Compositions containing polysaccharides or extracts derived from Lactobacillus plantarum

Abstract

The present invention relates to a composition containing polysaccharides or extract derived from Lactobacillus plantarum. More specifically, by containing polysaccharides derived from Lactobacillus plantarum or an extract of Lactobacillus plantarum, the composition can exhibit the effects of suppressing inflammatory responses, suppressing lipogenesis, suppressing intracellular glucose uptake, reducing insulin resistance and reducing hepatic steatosis, and can exhibit the effects of treating or preventing metabolic diseases.

Description

[Technical field]

[0001] The present invention relates to compositions comprising polysaccharides derived from Lactobacillus plantarum or an extract of Lactobacillus plantarum. [Background technology]

[0002] According to the World Health Organization (WHO), probiotics are defined as "live microorganisms that, when administered in adequate amounts, confer a health benefit on the host." The probiotics market is expected to grow 37% globally from 2016 to 2020 due to benefits such as improving gut health and preventing insulin resistance. However, recent studies have shown that regular consumption of probiotics can result in unexpected side effects. Probiotic administration can, for example, cause infections, unwanted inflammatory responses, and gene delivery from probiotics to the native host microorganisms.

[0003] To minimize the aforementioned side effects of probiotics, there has been an increased interest in postbiotics in recent years. Postbiotics, also known as "simple metabolites" or "cell-free supernatants", are identified as bioactive compounds secreted by live lactic acid bacteria (LAB). Postbiotics can include functional bioactive compounds such as metabolites, functional proteins, cell lysates and short-chain fatty acids. Postbiotics can be utilized as a substitute for probiotics since they can exhibit the effects of probiotics without the risk of delivering antibiotic resistance genes to the host. Postbiotics can also be recommended for children under the age of 5, as they pose less risk of LAB-related infections such as pneumonia and meningitis in infants and young children. Moreover, postbiotics are stable over a wide range of pH and temperature and can be separated into individual components, making them suitable for in vitro and in vivo studies and easy to commercialize. Recent studies have shown that exopolysaccharides (EPS), a postbiotic secreted by lactic acid bacteria, exhibit antiviral effects in intestinal epithelial cells. Summary of the Invention [Problem to be solved by the invention]

[0004] An object of the present invention is to provide a polysaccharide derived from Lactobacillus plantarum or a Lactobacillus plantarum extract.

[0005] An object of the present invention is to provide a pharmaceutical composition for preventing or treating metabolic diseases, comprising a polysaccharide derived from Lactobacillus plantarum or an extract of Lactobacillus plantarum.

[0006] An object of the present invention is to provide a food composition for preventing or improving metabolic diseases, which contains a polysaccharide derived from Lactobacillus plantarum or a Lactobacillus plantarum extract. [Means for solving the problem]

[0007] 1. A pharmaceutical composition for preventing or treating a metabolic disease comprising a polysaccharide derived from Lactobacillus plantarum or an extract of Lactobacillus plantarum.

[0008] 2. The pharmaceutical composition according to item 1, wherein the Lactobacillus plantarum is at least one selected from the group consisting of Lactobacillus plantarum L-14 (accession number KCTC13497BP), Lactobacillus plantarum ATCC10241, Lactobacillus plantarum NCDO704, and Lactobacillus plantarum NCDO1193.

[0009] 3. The pharmaceutical composition according to item 1, wherein the polysaccharide is at least one of an intracellular polysaccharide and an extracellular polysaccharide.

[0010] 4. The pharmaceutical composition according to item 1, wherein the polysaccharide is glucose.

[0011] 5. The pharmaceutical composition according to item 1, wherein the Lactobacillus plantarum extract is an ultrasonic disruptant of the Lactobacillus plantarum.

[0012] 6. The pharmaceutical composition according to item 1, wherein the metabolic disease is at least one selected from the group consisting of insulin resistance, type 2 diabetes, hyperlipidemia, fatty liver, obesity and inflammation.

[0013] 7. A food composition for preventing or improving a metabolic disease, comprising a polysaccharide derived from Lactobacillus plantarum or an extract of Lactobacillus plantarum. Effect of the Invention

[0014] The polysaccharides derived from Lactobacillus plantarum or Lactobacillus plantarum extract of the present invention can suppress inflammatory reaction.The polysaccharides derived from Lactobacillus plantarum or Lactobacillus plantarum extract of the present invention can show anti-inflammatory effect by suppressing the interaction between LPS and TLR4.

[0015] In addition, the polysaccharides derived from Lactobacillus plantarum or Lactobacillus plantarum extract of the present invention can show the effects of suppressing fat production, suppressing intracellular glucose uptake, reducing insulin resistance and reducing hepatic steatosis, and can show the effect of improving metabolic disease.The polysaccharides derived from Lactobacillus plantarum or Lactobacillus plantarum extract of the present invention can inhibit fat production by interacting with TLR2 and activating AMPK pathway. [Brief description of the drawings]

[0016] [Figure 1] FIG. 1 shows the results of analyzing the characteristics of exopolysaccharides derived from Lactobacillus plantarum according to one embodiment. [Diagram 2] FIG. 2 shows the results of confirming the inhibitory effect of metabolites secreted from Lactobacillus plantarum L-14 strain on inflammatory responses in mouse macrophages. [Diagram 3] FIG. 3 shows the results of confirming the inhibitory effect of an embodiment of EPS derived from Lactobacillus plantarum on morphological changes in cells induced by LPS. [Figure 4] FIG. 4 shows the results of confirming the inhibitory effect of an embodiment of EPS derived from Lactobacillus plantarum on an inflammatory response induced by LPS. [Diagram 5] FIG. 5 shows the results of confirming the inhibitory effect of an embodiment of EPS derived from Lactobacillus plantarum on LPS-induced nuclear translocation of NF-κB. [Figure 6]FIG. 6 shows the results of confirming that EPS derived from Lactobacillus plantarum according to one embodiment regulates the MAPK and NRF2 / HO-1 pathways, which are major inflammatory response regulatory pathways. [Figure 7] FIG. 7 shows the results confirming that EPS derived from Lactobacillus plantarum according to one embodiment suppresses inflammatory responses via the TLR4 pathway. [Figure 8] FIG. 8 shows the results of confirming the inhibitory effect of an embodiment of a Lactobacillus plantarum extract on differentiation from preadipocytes to mature adipocytes. [Figure 9] FIG. 9 shows the results of confirming the effect of an embodiment of a Lactobacillus plantarum extract in suppressing weight gain and the effect of inhibiting the expression of pro-inflammatory markers in a high-fat diet mouse model. [Figure 10] FIG. 10 shows the results of confirming the insulin resistance marker and liver fat inhibiting effects of one embodiment of a Lactobacillus plantarum extract. [Figure 11] FIG. 11 shows the results of confirming the effect of an embodiment of a Lactobacillus plantarum extract on upregulating the AMPK signaling pathway and inhibiting adipogenesis. [Figure 12] FIG. 12 shows the inhibitory effect of an embodiment of a Lactobacillus plantarum extract on the differentiation of human bone marrow mesenchymal stem cells (BM-MSCs) into adipocytes. [Figure 13] FIG. 13 shows the results of confirming the adipogenesis-inhibiting effect of an embodiment of a Lactobacillus plantarum extract under harsh temperature or pH change conditions. [Figure 14] FIG. 14 shows the results of confirming the adipogenesis inhibitory effect of a polysaccharide derived from Lactobacillus plantarum according to one embodiment. [Figure 15] FIG. 15 shows the results of confirming the adipogenesis inhibitory effect of a polysaccharide derived from Lactobacillus plantarum according to one embodiment. [Figure 16] FIG. 16 shows the results of confirming the adipogenesis inhibitory effect of one embodiment of a Lactobacillus plantarum extract. [Figure 17]FIG. 17 is a schematic diagram illustrating that polysaccharides from Lactobacillus plantarum suppress TLR4 and MyD88 signaling, thereby inhibiting pro-inflammatory mediators such as NF-B and MAPK pathways. [Figure 18] FIG. 18 is a schematic diagram that polysaccharides from Lactobacillus plantarum regulate lipid accumulation and glucose uptake via the TLR2 and AMPK signaling pathways. [Figure 19] FIG. 19 shows the FPLC results analyzing the characteristics of a polysaccharide derived from Lactobacillus plantarum according to one embodiment. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0017] The present invention provides a pharmaceutical composition for preventing or treating an inflammatory or metabolic disease, comprising a polysaccharide derived from Lactobacillus plantarum or an extract of Lactobacillus plantarum.

[0018] Polysaccharides derived from Lactobacillus plantarum may be one of the postbiotics. The term "postbiotics" refers to substances produced by the metabolism, fermentation, etc. of probiotics, and may include short-chain fatty acids, antimicrobial peptides, vitamin B, vitamin K, complex amino acids, peptides, neurotransmitters, enzymes, minerals, teichoic acids, polysaccharides, cell surface proteins, etc., each of which may exhibit different functions.

