobacillus plantarum NCHBL-004 strain was 1.48*10 9 CFU / ml was confirmed to exist. Considering that each mouse was fed at a concentration of 0.2 ml per day per strain at 1*10 7 CFU / mouse, 1*10 9 CFU / mouse, viable bacterial media were prepared by centrifugation at 3,000 rpm for 15 minutes.

[0098] Example 4: Experiment on the effect of improving obesity (in vivo)

[0099] 4-1. Measurement of body weight changes in obesity-induced mice

[0100] When a high-fat diet (60% composed of fat) is administered to mice, it is known that the body weight increases compared to mice administered a normal diet. Therefore, an obese mouse animal model induced by administration of a high-fat diet is widely used in obesity disease research.

[0101] 7-week-old male C57BL / 6 mice were administered a normal diet or a high-fat diet together with the viable bacterial media. Body weight was measured 2 weeks after administration of the normal diet or high-fat diet, and the body weights of each group were made the same. Body weight was measured at 1-week intervals from 3 weeks after administration of the normal diet or high-fat diet. A glucose tolerance test was performed at week 10, an insulin tolerance test was performed at week 15, and dissection was performed at week 16 to measure the weights of the organs.

[0102] The information of the groups is as follows. The number of mice in all groups was set to 10.

[0103]

Table 2

[0104] As a result of the experiment conducted according to Table 2, it was observed that the group fed a high-fat diet (G2) showed a significantly increased body weight compared to the group fed a normal diet (G1).

[0105]

Table 3

[0106] As can be confirmed from Table 3 and Figure 4A, in the group (G4) administered live Lactobacillus kunkeei NCHBL-003 at a concentration of 1*10 9 CFU together with a high-fat diet, a significant decrease in body weight was also observed compared to G2. In the group (G3) administered live Lactobacillus kunkeei NCHBL-003 at a low concentration of 1*10 7 CFU, the body weight decreased on average, but not at a significant level.

[0107] As can be confirmed from Table 2 and Figure 4B, in two groups (G5, G6) fed live Lactobacillus plantarum NCHBL-004 at concentrations of 1*10 7 and 1*10 9 CFU respectively together with a high-fat diet, the body weight decreased significantly compared to G2.

[0108] From this, it was confirmed that live Lactobacillus kunkeei NCHBL-003 and live Lactobacillus plantarum NCHBL-004 induce a body weight loss effect.

[0109] 4-2. Glucose Tolerance Test in Obesity-Induced Mice

[0110] When glucose is orally administered to mice, the blood glucose level temporarily increases, but then tends to decrease again due to normal insulin secretion in the body. However, it is known that in a mouse animal model fed a high-fat diet, the blood glucose level remains continuously high.

[0111] The mice in each group prepared in Example 4-1 were utilized for the glucose tolerance test after fasting for 12 hours from 9:00 p.m. the day before the experiment to 9:00 a.m. the next day. A 10% glucose (Glucose in PBS) solution sterilized by 0.2μm filtration was prepared, and 2mg (glucose / g, volume (μl) = body weight (g) x 20) was orally administered to each individual. The mice were put back into the cage and blood was collected from the tail, and the blood glucose levels were measured at 0, 15, 30, 60, 90, and 120 minutes.

[0112] In this study, it was observed that the G2 group fed a high-fat diet maintained significantly higher blood glucose levels compared to the G1 group fed a normal diet.

[0113]

Table 4

[0114] As can be confirmed from Table 4, Figure 5A, and Figure 5B, in the groups (G3-6) fed live Lactobacillus kunkeei NCHBL-003 and live Lactobacillus plantarum NCHBL-004 together with a high-fat diet, the blood glucose levels decreased compared to G2. In particular, in the case of Lactobacillus plantarum NCHBL-004, it decreased to a significant level at each time point when blood glucose was measured.

[0115] As can be confirmed from Figure 5C and Figure 5D, in the AUC results calculated for the area under the glucose tolerance test curve, in the group (G3) fed live Lactobacillus kunkeei NCHBL-003 at a concentration of 1*10 7 CFU, it decreased significantly compared to G2. In the two groups (G3, G4) administered live Lactobacillus plantarum NCHBL-004 at concentrations of 1*10 7 and 1*10 9 CFU respectively, it was observed that the levels decreased significantly compared to G2.

[0116] That is, from the glucose tolerance test, it was confirmed that Lactobacillus kunkeei NCHBL-003 and Lactobacillus plantarum NCHBL-004 have a blood glucose-lowering effect in an obesity model induced by a high-fat diet.