[0019] Probiotics such as Lactobacillus plantarum bacteria and cultures containing said bacteria contain live bacteria, which may cause safety problems such as bacteremia in individuals with weakened immune systems, whereas polysaccharides derived from Lactobacillus plantarum can avoid side effects that may occur due to the inclusion of bacteria and can show excellent safety. In addition, probiotics do not maintain a constant number of live bacteria (CFU) from the time of production to the expiration date, and the number of live bacteria decreases continuously, making it difficult to maintain its functionality, whereas polysaccharides derived from Lactobacillus plantarum can consistently maintain the functionality by maintaining the volume of functional components constant from the time of production. Specifically, polysaccharides derived from Lactobacillus plantarum are not affected by changes in external environments such as heat, humidity, and pH, compared to Lactobacillus plantarum bacteria and cultures containing bacteria, and can maintain their functionality under various conditions. Therefore, polysaccharides isolated from Lactobacillus plantarum are suitable for commercialization as products. According to one embodiment, it has been confirmed that polysaccharides derived from Lactobacillus plantarum are stable without being affected by changes in external environments such as heat, humidity, and pH.

[0020] Lactobacillus plantarum may be a known strain, for example, at least one selected from the group consisting of Lactobacillus plantarum L-14 (accession number KCTC13497BP), Lactobacillus plantarum ATCC10241, Lactobacillus plantarum NCDO704, and Lactobacillus plantarum NCDO1193, but is not limited thereto.

[0021] Lactobacillus plantarum L-14 (accession number KCTC13497BP) was deposited at the Biological Resource Center of the Korea Institute of Bioscience and Biotechnology on March 15, 2018 under the accession number KCTC13497BP (name of depository institution: Korea Institute of Bioscience and Biotechnology, accession number: KCTC13497BP, date of deposit: March 15, 2018).

[0022] The term "polysaccharide" is a general term for sugars that are made up of three or more monosaccharides linked together by glycosidic bonds to form large molecules, which when hydrolyzed become monosaccharides.

[0023] The polysaccharide is not limited to a particular type, so long as it is a high molecular weight polysaccharide that is produced and excreted during the growth process of microorganisms, including lactic acid bacteria. For example, the polysaccharide may be an exopolysaccharide (EPS), an intracellular polysaccharide, or a structural polysaccharide, and more specifically, the polysaccharide is an exopolysaccharide or an intracellular polysaccharide.

[0024] For example, polysaccharides may be collected from the culture solution of Lactobacillus plantarum.Polysaccharides may be the metabolic products that Lactobacillus plantarum excretes into culture solution during fermentation, and collected from culture solution.According to one embodiment, it can be separated from the culture solution obtained by culturing Lactobacillus plantarum strain in a medium, specifically, protein is denatured in the culture solution from which the strain is removed, and then ethanol is added to separate polysaccharides.

[0025] The polysaccharide from Lactobacillus plantarum may be separated from the extract of Lactobacillus plantarum strain.For example, the polysaccharide may be separated from the disrupted product of Lactobacillus plantarum strain.In one embodiment, the polysaccharide may be separated by denaturing protein in the extract (also called "disrupted product") obtained by ultrasonicating Lactobacillus plantarum strain, collecting the supernatant, and adding ethanol to separate.

[0026] Polysaccharide production using microorganisms can be affected by environmental factors such as culture time, culture pH, culture temperature, O2 concentration, and agitation, as well as the content of nutrients such as the type and concentration of carbon source, type and concentration of nitrogen source, phosphate, sulfur, potassium, magnesium, iron, and calcium, and the culture method.

[0027] The polysaccharide derived from Lactobacillus plantarum is sufficient as long as it is derived from Lactobacillus plantarum, and there are no limitations on the type of strain.

[0028] The polysaccharide derived from Lactobacillus plantarum may be a homogenous polysaccharide.

[0029] The polysaccharide derived from Lactobacillus plantarum may be glucose.

[0030] The polysaccharide derived from Lactobacillus plantarum may be a glucose homogeneous polysaccharide.

[0031] Polysaccharides derived from Lactobacillus plantarum were 3×10 4 Da~12×10 4 Da, 4 × 10 4 Da~11×10 4 Da, 5 × 10 4 Da~10×10 4 Da, 6 × 10 4 Da~9×10 4 Da, or 7 × 10 4 Da~8×10 4 According to one embodiment, the polysaccharide from Lactobacillus plantarum may have a weight average molecular weight (Mw) of 7.57×10 Da. 4 It may have a weight average molecular weight of 10 Da.

[0032] The polysaccharide derived from Lactobacillus plantarum is 0.01 × 10 4 Da~6×10 4 Da, 0.05 × 10 4 Da~5×10 4 Da, 0.1 × 10 4 Da~4×10 4 Da, 0.5 × 10 4 Da~3×10 4 Da, or 1 × 10 4 Da~2×10 4According to one embodiment, the polysaccharide from Lactobacillus plantarum has a number average molecular weight (Mn) of 1.84×10 Da. 4 It may have a number average molecular weight of 10 Da.

[0033] The polysaccharides derived from Lactobacillus plantarum may have a polydispersity index (PDI) of 2.5 to 6, 3 to 5.5, 3.5 to 5, or 4 to 4.5. According to one embodiment, the polysaccharides derived from Lactobacillus plantarum may have a polydispersity index of 4.12.

[0034] In one embodiment, the unique properties of the polysaccharides isolated from Lactobacillus plantarum were analyzed by fast protein liquid chromatography (FPLC), thin layer chromatography (TLC), Fourier-Transform Infrared Spectroscopy (FTIR), gel permeation chromatography (GPC), and the like.

[0035] The polysaccharides derived from Lactobacillus plantarum of the present invention show excellent anti-inflammatory effects. According to one embodiment, the polysaccharides derived from Lactobacillus plantarum can suppress inflammatory cytokines. For example, the extracellular polysaccharides derived from Lactobacillus plantarum can reduce the expression levels of IL-6, TNF-α and IL-1α. According to one embodiment, the polysaccharides derived from Lactobacillus plantarum can reduce the expression levels of COX-2 and inducible nitric oxide synthase (iNOS), which are known as the main mediators of inflammation. According to one embodiment, the polysaccharides derived from Lactobacillus plantarum can suppress the phosphorylation and translocation of NF-κB to the nucleus. According to one embodiment, the polysaccharides derived from Lactobacillus plantarum can suppress the phosphorylation of MAPK family proteins, such as JNK, ERK or p38. According to one embodiment, the polysaccharide derived from Lactobacillus plantarum can suppress the expression of Nuclear Factor E2-Related Factor 2 (NRF2) and heme oxygenase-1 (HO-1).The polysaccharide derived from Lactobacillus plantarum of the present invention can suppress the interaction between LPS and TLR4 to exhibit anti-inflammatory effects.

[0036] The polysaccharides derived from Lactobacillus plantarum of the present invention show excellent anti-obesity effect.According to one embodiment, the polysaccharides derived from Lactobacillus plantarum can suppress differentiation of preadipocytes into adipocytes.According to one embodiment, the polysaccharides derived from Lactobacillus plantarum can show the effect of suppressing adipogenesis and intracellular glucose uptake.The polysaccharides derived from Lactobacillus plantarum of the present invention can suppress adipogenesis by interacting with TLR2 and activating AMPK pathway.

[0037] The polysaccharides derived from Lactobacillus plantarum of the present invention can show excellent insulin resistance or hepatic fat inhibition effect.According to one embodiment, the polysaccharides derived from Lactobacillus plantarum can reduce fasting blood glucose, fasting insulin, leptin, resistin, and inhibit fat accumulation in liver.

[0038] The polysaccharides derived from Lactobacillus plantarum of the present invention can be purified using alcohol precipitation.

[0039] For example, polysaccharides derived from Lactobacillus plantarum can be obtained by a method comprising the steps of adding acetic acid to a Lactobacillus plantarum culture solution, and then adding alcohol to separate the precipitate.

[0040] In another example, polysaccharides derived from Lactobacillus plantarum can be obtained by a method comprising the steps of adding acetic acid to a Lactobacillus plantarum extract, followed by adding alcohol and isolating the precipitate.

[0041] The method may further include, after the step of adding alcohol to separate the precipitate, a step of dialyzing the precipitate against purified water.

[0042] The acetic acid may be trichloroacetic acid. The acetic acid treatment can denature the protein content in the culture medium.

[0043] The alcohol may be a C1-C4 alcohol, and according to one embodiment may be ethanol. The alcohol may be added to decompose nucleic acids. The alcohol may be absolute ethanol.

[0044] The Lactobacillus plantarum extract may be a disrupted product of Lactobacillus plantarum, for example, a powder obtained by freeze-drying a disrupted product of Lactobacillus plantarum, or a solution or dispersion thereof.

[0045] The Lactobacillus plantarum extract may be a sonicated product of Lactobacillus plantarum.

[0046] As long as the Lactobacillus plantarum extract is obtained by ultrasonicating the Lactobacillus plantarum strain with an ultrasonic crusher, the type of the strain is not limited.According to one embodiment, the Lactobacillus plantarum extract can be Lactobacillus plantarum L-14 extract, Lactobacillus plantarum ATCC10241 extract, Lactobacillus plantarum NCDO704 extract, or Lactobacillus plantarum NCDO1193 extract.

[0047] The Lactobacillus plantarum extract may be obtained by subjecting Lactobacillus plantarum cultured in a culture medium to ultrasonic treatment. The Lactobacillus plantarum extract may be obtained by removing cell wall components and other residues from the cultured and ultrasonically treated Lactobacillus plantarum. The ultrasonic treatment may be carried out at 0°C or lower.

[0048] The Lactobacillus plantarum extract of the present invention can show excellent anti-inflammatory effect.According to one embodiment, the Lactobacillus plantarum extract can reduce the expression of pro-inflammatory markers such as leptin, interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α), resistin in liver or adipose tissue, and can increase the expression of anti-inflammatory markers such as adiponectin and arginase 1 (Arg1).

[0049] The Lactobacillus plantarum extract of the present invention can show excellent anti-obesity effect.According to one embodiment, the Lactobacillus plantarum extract can suppress the differentiation of preadipocyte into adipocyte.According to one embodiment, the Lactobacillus plantarum extract can show the effect of suppressing adipogenesis and suppressing intracellular glucose uptake.

[0050] The Lactobacillus plantarum extract of the present invention can show excellent insulin resistance or hepatic fat inhibition effect.According to one embodiment, the Lactobacillus plantarum extract can reduce fasting blood glucose, fasting insulin, leptin, resistin, and inhibit fat accumulation in liver.

[0051] The metabolic disease may be at least one selected from the group consisting of insulin resistance, type 2 diabetes, hyperlipidemia, fatty liver, obesity and inflammation, but is not limited thereto.

[0052] The pharmaceutical compositions can be administered in various oral and parenteral formulations, and when they are formulated, they can be produced using diluents or excipients such as commonly used fillers, extenders, binders, wetting agents, disintegrants, and surfactants.

[0053] The term "treatment" refers to a procedure that provides a beneficial effect to a subject or patient suffering from the condition being treated, including any degree of alleviation, including mild alleviation, substantial alleviation, and extensive alleviation, as well as cure, and the degree of alleviation is at least mild alleviation.

[0054] The term "prevention" includes not only total prevention, but also a mild, substantial, or large reduction in the likelihood of onset or recurrence of the condition, and refers to a prophylactic measure that results in any degree of reduction in the likelihood of the condition or recurrence to be prevented, where the degree of reduction in likelihood is at least a mild reduction.

[0055] The present invention also provides a food composition for preventing or improving inflammation or metabolic diseases, comprising a polysaccharide derived from Lactobacillus plantarum or a Lactobacillus plantarum extract.

[0056] The polysaccharides derived from Lactobacillus plantarum, the Lactobacillus plantarum extract, and the inflammatory or metabolic diseases are as described above, and therefore detailed explanations thereof will be omitted.

[0057] The form of the food composition is not particularly limited, and may be, for example, drinks, meat, sausages, bread, biscuits, rice cakes, chocolate, candies, snacks, confectionery, pizza, ramen, other noodles, gums, dairy products including ice cream, various soups, drinking water, alcoholic beverages, vitamin complexes, dairy products and dairy products.

[0058] Furthermore, the present invention provides a method for preventing or treating an inflammatory or metabolic disease, comprising the step of administering a polysaccharide derived from Lactobacillus plantarum or an extract of Lactobacillus plantarum to an individual.

[0059] The polysaccharides derived from Lactobacillus plantarum, the Lactobacillus plantarum extract, and the inflammatory or metabolic diseases are as described above, and therefore detailed explanations thereof will be omitted.

[0060] The individual may be a mammal, for example a human, cow, horse, pig, dog, sheep, goat, or cat.

[0061] Administration can be by methods known in the art. Administration can be by any means, for example, by oral, intravenous, intramuscular, transdermal, mucosal, intranasal, intratracheal or subcutaneous administration, directly to the individual. Administration can be systemic or local. Administration can be local.

[0062] The configuration and effects of the present invention will be described in more detail below with reference to examples. However, these examples are provided for the purpose of illustration to aid in understanding the present invention, and are not intended to limit the scope of the present invention.

[0063] I. Preparation of Lactobacillus plantarum Extract 1. Example 1: Lactobacillus plantarum L-14 extract Lactobacillus plantarum L-14 strain (also referred to as "L-14" in the present description) (KTCT13497BP) obtained from Neoregen Biotech (Gyeonggi-do, Korea) was precultured in MRS medium at 37°C for 18 hours. Then, they were inoculated into 500 mL MRS broth at 1% and cultured at 37°C for 18 hours. The cultured L-14 was collected by centrifugation (10,000g for 10 minutes at 4°C) and washed twice with PBS. Then, it was washed with distilled water to completely remove MRS broth and PBS. L-14 resuspended in 20 mL distilled water was sonicated on ice for 30 minutes using an ultrasonic disintegrator. To remove cell wall components and other residues, the pellet was discarded after centrifugation at 10,000g for 20 minutes at 4°C. The supernatant was filtered (0.2 μm) and frozen at -80°C overnight. Then, the L-14 extract was obtained by lyophilization (Example 1). The obtained L-14 extract was reconstituted with PBS before use. In addition, the L-14 extract (N60, Example 2-1) was adjusted to pH 7.0 (hereinafter, "P60") or incubated at 90°C for 30 minutes (hereinafter, "H60") to confirm the properties of the molecules effective in the L-14 extract.

[0064] 2. Example 2: Lactobacillus plantarum ATCC10241 extract A Lactobacillus plantarum ATCC10241 extract (Example 2) was produced in the same manner as in Example 1, except that Lactobacillus plantarum subsp. plantarum (strain number: ATCC10241) was used instead of Lactobacillus plantarum L-14 strain (KTCT13497BP).

[0065] 3. Example 3: Lactobacillus plantarum NCDO704 extract A Lactobacillus plantarum NCDO704 extract (Example 3) was produced in the same manner as in Example 1, except that Lactobacillus plantarum (strain number: NCDO704) was used instead of Lactobacillus plantarum L-14 strain (KTCT13497BP).

[0066] 4. Example 4: Lactobacillus plantarum NCDO1193 extract A Lactobacillus plantarum NCDO1193 extract (Example 4) was produced in the same manner as in Example 1, except that Lactobacillus plantarum (strain number: NCDO1193) was used instead of Lactobacillus plantarum L-14 strain (KTCT13497BP).

[0067] II. Production of polysaccharides from Lactobacillus plantarum 1. Example 5: Extracellular polysaccharides derived from Lactobacillus plantarum L-14 (KTCT13497BP) Lactobacillus plantarum L-14 strain (KTCT13497BP, hereafter also referred to as "L-14") obtained from Neoregen Biotech (Gyeonggi-do, Korea) was cultured for 18 hours at 30°C in MRS medium (Hardy Diagnostics, Santa Maria, CA, USA) containing 2% dextrose, 1% peptic digest of animal tissue, 1% beef extract, 0.5% yeast extract, 0.5% sodium acetate, 0.2% disodium phosphate, 0.2% ammonium citrate, 0.1% polysorbate 80, 0.01% magnesium sulfate, 0.005% manganese, and 0.005% sulfate. The L-14 culture medium was separated by centrifugation at 10,000g for 20 minutes. The supernatant of the medium was then separated, and trichloroacetic acid was added to denature the proteins in the L-14 culture medium at 37°C for 1 hour. Next, the mixture was centrifuged at 10,000 g for 20 minutes to remove denatured proteins, and the supernatant was mixed with absolute ethanol. The precipitate formed by mixing with absolute ethanol was collected and dialyzed against distilled water (DW) at 4°C for 24 to 48 hours to completely remove medium components and other substances. The dialyzed solution was then lyophilized under reduced pressure to obtain EPS derived from L-14 (Example 5), which was then resuspended in distilled water and stored at -80°C for subsequent experiments.