[0117] 4-3. Insulin Load Blood Glucose Evaluation of Obesity-Induced Mice

[0118] In a mouse animal model fed a high-fat diet, it is known that blood glucose is maintained at a very high level. When insulin is intraperitoneally administered to such a mouse animal model, blood glucose temporarily decreases, but in the mouse animal model fed a high-fat diet, the decrease in blood glucose is relatively small.

[0119] The mice in each group prepared in Example 4-1 above were utilized for an insulin load test after maintaining a 4-hour fast from 9:00 am to 1:00 pm. An insulin (Insulin in PBS) solution sterilized by 0.2 μm filtration was prepared, and each individual was intraperitoneally administered 0.5 IU / kg (BW.Vol = 20 μl) each (27-gauge sterile needle). The mice were put back into the cage and blood was collected from the tail, and blood glucose values were measured every 0, 15, 30, 60, 90, 120, and 150 minutes.

[0120] In this study, it was observed that the G2 group administered a high-fat diet maintained a much higher blood glucose value compared to the G1 group administered a normal diet.

[0121]

Table 5

[0122] As can be confirmed from Table 5, Figures 6A and 6B, in particular, in the groups (G3-6) fed live Lactobacillus kunkeei NCHBL-003 and live Lactobacillus plantarum NCHBL-004 together with a high-fat diet, the blood glucose value decreased compared to G2. In particular, in the case of Lactobacillus plantarum NCHBL-004, it significantly decreased at each time point when blood glucose was measured.

[0123] As can be confirmed from FIGS. 6C and 6D, in the AUC results of calculating the area under the insulin tolerance test curve, in the case of Lactobacillus kunkeei NCHBL-003 (G3, G4), the blood glucose levels decreased compared to G2, but not at a significant level. From the AUC results, in two groups (G5, G6) administered with viable cells of Lactobacillus plantarum NCHBL-004 at concentrations of 1*10 7 、1*10 9 CFU, respectively, it was confirmed that the levels were significantly decreased compared to G2.

[0124] That is, from the insulin tolerance test, it was confirmed that Lactobacillus kunkeei NCHBL-003 and Lactobacillus plantarum NCHBL-004 have a blood glucose-lowering effect in the obesity model induced by a high-fat diet.

[0125] 4-4. Measurement of Organ Weights of Obesity-Induced Mice

[0126] Mice animal models induced to obesity by a high-fat diet were dissected at the 16th week, and the organ weights were measured and compared.

[0127] For histochemical analysis, the liver and adipose tissues fixed in 10% formalin solution were embedded in paraffin, then sectioned into 3-μm-thick slices to make section slides. After staining the nucleus and cytoplasm with H&E staining solution composed of hematoxylin and eosin, images were developed at a magnification of 100 using an optical microscope and a digital camera attached to the microscope. For the evaluation of histological clinical symptoms of non-alcoholic fatty liver, the following criteria were established for evaluation: steatosis (0-3), hepatocellular ballooning (0-3), lobular inflammatory cell infiltration (0-2). The adipocyte size of adipose tissue was measured using the Image J program.

[0128] Both the liver weight and the subcutaneous fat weight increased in Group G2 fed a high-fat diet compared to the normal diet Group G1.

[0129] As can be confirmed from Fig. 7A, a decrease in liver weight was observed in Group G4 fed Lactobacillus kunkeei NCHBL-003 viable bacteria at a concentration of 1*10 9 CFU, but it was confirmed that this was not at a significant level. A decrease in liver weight was seen in Groups (G5, G6) fed Lactobacillus plantarum NCHBL-004 viable bacteria together with a high-fat diet, and it was confirmed that the decrease was at a significant level in Group G6, which was fed at a concentration of 1*10 9 CFU.

[0130] As can be confirmed from Fig. 7B, a decrease in subcutaneous fat weight was seen in Groups (G3, G4) fed Lactobacillus kunkeei NCHBL-003 viable bacteria together with a high-fat diet, and it was confirmed that the decrease was at a significant level in Group G4, which was administered at a concentration of 1*10 9 CFU. A decrease in subcutaneous fat weight was seen in Groups (G5, G6) fed Lactobacillus plantarum NCHBL-004 viable bacteria together with a high-fat diet, and it was confirmed that the decrease was at a significant level in Group G5, which was administered at a concentration of 1*10 7 CFU.