[0068] 2. Example 6: Polysaccharides derived from Lactobacillus plantarum L-14 (KTCT13497BP) Trichloroacetic acid was added to the L-14 extract (Example 1) produced by the method of "I.1." above to a final concentration of 14% (v / v) to denature the protein content. The L-14 extract was incubated at 37°C for 30 minutes in a shaking incubator at 90 rpm and centrifuged at 8,000 g for 20 minutes. The supernatant was collected and cold absolute ethanol was added to a final concentration of 67% (v / v). The mixture was incubated at 4°C for 24 hours and the precipitate was collected. The same volume of distilled water (DW) as the precipitate was added. The solution was dialyzed using Standard RC tubing (molecular weight cut-off: 3.5 kDa; Spectrum Chemical, New Brunswick, NJ, USA) for 2 days, changing the water twice a day, and the dialysate was filtered (0.2 μm). It was then lyophilized to obtain polysaccharides derived from L-14 (Example 6). The obtained polysaccharides of Example 6 were reconstituted with PBS before use.

[0069] 3. Example 7: Polysaccharides from Lactobacillus plantarum ATCC10241 A polysaccharide derived from Lactobacillus plantarum ATCC10241 (Example 7) was produced in the same manner as in Example 6, except that the L-14 extract produced by the method "I.1." above (Example 1) was replaced with the extract produced by the method "I.2." above (Example 2).

[0070] 4. Example 8: Polysaccharides from Lactobacillus plantarum NCDO704 A polysaccharide derived from Lactobacillus plantarum NCDO704 (Example 8) was produced in the same manner as in Example 6, except that the L-14 extract produced by the method "I.3." (Example 3) was used instead of the L-14 extract produced by the method "I.1." (Example 1).

[0071] 5. Example 9: Polysaccharides from Lactobacillus plantarum NCDO1193 A polysaccharide derived from Lactobacillus plantarum NCDO1193 (Example 9) was produced in the same manner as in Example 6, except that the L-14 extract produced by the method "I.4." (Example 4) was used instead of the L-14 extract produced by the method "I.1." (Example 1).

[0072] III. Analysis of polysaccharides from Lactobacillus plantarum 1. Fast Protein Liquid Chromatography (FPLC) In order to determine whether the polysaccharides derived from Lactobacillus plantarum separated by the method of "II." are homogeneous polysaccharides, the polysaccharides were analyzed by fast protein liquid chromatography (FPLC) size exclusion chromatography. Specifically, EPS (30 mg / mL) was separated by size exclusion chromatography using PBS on a HiLoad (registered trademark) 16 / 600 Superdex 200 pg column (GE Healthcare), and analyzed by AKTA fast protein liquid chromatography (GE Healthcare). As a result, the separated polysaccharides derived from Lactobacillus plantarum produced a single symmetrical peak. This indicates that the polysaccharides are homogeneous polysaccharides (Figure 1A (Example 5) and Figure 19 (Example 6)).

[0073] 2. Thin layer chromatography (TLC) and Benedict's test To confirm the monosaccharide composition of the polysaccharides isolated by the method in "II." above, 10 mg of polysaccharide was hydrolyzed with 1 mL sulfuric acid (2N) at 100°C for 4 h. Residual sulfuric acid was neutralized with sufficient BaCO3 for 12 h. The EPS hydrolysate was adjusted to pH 7 and then lyophilized for analysis. The EPS hydrolysate was treated with TLC silica gel (Merck, Darmstadt, Germany) and transferred to a buffer consisting of n-butanol:methanol:25% ammonia solution:DW (5:4:2:1). To visualize the composition of EPS, the gel was immersed in aniline-diphenylamine reagent and baked in an oven at 110°C for 5 min. To perform Benedict's test, the polysaccharide and polysaccharide hydrolysate were mixed with the same amount of Benedict's reagent (BIOZOA Biological Supply, Seoul, Korea) and then heated to boiling water. The monosaccharide components of the polysaccharides were determined by thin layer chromatography (TLC). The polysaccharide hydrolysate appeared at the same point as the standard substance glucose (Figure 1B, Example 5). As a result of the Benedict's test, the polysaccharide hydrolysate changed the color of the reagent to orange-red. Furthermore, the polysaccharide changed the color of the reagent to green (Figure 1C, Example 5). These results showed that the isolated polysaccharides were mainly composed of glucose.

[0074] 3. Fourier-transform infrared spectroscopy (FTIR) and gel permeation chromatography (GPC) Seoul National University's National Inter-University Research Facility Center has a 4000-500 cm -1The structural characteristics of the polysaccharides were analyzed using a TENSOR27 FTIR (Bruker, Bilerica, MA, USA) in the absorption range of 100 nm to 150 nm. GPC analysis was also performed to determine the molecular weight of the polysaccharides using a Dionex HPLC Ultimate3000 RI System (Thermo Scientific, Waltham, MA, USA) with 120 Å, 500 Å, and 1000 Å columns (Waters, Milford, MA, USA) at 40 °C. The experimental data were corrected with pullulan and processed with a chromatography data system (Chromeleon 6.8 Extention-pak). The polysaccharides were eluted with Sodium azide 0.1M in water and run at a flow rate of 1 mL / min.

[0075] The FTIR result of the polysaccharide of Example 5 was 3500 cm -1 From 500cm -1 A complex peak pattern was observed, ranging from 3307.31 cm -1 The peak of the OH group is 2935.1 cm -1 The peak at 1648.88 cm is a weak CH stretching peak of the methyl group. -1 The peaks at 1032.58 cm show characteristic groups of glucose such as the C=O stretching peak. Also, the strongest absorption band is at 1032.58 cm -1 The peak at 911.98 cm indicates the CO and OH bonds. -1 and 812.28 cm -1 The peak indicates the carbohydrate side group (FIG. 1D, Example 5). From these results, it was confirmed that the isolated polysaccharide had an absorption peak of a polysaccharide mainly composed of glucose. In addition, the molecular weight of the polysaccharide of Example 5 was calculated by GPC to be 1.84×10 4 Number average molecular weight (Mn) in Da: 7.57×10 4 Weight average molecular weight (Mw) in Da, 3.74 × 10 5 The polysaccharides showed a Z-average molecular weight (Mz) of 1.0 Da and a polydispersity index (PDI) of 4.12 (Figure 1E). These results confirmed that the polysaccharides derived from Lactobacillus plantarum were homogeneous polysaccharides composed mainly of glucose.

[0076] IV. Confirmation of the anti-inflammatory effect of Lactobacillus plantarum extract or polysaccharides derived from Lactobacillus plantarum 1. Experimental Method (1) Cell culture, materials and statistics Mouse macrophages RAW264.7 were obtained from ATCC (American Type Culture Collection). Cells were cultured in Dulbecco's modified Eagle's media (DMEM) (WELGENE, Daegu, Korea) containing 10% fetal bovine serum (HyClone, Logan, UT, USA) and 1% penicillin / streptomycin (Gibco, Grand Island, NY, USA) in a 5% CO2 humidified incubator at 37°C. Antibodies were purchased from the following sources: phospho-NF-κB, NF-κB, phospho-ERK, ERK, phospho-p38, p38, phospho-JNK, JNK, and COX-2 from Cell Signaling Technology (Danvers, MA, USA), HO-1 from Abcam (Cambridge, UK), NRF2 and TLR4 from Cusabio (Wuhan, China), iNOS from Invitrogen (Carlsbad, CA, USA), MyD88 from Novus Biologicals (Centennial, CO, USA), and GAPDH from BioLegend (San Diego, CA, USA). All data were obtained from three independent experiments and are shown as mean ± standard deviation (SD). Statistical analysis was determined using unpaired ANOVA, and significance was defined as *p<0.05, **p<0.01, and ***p<0.001.

[0077] (2) Cell viability analysis To confirm the effect of Lactobacillus plantarum L-14 on cell viability, RAW264.7 cells were plated in a 96-well plate at 2.0 × 10 3Cells were seeded at a density of 1000 x 1000 cells / well. After 1 day, the medium was replaced with medium containing L-14 strain and cultured for another 6 h. The medium containing L-14 strain was then replaced with fresh medium containing LPS at the indicated concentrations for 6 h to induce inflammatory responses. Cell viability was confirmed using a Quanti-Max WST-8 cell viability kit (BIOMAX, Seoul, Korea).

[0078] In addition, to confirm the effect of L-14-derived EPS (Example 5) on cell viability, the inoculated cells were cultured for 1 day in media containing various concentrations of L-14-derived EPS (Example 5). Viability was confirmed using the same kit.

[0079] (3) ELISA 2.0 x 10 RAW264.7 cells per well 5 The cells were seeded into 12-well plates at a density of 1.0 × 10 6 The medium was replaced with one inoculated with L-14 strain at a concentration of CFU / mL and maintained for 6 h. The medium was then removed and the cells were thoroughly washed three times with DMEM. To induce inflammation, the washed cells were cultured in medium containing LPS (1 g / mL) and maintained for 6 h. The culture medium obtained in each well was centrifuged at 10,000 g for 3 min, and the supernatant was collected. Cytokines were quantified by ELISAMAX-Deluxe Set (BioLegend) according to the manufacturer's guidelines.

[0080] Furthermore, to examine whether pretreatment with L-14-derived EPS (Example 5) reduced the induction of cytokines by LPS, RAW264.7 cells were seeded in 12-well plates for 24 hours. After pretreatment with L-14-derived EPS (Example 5) for 6 hours, the cultured medium was replaced with fresh medium containing LPS for 18 hours to induce an inflammatory response. Cytokines in the culture medium were quantified as described above.

[0081] (4) Crystal violet staining RAW264.7 cells were seeded in 12-well plates and cultured for 1 day. To examine whether EPS pretreatment affects cell morphology and suppresses morphological changes induced by LPS, cells were treated with EPS (Example 5) for 6 h and the culture medium was replaced with fresh medium containing LPS (1 g / mL) for 18 h. Cells were then washed with PBS and stained with crystal violet solution (Sigma-Aldrich, Saint Louis, MO, USA). Morphological changes were measured at 100x magnification using an EVOS CL Core microscope (Life Technologies, Carlsbad, CA, USA).

[0082] (5) Western blot Proteins were isolated from LPS-treated RAW264.7 cells using Cell Culture Lysis 1× Reagent (Promega, Fitchburg, WI, USA) with protease and phosphatase inhibitor cocktails. Cytoplasmic and nuclear proteins were obtained using the ExKine-Nuclear and Cytoplasmic Protein Extraction Kit (Abbkine, Wuhan, China) according to the manufacturer's guidelines. Total protein concentration was quantified with a Pierce-BCA protein analysis kit (Thermo Scientific, Waltham, MA, USA). Denatured proteins were then separated by 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride membranes. After blocking for 1 h at room temperature (RT), the membranes were incubated overnight at 4°C in nonfat milk containing the appropriate primary antibodies (1:1000). The membranes were washed with 0.1% Tris-buffered saline. The sections were further incubated in nonfat milk containing Tween 20 (Sigma) and secondary antibody (1:2000) at room temperature for 1 h. Protein signals were detected using ECL Western Blot Substrate (Daeil Lab Service, Seoul, Korea).

[0083] (6) Immunofluorescence (IF) analysis RAW264.7 cells were plated in a 6-well plate at 1.0 x 10 cells per well. 6 The cells were seeded at a density of 100x100 cells and incubated overnight. Cells pretreated with EPS (Example 5) were cultured in fresh medium containing LPS and harvested by scraping. After fixation with 4% paraformaldehyde and permeabilization with 0.1% Triton X-100 (Sigma), the cells were blocked with 3% bovine serum albumin (BSA, Bovogen, East Keilor, Australia) for 1 h. They were then incubated with PE-conjugated iNOS antibody. The washed cells were mounted with ProLong-Glass Antifade Mountant (Invitrogen) containing NucBlue-Stain. Also, to visualize the translocation of NF-κB, RAW264.7 cells were pretreated with EPS for 2 h and the inflammatory response was stimulated with LPS (1 g / mL) for 1 h. The isolated cells were produced in a similar manner. After blocking for 1 h, the cells were cultured with NF-B antibody (1:200) for 18 h at 4 °C. The cells were then washed extensively and incubated with Alexa Fluor 488-conjugated secondary antibodies in 3% BSA for 30 min at room temperature. The cells were mounted using the same strain. All slides were analyzed using an LSM 800 confocal laser scanning microscope (Carl Zeiss, Oberkochen, Germany).

[0084] 2. Experimental Results (1) Confirmation of the inhibitory effect of metabolites secreted from Lactobacillus plantarum on LPS-induced inflammatory responses in mouse macrophages We quantified LPS-induced cytokines in mouse macrophages RAW264.7 (American Type Culture Collection) by enzyme-linked immunosorbent assay (ELISA) to confirm the inhibitory effect of Lactobacillus plantarum L-14 strain (KTCT13497BP) on inflammatory responses. Cells seeded in 12-well plates were cocultured with L-14 strain for 6 hours, and the proinflammatory markers IL-6, TNF-α, and MCP-1 (monocyte chemoattractant protein-1) were induced by LPS treatment. As a result, the expression levels of IL-6, TNF-α, and MCP-1 increased with LPS treatment, but not with L-14 treatment alone. The release of inflammatory cytokines was significantly reduced in cells cocultured with L-14 compared to the control group (LPS-treated group) (Figure 2A). We also quantified the viability of RAW264.7 cells cultured under the same culture conditions to confirm whether the L-14 strain had an effect on cell proliferation and thus reduced inflammatory cytokines. As a result, cell viability was not affected by L-14 or LPS (Figure 2B). These results suggest that metabolites secreted by Lactobacillus plantarum L-14 directly interact with immune cells to exert anti-inflammatory effects.

[0085] (2) Confirmation of the inhibitory effect of EPS derived from Lactobacillus plantarum on morphological changes induced by LPS in mouse macrophages To confirm whether EPS derived from Lactobacillus plantarum suppresses morphological changes induced by LPS, mouse macrophages were pretreated with EPS of Example 5 for 6 hours, and then stimulated with LPS for 18 hours to confirm the morphology of the cells. Crystal violet staining showed that EPS derived from L-14 (Example 5) alleviated the morphological changes of cells caused by LPS treatment (Figure 3A, in Figure 3, EPS100 means 100 μg / ml treatment of Example 5, and EPS200 means 200 μg / ml treatment of Example 5). In addition, when RAW264.7 cells were treated with higher concentrations of EPS for 1 day, cell viability was maintained without being affected (Figure 3B). These results indicate that EPS derived from Lactobacillus plantarum suppresses morphological changes induced by LPS in mouse macrophages without affecting cell viability.

[0086] (3) Confirmation of the inhibitory effect of EPS derived from Lactobacillus plantarum on LPS-induced inflammatory response in mouse macrophages To confirm whether Lactobacillus plantarum-derived EPS suppresses inflammatory responses stimulated by LPS, the proinflammatory cytokines produced in RAW264.7 cells pretreated with EPS of Example 5 were quantified. As a result of cytokine quantification, pretreatment with EPS from L-14 weakened the levels of IL-6, TNF-α and IL-1β, and in particular reduced IL-1β to a level similar to the expression level of the control group (FIG. 4A, in FIG. 4, EPS means Example 5). In addition, the expression levels of COX-2 and inducible nitric oxide synthase (iNOS), which are known as the main mediators of inflammation, were analyzed by Western blot in RAW264.7 cells pretreated with EPS of Example 5. After treatment with LPS, the expression levels of COX-2 and iNOS proteins increased, but these expression levels were suppressed in EPS-pretreated RAW264.7 cells (FIG. 4B). Furthermore, the expression level of LPS-induced iNOS in RAW264.7 cells pretreated with EPS of Example 5 was analyzed by immunofluorescence (IF) analysis. The analysis results showed that the expression of LPS-induced iNOS increased after LPS treatment, but the expression of LPS-induced iNOS decreased in RAW264.7 cells pretreated with EPS (Figure 4C). From these results, it was confirmed that EPS derived from Lactobacillus plantarum exhibits an inhibitory effect on LPS-induced inflammatory response.

[0087] (4) Confirmation of the inhibitory effect of EPS derived from Lactobacillus plantarum on the nuclear translocation of NF-κB induced by LPS in mouse macrophages To confirm whether Lactobacillus plantarum-derived EPS inhibits the translocation and phosphorylation of NF-κB induced by LPS, RAW264.7 cells pretreated with EPS of Example 5 were subjected to LPS-induced inflammatory response, and then the expression level and location of phosphorylated form of NF-κB were analyzed. As a result, the p-NF-κB / NF-κB ratio was decreased by pretreatment with EPS of L-14 (Example 5) (Figure 5A, in Figure 5, EPS means Example 5). EPS of L-14 (Example 5) itself did not promote the phosphorylation of NF-κB. Meanwhile, EPS of L-14 (Example 5) inhibited LPS-induced nuclear translocation of NF-κB at all concentrations (Figure 5B). Consistently, NF-κB translocation to the nucleus was induced by LPS but was reduced by pretreatment with EPS derived from L-14 (Example 5) (Figure 5C, in Figure 5C, EPS100 means 100ug / ml treatment of Example 5, and EPS200 means 200μg / ml treatment of Example 5).