[0131] 4 - 5. Evaluation of Clinical Symptoms of Fatty Liver in Obesity-Induced Mice

[0132] In a mouse animal model fed a high-fat diet, it is known that lipids accumulate in the liver tissue and clinical symptoms of non-alcoholic fatty liver appear.

[0133] Adipose tissue was used to isolate total RNA using TRIzol reagent solution. The isolated total RNA was quantified using a Nanodrop spectrophotometer, and the reverse transcription reaction was analyzed by synthesizing cDNA from the RNA using TOPscript RT DryMIX (Enzynomics, Korea). Using the synthesized cDNA as a template, each primer and kit (TOPreal SYBR Green PCR kit, Enzynomics, Korea) were added, and after amplification using a real-time PCR (Rotor-Gene Q, QIAGEN) instrument, it was analyzed using quantification software.

[0134] The products amplified by real-time PCR were quantified using the Ct (comparative cycle threshold) method, and for each sample, the expression level was corrected using the housekeeping gene 18S rRNA as an internal control factor. The primer sequences for each gene used in the PCR are presented in Table 6.

[0135]

Table 6

[0136] As can be confirmed from Figures 8A and 8B, as a result of feeding a high-fat diet for 16 weeks, it was confirmed that steatosis, hepatocellular ballooning, and lobular inflammatory cell infiltration occurred in the liver tissue. As a result of feeding live Lactobacillus kunkeei NCHBL-003 together with the high-fat diet, such clinical symptoms decreased, and 1*10 9A significant decrease was shown in the group (G4) fed at the concentration of CFU. In both of the two groups (G5, G6) fed with live Lactobacillus plantarum NCHBL-004 together with the high-fat diet, the clinical symptoms decreased, and it was confirmed that this was at a significant level compared to G2 fed only with the high-fat diet.

[0137] 4 - 6. Histological evaluation of adipose tissue in obesity-induced mice

[0138] In a mouse animal model fed with a high-fat diet, it is known that while lipid accumulation in adipose tissue increases, the size of adipocytes also increases.

[0139] As can be confirmed from FIGS. 9A and 9B, for the group (G4) fed with live Lactobacillus kunkeei NCHBL-003 at a concentration of 1*10 9 CFU, the average adipocyte size decreased. As a result of feeding with live Lactobacillus plantarum NCHBL-004 together with the high-fat diet, the average adipocyte size decreased, and it was confirmed that in the group (G6) fed at a concentration of 1*10 9 CFU, it decreased to a significant level.

[0140] 4 - 7. Gene expression evaluation of epididymal adipose tissue by Lactobacillus kunkeei NCHBL-003 in obesity-induced mice

[0141] The mice grouped as prepared in Example 4-1 were dissected at the 16th week, RNA was extracted from the epididymal fat, and the expression levels of metabolism-related genes were evaluated. The primers used for the expression level evaluation are as shown in Table 7 below.

[0142]

Table 7

[0143] Adiponectin is mostly produced in adipocytes and is well known to regulate glucose and lipid metabolism, increase insulin sensitivity, and decrease in metabolic syndrome. GLUT4 (glucose transporter type 4) plays a role in mediating intracellular glucose uptake in peripheral tissues upon insulin stimulation, and when insulin resistance occurs, the translocation of GLUT4 from the cytoplasm to the cell membrane is inhibited.

[0144] Hormone-sensitive lipase (HSL) and adipose triglyceride lipase (ATGL), which are important in lipid metabolism, are mainly activated in adipose tissue and act to break down triglycerides. Carnitine palmitoyl transferase 1A (CPT1A) plays a role in promoting mitochondrial fatty acid beta-oxidation.

[0145] As can be confirmed from Figure 10, as a result of feeding Lactobacillus kunkeei NCHBL-003 live bacteria together with a high-fat diet, it was confirmed that the expression of the gene increased to a significant level in epididymal fat. However, the degree of increase in GLUT4 gene expression in the group administered at a concentration of 1*10 7 CFU (G3) and the degree of increase in CPT1α gene expression in the two groups G3 and G42 were not at a significant level. From this, the fact that Lactobacillus kunkeei NCHBL-003 live bacteria administration regulates the expression of metabolism-related genes in adipose tissue was confirmed.

[0146] 4 - 8. Evaluation of gene expression in epididymal adipose tissue by Lactobacillus plantarum NCHBL-004 in obesity-induced mice

[0147] Mice induced to be obese by a high-fat diet were dissected at the 16th week, and RNA was extracted from epididymal fat to evaluate the expression levels of adipocyte differentiation-related genes and inflammation-related genes.