[0088] (5) Confirmation of the inflammation suppression pathway of EPS derived from Lactobacillus plantarum in mouse macrophages 1) Regulation of mitogen-activated protein kinase (MAPK) and the Nuclear Factor E2-Related Factor 2 / Heme Oxygenase-1 (NRF2 / HO-1) pathway The MAPK and NRF2 / HO-1 pathways are known to be major regulators of inflammatory responses in mouse macrophages. First, to confirm whether Lactobacillus plantarum-derived EPS can suppress the MAPK pathway in LPS-induced RAW264.7 cells, the phosphorylation of MAPK family proteins (JNK, ERK and p38) was analyzed by Western blot. As a result, the EPS of Example 5 significantly suppressed the phosphorylation of JNK and ERK even at a concentration of 100 g / mL (Figure 6A, EPS in Figure 6 means Example 5). The phosphorylation of p38 was suppressed when the EPS of Example 5 was treated at a concentration of 200 g / mL. In addition, to confirm whether the anti-inflammatory effect of Lactobacillus plantarum-derived EPS is mediated by the NRF2 / HO-1 pathway, the protein expression levels of NRF2 and HO-1 markers were confirmed. As a result, the expression levels of HO-1 and NFR2 increased regardless of the presence or absence of LPS (Figure 6B). Furthermore, the EPS of Example 5 increased the translocation of NRF2 to the nucleus, which is known as a major pathway mediating inflammatory responses (FIG. 6C). That is, the EPS derived from Lactobacillus plantarum suppressed the phosphorylation of MAPK proteins and enhanced the expression of NRF2 / HO-1 in LPS-induced RAW264.7 cells.

[0089] 2) Inhibition of LPS-TLR4 interaction To investigate whether EPS derived from Lactobacillus plantarum suppresses inflammatory responses induced by TLR4, RAW264.7 cells were treated with EPS and LPS from Example 5, and the expression level of TLR4 was analyzed. As a result, TLR4 upregulated by LPS was reduced by EPS (all concentrations) (Figure 7A, EPS in Figure 7 means Example 5). In addition, the way in which EPS derived from Lactobacillus plantarum interacts with TLR4 was confirmed using TAK-242, which was confirmed to block the TLR4 pathway. Interestingly, when RAW264.7 cells were treated with EPS alone, the protein expression of TLR4 was further suppressed than that of the untreated control group (Figure 7B). EPS also suppressed the expression levels of TLR4 and MyD88 in the LPS-induced group, similar to that observed in the TAK-242-treated group. The expression of COX-2 was suppressed by TAK-242 and in a similar manner by EPS. Consistently, EPS significantly decreased the expression of cytokines IL-1, IL-6 and TNF-α secreted from the medium, while TAK-242 down-regulated them (Figure 7C).The above results suggest that EPS from Lactobacillus plantarum exerts anti-inflammatory effects through TLR4 in LPS-treated RAW264.7 cells.

[0090] V. Confirmation of the anti-obesity, insulin resistance, and fatty liver inhibitory effects of Lactobacillus plantarum extract or polysaccharides derived from Lactobacillus plantarum 1. Experimental Method (1) Cell culture, materials and statistics 3T3-L1 cell line and hBM-MSCs were obtained from ATCC (American Type Culture Collection, Manassas, VA, USA) and PromoCell (Heidelberg, Germany), respectively. 3T3-L1 cells were cultured in Dulbecco's modified Eagle's medium (DMEM) (GE Healthcare, Chicago, IL, USA) containing 4.5 g / L D-glucose, 10% fetal bovine serum (FBS), 1% penicillin / streptomycin (P / S), 25 mM HEPES, 3.7 g / L sodium bicarbonate, 4 mM L-glutamine, and 1 mM sodium pyruvate. 3T3-L1 cells were cultured at 37°C in an incubator containing a humidified atmosphere of 5% CO2.

[0091] Rodent Diet containing 60% kcal fat was purchased from Research Diets (New Brunswick, NJ, USA). AICAR and CC were purchased from Selleckchem (Houston, TX, USA), and C29 was purchased from Cayman Chemical (Ann Arbor, MI, USA). Insulin, leptin, adiponectin, and resistin ELISA kits were purchased from CUSABIO (Hubei, China), and IFN-γ, IL-6, and MCP1 ELISA kits were purchased from BioLegend (San Diego, CA, USA). Antibodies were purchased from the following sources: Akt, rabbit IgG isotype, and goat IgG isotype antibodies from Bioss (Woburn, MA, USA); PPARγ, C / EBPα, FABP4, t-AMPKα, p-AMPKα, t-ACC, p-ACC, FAS, p-NF-κB, t-NF-κB, p-AKT, t-AKT, t-AS160, p-AS160, and MyD88 antibodies from Cell Signaling Technology (Danvers, MA, USA); Arg1, ATGL, IL-6, TNF-α, and TLR2 antibodies from CUSABIO; SREBP-1c antibody from Novus Biologicals (Centennial, CO, USA); and leptin and resistin antibodies from R&D. Systems (Minneapolis, MN, USA); β-actin, GAPDH and SCD1 antibodies were purchased from Santa Cruz Biotechnology (Dallas, TX, USA).

[0092] Statistical analysis was performed using SPSS software (version 25.0; SPSS Inc., Chicago, Illinois) and GraphPad Prism 5 (GraphPad, CA, USA). Data were presented as mean ± standard deviation. Statistical analysis was determined using ANOVA, and significance was defined as *p<0.05, **p<0.01, ***p<0.001.

[0093] (2) Differentiation of 3T3-L1 cells and hBM-MSCs; and analysis of fat accumulation and triacylglycerol 3T3-L1 cells or hBM-MSCs were plated in culture medium at 1.0 x 10 cells per well in a 24-well plate. 5 The cells were seeded at a density of 1000 cells / ml. Two days after the cells were fused, the medium was replaced with adipogenic induction medium (MDI) containing α-MEM, 10% FBS, 1% P / S, 1uM dexamethasone, 0.5mM isobutylmethylxanthine, 100uM indomethacin, 10mg / mL insulin, and Lactobacillus plantarum extract (Examples 1-4), Lactobacillus plantarum extract adjusted to pH 7.0 (P60), and Lactobacillus plantarum extract incubated at 90°C for 30 minutes (H60). Four days after the initial adipogenic induction, the medium was replaced with adipogenic maintenance medium containing α-MEM, 10% FBS, 1% penicillin / streptomycin, 10mg / mL insulin, and extract. The medium was replaced every two days during adipogenic differentiation, and 3T3-L1 cells and hBM-MSCs were maintained for 12 and 7 days, respectively. After 12 days, to compare lipid accumulation, 3T3-L1 cells and hBM-MSCs were washed with PBS, fixed with 4% formaldehyde, and then stained with Oil Red O solution for 30 min. Stained adipocytes were observed under 200x magnification with an EVOS CL Core microscope (Life Technologies). To compare relative lipid accumulation, Oil Red O in 3T3-L1 cells and hBM-MSCs was dissolved in isopropanol and quantified by measuring absorbance at 500 nm using a microplate reader. Results were analyzed as percentages relative to the control group, which was set to 1.0. The formula for relative lipid accumulation was (Asample-Ablank) / (Acontrol-Ablank). Triacylglycerol (TAG) was also quantified with a TAG analysis kit (Cayman Chemical) according to the manufacturer's guidelines.

[0094] (3) Cell viability analysis 3T3-L1 cells were plated in a 96-well plate at 1.0 x 10 cells / well. 3The cells were seeded at a density of 1000 cells / well. After 24 hours, the medium was replaced with various concentrations of Lactobacillus plantarum extracts (Examples 1 to 4) and maintained for 4 days. Cell viability was confirmed using a WST-1 cell viability assay kit (DONGIN LS CO., LTD, Seoul, Korea).

[0095] (4) Western blot analysis 3T3-L1 cells and hBM-MSCs were harvested according to the conditions and lysed in Cell Culture Lysis 1X Reagent (Promega) containing a mixture of protease and phosphatase inhibitors (MCE) for 5 min on ice. Insoluble debris was removed by centrifugation at 15,000 g for 15 min at 4°C. A total of 10–40 μg of protein from the separated supernatant was separated using 8–12% SDS-PAGE and transferred to a polyvinylidene difluoride membrane. The membrane was incubated in 0.1% Tween 20 Tris-buffered saline (TBST) containing 5% bovine serum albumin (BSA) for 1 h at room temperature. The membrane was incubated overnight in 5% BSA-TBST with primary antibodies at 4°C. After washing three times with TBST, the membrane was incubated in 5% BSA-TBST conjugated with horseradish peroxidase for 1 h at room temperature. The membrane protein signals were analyzed by developing with ECL Western Blot Substrate (DAEILLAB SERVICE). All experiments were repeated three times.

[0096] (5) Real-time quantitative PCR Isolate mRNA from 3T3-L1 cells and use PureLink TM Using an RNA mini kit (Invitrogen, Carlsbad, CA, USA), the Lactobacillus plantarum extracts (Examples 1 to 4) were cultured and reverse transcribed into cDNA using a cDNA kit (Promega, Madison, WI, US). TMcDNA was amplified and analyzed using Real-Time PCR Systems (Applied Biosystems, Foster City CA, USA) with TB Green Mix (TAKARA, Shinga, Japan). The primers used for RT-qPCR are shown in Table 1 below.

[0097] [Table 1] JPEG0007649070000002.jpg181145

[0098] (6) Animals, diets and study design Animal studies were performed in the animal facility center accredited by Seoul National University College of Dentistry. Procedures involving animals were performed in accordance with the Korean regulations on animal experiments, and the study was approved by the Seoul National University Animal Care Committee (SNU-180309-1).

[0099] Four-week-old C57BL / 6J male mice were purchased from Orient Bio (Seongnam, Korea). Mice were randomly divided into three groups: (i) normal diet (ND, n=6); (ii) high fat diet (HFD) (n=7); (iii) L-14 diet (high fat diet (HFD) treated with L-14 extract (Example 1)) (n=8). Mice were fed ND or HFD for another 7 weeks, and mice were allowed free access to water. L-14 diet group was orally administered L-14 extract (500 mg / kg body weight) through a needle catheter every 2 days during the animal study period. The other two groups were orally administered PBS under the same conditions to provide the same stress. Body weight and food intake were measured every 2 days. After 7 weeks of feeding and administration, mice were fasted overnight and euthanized. Epididymal and inguinal white adipose tissues were harvested and weighed. Liver and serum were immediately isolated for further study. TMTotal protein was isolated from mouse adipose tissue using a ELISA kit (MP Biomedicals, Irvine, CA, USA) and Western blot analysis was performed. Serum biochemical analysis was performed at the Korea Mouse Phenotyping Center (Seoul, Korea), and hormones and cytokines in mouse serum were quantified using ELISA kits according to the manufacturer's guidelines.

[0100] (7) Mouse epidermal white adipose tissue (eWAT) and liver histology Mouse eWAT and liver were rinsed twice with sterile PBS and fixed overnight in 4% paraformaldehyde in PBS. Tissues were cut into 5 μm sections, inserted into paraffin blocks, and placed on an adhesion microscope (Paul Marienfeld, Lauda-Konigshofen, Germany). Wax was removed, and rehydrated sections were stained with hematoxylin and eosin. For immunohistochemistry, antigens in sections were retrieved in a pressure vessel filled with citrate buffer (pH 7.0) for 10 min. Sections were immersed in BLOXALL® Endogenous Peroxidase Solution (Vector Laboratories, Burlingame, CA, USA) for 20 min at room temperature and incubated in 2.5% normal horse serum to reduce nonspecific binding. Antibodies were diluted 1:100-200 in 2.5% normal horse serum, and sections were incubated with the diluted primary antibodies overnight at 4°C. Rabbit IgG and goat IgG antibodies were used as negative controls. Sections were then incubated with ImmPRESS polymer anti-rabbit IgG and anti-goat IgG reagents for 30 minutes at room temperature. Sections were stained with ImmPACT® DAB Peroxidase (HRP) substrate (Vector Laboratories) and then lightly counterstained with hematoxylin. Images were obtained under a microscope (BX50, Olympus, Tokyo, Japan).

[0101] (8) Analysis of the effects of Lactobacillus plantarum extract and polysaccharides derived from Lactobacillus plantarum on AMPK and TLR2 signaling pathways 3T3-L1 cells were plated in culture medium at 1.0 x 10 cells per well in a 24-well plate. 5 The cells were seeded at a density of 1000 cells / ml. Two days after the cells were fused, the medium was replaced with starvation medium containing only DMEM and 1% P / S for 1 hour. After starvation, the medium was replaced with general medium containing 250 μM 5-aminoimidazole-4-carboxyamide ribonucleotide (AICAR), 5 μM Compound C (CC) and 50 μM C29, and the cells were cultured for 2 hours. The cells were induced to differentiate into mature adipocytes in MDI using L-14 extract (Example 1) or polysaccharides derived from Lactobacillus plantarum (Examples 6-9). AICAR and C29 were treated every 2 days, and CC was not further treated throughout the entire period. After 12 days, the adipogenesis inhibitory effect of L-14 extract or polysaccharides derived from Lactobacillus plantarum through AMPK and TLR2 signaling pathways was analyzed by Oil Red O staining and TAG analysis. Furthermore, to confirm the effect of L-14 extract or polysaccharides derived from Lactobacillus plantarum on the early stage of adipogenesis, proteins were isolated on day 4 and analyzed by Western blot.

[0102] 2. Experimental Results (1) Confirmation of the inhibitory effect of Lactobacillus plantarum extract on the differentiation of 3T3-L1 preadipocytes and hBM-MSCs into mature adipocytes In order to confirm whether Lactobacillus plantarum extract shows an effect of suppressing adipogenesis, the following experiment was carried out. 3T3-L1 cells were treated with Lactobacillus plantarum extract (Examples 1-4) at concentrations of 20 and 60 μg / mL every 2 days during the adipogenesis process (FIG. 8A). Oil Red O staining showed that lipid accumulation was dose-dependently suppressed by L-14 extract (FIGS. 8B and 8C, scale bar=100 μm, FIGS. 8B and 8C show the experimental results of Example 1). In addition, TAG assay confirmed that Lactobacillus plantarum extract (Examples 1-4) suppressed TAG preservation (FIGS. 8D and 16, 8D and 16 show the experimental results of Example 1 and Examples 2-4, respectively). Based on the bacterial cell protein content extracted from 1L culture medium of each lactic acid bacteria strain, the following amounts were obtained: L-14 (32.015mg), ACTT10241 (23.247mg), NCDO71 (19.181mg), and NCDO1193 (17.532mg), with L-14 having the fastest growth rate and the highest bacterial cell mass at the same culture volume. Based on the protein quantified from the bacterial cell extract, it was confirmed that when the same protein amount was treated, a reduction effect of about 75% was observed compared to the control group. That is, the amount required to show a reduction effect of about 75% is the dilution ratio (corresponding to a concentration of 25 μg / ml) based on the protein of 1 L cultured cells, as follows: L-14 (1280-fold dilution_1280x), ACTT10241 (930-fold dilution_930x), NCDO71 (767-fold dilution_767x), NCDO1193 (701-fold dilution_701x). From this, it was found that among Lactobacillus plantarum strains, L-14 seed strain can produce the most anti-obesity substances. The L-14 extract (Example 1) did not affect the cell viability of 3T3-L1 cells (Figure 8E). This means that the inhibitory effect on adipocyte differentiation is not due to the cytotoxicity of the L-14 extract (Example 1).Furthermore, Western blot analysis confirmed that the L-14 extract (Example 1) reduced the protein expression of peroxisome proliferator-activated receptor γ (PPARγ), CCAAT / enhancer-binding proteins α (C / EBPα), and fatty acid-binding protein 4 (FABP4), which are known to be major adipogenic markers (Figure 8F). RT-qPCR confirmed that the L-14 extract (Example 1) significantly reduced the gene expression of adipogenic differentiation markers, including PPARγ, C / EBPα, FAB4, lipoprotein lipase (LPL), fatty acid synthase (FAS), glycerol-3-phosphate dehydrogenase (GPDH), and CD36, in 3T3-L1 cells, consistent with the Western blot results (Figures 8G and 8H). The markers were already decreased by L-14 extract (Example 1) at the early stage of adipogenic differentiation (days 0-4). These results indicate that the inhibition of adipogenesis by Lactobacillus plantarum extract may occur by decreasing the expression of adipogenic factors at the early stage of differentiation. "NC" in Figure 8 refers to cells cultured in normal medium as a negative control group.

[0103] (2) Confirmation of the effect of Lactobacillus plantarum extract in suppressing weight gain and expression of pro-inflammatory markers in HFD mouse model To confirm the effect of Lactobacillus plantarum extract intake in vivo, C57BL / 6J mice were randomly divided into three groups: normal diet (n=6), high fat diet (HFD) (n=7) and L-14 diet (high fat diet (HFD) treated with L-14 extract (Example 1)) (n=8). The L-14 diet group was orally administered L-14 extract (Example 1) (500 mg / kg body weight) through a needle catheter every two days during the animal study period. The other two groups were orally administered PBS under the same conditions to give the same stress. As a result of oral administration, the average body weight of the mice showed significant differences after 36 days. In particular, the body weight of the L-14 diet group (31.51 ± 1.96 g) was significantly different from that of the HFD group (35.14 ± 3.18 g) (Figures 9A and 9B; in Figure 9, ND means normal diet group, HFD means high-fat diet group, and HFD + L-14 means L-14 diet group). Food intake did not change significantly after oral administration of the L-14 extract (Figure 9C). The L-14 diet group significantly reduced the fat mass of epidermal white adipose tissue (eWAT) (1.10 ± 0.14 vs. 0.81 ± 0.10 g) and inguinal white adipose tissue (iWAT) (0.99 ± 0.19 vs. 0.67 ± 0.12 g) compared with the HFD group (Figures 9D and 9E). H&E staining confirmed that adipocyte size in eWAT was significantly smaller in the L-14 diet group than in the HFD group (Figure 9F, scale bar = 100 μm). In addition, the expression of pro-inflammatory markers such as leptin, interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α), and resistin was decreased, and the expression of anti-inflammatory markers such as adiponectin and arginase 1 (Arg1) was increased in adipose tissue of the L-14 diet group (Figure 9F and Figure 9G).