[0148]

Table 8

[0149] PPARγ (peroxisome proliferator-activated receptor-γ) and C / EBPα (CCAAT-enhancer binding protein), which are transcription factors acting on adipocyte differentiation, regulate the expression of their respective target genes to promote adipocyte differentiation, and aP2 (adipocyte protein 2) is a gene found in mature adipocytes.

[0150] As can be confirmed from Figure 11, as a result of administering live Lactobacillus plantarum NCHBL-004 at a concentration of 1*10 7 CFU (G5), the expression of the above genes decreased, and C / EBPα and aP2 decreased to significant levels. When live Lactobacillus plantarum NCHBL-004 was administered at a concentration of 1*10 9 CFU (G6), the gene expressions of PPARγ and aP2 decreased, but not at significant levels.

[0151] In the occurrence of metabolic diseases, the inflammatory response appears chronically at a low level, induces insulin resistance, and is presented as a pathophysiological mechanism of metabolic diseases caused by obesity. TNFα (tumor necrosis factor α) and MCP1 (monocyte chemoattractant protein-1), which are inflammation-related genes in adipocytes, are inflammatory cytokines mainly secreted from adipocytes, and caspase-1 is known to have a major function in macrophage-derived inflammation in adipose tissue.

[0152] As can be confirmed from Figure 12, as a result of administering live Lactobacillus plantarum NCHBL-004 at a concentration of 1*10 7 CFU (G5), the expressions of the above genes all decreased to significant levels. When live Lactobacillus plantarum NCHBL-004 was administered at a concentration of 1*10 9When administered at the concentration of CFU (G6), the expression of the gene decreased, and in particular, the decrease in the expression of caspase-1 was at a significant level.

[0153] From this, it was confirmed that when live Lactobacillus plantarum NCHBL-004 was administered, the gene expression related to adipocyte differentiation and inflammation in adipose tissue was regulated.

Industrial Applicability

[0154] The present invention relates to a composition for preventing, treating or improving metabolic diseases, comprising Lactobacillus plantarum NCHBL-004 strain or its culture solution. More specifically, the present invention relates to a technique for using the culture solution or live bacteria of Lactobacillus plantarum NCHBL-004 strain for the prevention, treatment or improvement of metabolic diseases such as obesity, diabetes, non-alcoholic fatty liver disease (NAFLD).

[0155] TIFF2025518584000010.tif187170

Deposit Number

[0156] Lactobacillus plantarum NCHBL-004 (Deposit Number KCTC14909BP)

Claims

1. A pharmaceutical composition for preventing or treating metabolic diseases, comprising Lactobacillus plantarum NCHBL-004 strain or a culture solution thereof.

2. The pharmaceutical composition for preventing or treating metabolic diseases according to claim 1, wherein the strain is deposited under the accession number KCTC14909BP.

3. The pharmaceutical composition for preventing or treating metabolic diseases according to claim 1, wherein the strain is viable bacteria, dead bacteria, or a mixture thereof.

4. The pharmaceutical composition for preventing or treating metabolic diseases according to claim 1, comprising the strain at a concentration of 5×10 ５ to 5×10 １１ CFU / ml.

5. The pharmaceutical composition for preventing or treating metabolic diseases according to claim 1, wherein the metabolic disease is one or more selected from the group consisting of obesity, diabetes, non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), hypertension, hyperlipidemia, cardiovascular diseases, and hyperinsulinemia.

6. A health functional food composition for improving metabolic diseases, comprising Lactobacillus plantarum NCHBL-004 strain or a culture solution thereof.

7. The health functional food composition for improving metabolic diseases according to claim 6, wherein the strain is deposited under the accession number KCTC14909BP.

8. The health functional food composition for improving metabolic diseases according to claim 6, wherein the strain is viable bacteria, dead bacteria, or a mixture thereof.

9. The health functional food composition for improving metabolic diseases according to claim 6, comprising the strain at a concentration of 5×10 ５ to 5×10 １１The health functional food composition for improving metabolic diseases according to claim 6, which is contained at a concentration of CFU / ml.

10. The metabolic disease is one or more selected from the group consisting of obesity, diabetes, non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), hypertension, hyperlipidemia, cardiovascular disease, and hyperinsulinemia. The health functional food composition for improving metabolic diseases according to claim 6.

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