[0104] (3) Confirmation of the insulin resistance marker and liver fat inhibition effects of Lactobacillus plantarum extract Blood chemistry test and ELISA confirmed the effect of Lactobacillus plantarum extract in reducing insulin resistance markers and hepatic steatosis. Blood chemistry test confirmed that the levels of low-density lipoprotein cholesterol (LDL-c) and TAG were significantly reduced in L-14 diet mice compared with HFD group, and the level of high-density lipoprotein cholesterol (HDL-c) was increased in parallel (p<0.056) (Figure 10A, in Figure 10, ND means normal diet group, HFD means high-fat diet group, and HFD+L-14 means L-14 diet group). Consistently, it was confirmed that L-14 extract (Example 1) reduced total cholesterol and significantly reduced the ratio of TAG / HDL-c, an insulin resistance marker in serum (Figure 10B). Fasting blood glucose, fasting insulin, leptin, and resistin were decreased, whereas GOT (AST), GPT (ALT), adiponectin, interferon gamma (IFN-γ), IL-6, and MCP1 (monocyte chemoattractant protein 1) showed no significant changes (Figures 10C and 10D). In addition, to confirm the effect of L-14 extract (Example 1) on the liver, liver tissue samples isolated from the HFD model were histologically analyzed. H&E staining confirmed that L-14 extract (Example 1) attenuated hepatic steatosis induced by HFD (Figure 10E, scale bar = 100 μm). Furthermore, SuperFastPrep-2 TM Total protein was isolated from mice and liver using a ELISA kit (MP Biomedicals, Irvine, CA, USA) and analyzed by Western blot. It was confirmed that the L-14 extract (Example 1) suppressed the expression of MCP1 and IL-6, while activating the AMPK signaling pathway. There was no significant change in the expression of Arg1 and ATGL (adipose triglyceride lipase) involved in lipolysis, but the expression of IL-6 in the liver was reduced by the L-14 extract (Figure 10F). As with the TAG inhibitory effect described above, it can be fully predicted that such insulin resistance markers and liver fat inhibitory effects will be shown in Examples 2 to 4, which are the same Lactobacillus plantarum extract as in Example 1.

[0105] (4) Confirmation of the effect of Lactobacillus plantarum extract in inhibiting adipogenesis at the early stage of adipogenesis To confirm whether the inhibitory effect of Lactobacillus plantarum extract on adipogenic differentiation is mediated by the AMPK signaling pathway, 3T3-L1 cells were treated with L-14 extract (Example 1) together with AICAR, an AMPK activator, and CC, an AMPK inhibitor. As a result, AICAR significantly reduced the lipid content of 3T3-L1 cells after differentiation, and additional L-14 extract (Example 1) treatment improved the inhibitory effect of AICAR. CC did not affect lipid accumulation in 3T3-L1 cells, but L-14 extract (Example 1) reduced TAG even under AMPK inhibitory conditions (Figures 11A and 11B, scale bar = 100 μm, L14 in Figure 11 means Example 1). These results suggest that the anti-adipogenic effect of Lactobacillus plantarum extract is mediated by the AMPK signaling pathway. To confirm whether Lactobacillus plantarum extract can activate the AMPK pathway at the early stage of adipogenic differentiation (Day 0-4), markers were analyzed by Western blot. The AMPK pathway was synergistically activated by AICAR and L-14 extract (Example 1) within 4 days. As a result, the expression of adipogenic markers such as PPARγ and FABP4 was significantly decreased (Figure 11C). In addition, L-14 extract (Example 1) increased the phosphorylation of AMPKα, which was suppressed by CC. Simultaneous treatment with AICAR and L-14 extract (Example 1) synergistically suppressed TAG accumulation. In addition, L-14 extract (Example 1) upregulated the AMPK signaling pathway at the early differentiation stage (day 4) to suppress adipogenic differentiation of human bone marrow mesenchymal stem cells (BM-MSCs) (Figure 12, in Figure 12, L14 means Example 1). Specifically, Oil Red O staining confirmed that L-14 extract (Example 1) suppressed lipid accumulation in hBM-MSCs (scale bar = 100 μm) (Figure 12C). TAG assay confirmed that L-14 extract (Example 1) suppressed TAG preservation (Figure 12D). Gene expression of fat differentiation markers (PPARγ, C / EBPα, FABP4, leptin, GPDH and CD36) was also decreased by L-14 extract (Example 1) (Figure 12E).The protein expression of major adipogenic markers and adiponectin in hBM-MSCs cultured with L-14 extract (Example 1) for 4 days was decreased, while the expression of AMPK signaling pathway markers was increased (Figure 12F). These results suggest that Lactobacillus plantarum extract upregulates the AMPK signaling pathway at the early stage of adipogenesis and inhibits the differentiation of mouse preadipocytes and hBM-MSCs into mature adipocytes.

[0106] (5) Confirmation of the effect of polysaccharides derived from Lactobacillus plantarum on adipogenesis In order to confirm what molecules in Lactobacillus plantarum extract have the effect of inhibiting adipogenesis, we confirmed the adipogenesis inhibitory effect of Lactobacillus plantarum extract (H60, P60) exposed to harsh temperature and pH conditions. As a result, the adipogenesis inhibitory effect of Lactobacillus plantarum extract did not change due to pH change or culture under harsh temperature conditions, which means that the active molecules with the adipogenesis inhibitory effect are heat stable (Figure 13, scale bar = 100 μm).

[0107] In order to confirm whether the polysaccharides derived from Lactobacillus plantarum in Examples 5 to 9 can suppress lipid accumulation, 3T3-L1 cells were cultured with EPS in MDI for 12 days and analyzed. As a result of lipid accumulation analysis and TAG analysis, it was confirmed that all of the polysaccharides derived from the four types of Lactobacillus plantarum strains have an effect of suppressing adipogenesis (Figures 14A to 14C and Figure 15, Figure 14 shows the experimental results of Example 6, and Figure 15 shows the experimental results of Examples 7 to 9). In addition, Western blot analysis confirmed that the polysaccharides derived from Lactobacillus plantarum upregulated the AMPK pathway and downregulated the expression of adipogenic markers and adiponectin. Furthermore, it was confirmed that the polysaccharides derived from Lactobacillus plantarum increased the phosphorylation of AKT (Protein kinase B) and 160 kDa Akt substrate (AS160), which are known to be related to intracellular glucose uptake (Figure 14D). Further studies were performed using the TLR2 inhibitor C29 to confirm which intracellular receptor interacts with polysaccharides derived from Lactobacillus plantarum. As a result, lipid accumulation was not affected by C29, but the adipogenesis-inhibitory effect of polysaccharides derived from Lactobacillus plantarum was significantly reduced in the C29-treated group (Figures 14E and 14F). Furthermore, the activation of the AMPK pathway by EPS was suppressed at the early stage (day 4) when the expression of TLR2 and MyD88 (myeloid differentiation primary response 88) was suppressed by C29. This means that polysaccharides derived from Lactobacillus plantarum interact with TLR2 to activate the AMPK pathway, thereby suppressing adipogenesis (Figure 14G).

[0108] [Microorganism deposit certificate] JPEG0007649070000003.jpg206151

Claims

1. A pharmaceutical composition for preventing or treating a metabolic disease, comprising a polysaccharide derived from Lactobacillus plantarum or an extract of Lactobacillus plantarum, The Lactobacillus plantarum is at least one selected from the group consisting of Lactobacillus plantarum ATCC10241, Lactobacillus plantarum NCDO704, and Lactobacillus plantarum NCDO1193; The pharmaceutical composition for preventing or treating a metabolic disease, wherein the metabolic disease is at least one selected from the group consisting of insulin resistance, type 2 diabetes, hyperlipidemia, fatty liver, obesity and inflammation.

2. The pharmaceutical composition of claim 1 , wherein the polysaccharide is at least one of an intracellular polysaccharide or an exopolysaccharide.

3. The pharmaceutical composition according to claim 1 , wherein the polysaccharide is a glucose homogeneous polysaccharide.

4. The pharmaceutical composition according to claim 1 , wherein the Lactobacillus plantarum extract is a disruption of the Lactobacillus plantarum.

5. A food composition for preventing or improving a metabolic disease, comprising a polysaccharide derived from Lactobacillus plantarum or an extract of Lactobacillus plantarum, The Lactobacillus plantarum is at least one selected from the group consisting of Lactobacillus plantarum ATCC10241, Lactobacillus plantarum NCDO704, and Lactobacillus plantarum NCDO1193; The food composition for preventing or ameliorating a metabolic disease, wherein the metabolic disease is at least one selected from the group consisting of insulin resistance, type 2 diabetes, hyperlipidemia, fatty liver, obesity and inflammation.

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