KR101494279B1 - Lactobacillus plantarum KY1032 having inhibitory activity against adipocyte-specific gene expression and adipocyte differentiation, and product containing thereof as an effective factor - Google Patents

Abstract

The present invention relates to Lactobacillus plantarum KY1032 which inhibits mRNA and protein expression of genes involved in induction of 3T3-L1 adipocyte differentiation and ultimately inhibits 3T3-L1 adipocyte differentiation and contains it as an active ingredient a pharmaceutical composition, fermented milk, beverage, relates to a dietary supplement, since Lactobacillus Planta room (Lactobacillus plantarum) KY1032 is a very excellent inhibitory effect differentiated 3T3-L1 adipocytes in accordance with the present invention Lactobacillus bacteria Planta room (Lactobacillus that plantarum ) KY1032 can be provided.

Description

Lactobacillus plantarum KW1032 having an inhibitory effect on adipocyte differentiation by inhibiting the expression of a gene involved in induction of adipocyte differentiation and a product containing the same as an active ingredient {Lactobacillus plantarum KY1032 having inhibitory activity against adipocyte-specific gene expression and adipocyte differentiation, and product containing as an effective factor.

The present invention relates to Lactobacillus plantarum KY1032 having inhibitory effect on adipocyte differentiation by inhibiting the expression of a gene involved in adipocyte differentiation induction and a pharmaceutical composition containing it as an active ingredient, (CCAAT / enhancer binding protein-alpha) and FAS (fatty acid synthase (FAS)), which are representative genes involved in the induction of 3T3-L1 adipocyte differentiation, such as peroxisome proliferator activated receptor gamma 2 ), Lactobacillus plantarum KY1032, which has ultimate inhibitory effect on 3T3-L1 adipocyte differentiation by inhibiting mRNA and protein expression of FABP4 (fatty acid binding protein 4), and a pharmaceutical composition containing it as an active ingredient , Fermented milk, beverage, and health functional food.

3T3-L1 cells are mouse embryonic fibroblasts, a cell line commonly used in biological studies of adipose tissue. When 3T3-L1 mouse embryonic fibroblasts are treated with hormones such as insulin, the fat synthesis signal pathway is activated and the amount of intracellular fat is increased and the shape of the cells changes to a circular shape. The differentiation process of 3T3-L1 cells, called 3T3-L1 pre-adipocyte, is known as "3T3-L1 pre-adipocyte" The completed 3T3-L1 cells are called " mature 3T3-L1 adipocytes ".

Researches are underway in various countries to search for substances that inhibit lipid differentiation of 3T3-L1 cells. For example, green tea polyphenol component epigallocatechin gallate (Obesity res. 2005; 13: 982-90), red wine ingredient resveratrol (Phytother Res. 2008; 22: 1367-71), garlic ingredient ajoene (Phytother Res. ; 23: 513-8) have been shown to inhibit the lipid differentiation of 3T3-L1 cells. Since 3T3-L1 cells inhibit the lipid differentiation, they are highly effective in food, medicine, etc.

Obesity is known to be a chronic disease that is caused by a variety of factors that are still unclear. Obesity is a cause of hypertension, diabetes, cardiovascular disease, gallstone, osteoarthritis, sleep apnea, respiratory disturbance, endometrium, prostate, breast or colorectal cancer.

Treatment methods for obesity are diet - exercise, surgery and drug therapy. Dietary-exercise therapy is a method of treating low-calorie-low-fat meals and oxygen-consuming physical activity, which is perceived to be difficult to see because of the need to be patient, repetitive, and continuous. Surgery is a method of physically removing body fat through surgical operations, which is advantageous in that it can be effective in a short period of time. However, it is limited due to the necessity of surgery, lack of persistence of effect, and cost . Pharmacotherapy is a method to treat or prevent obesity by using drugs that induce appetite reduction or inhibit fat absorption. So far, the drugs for prevention and treatment of obesity have various physiological mechanisms.

Pancreatic lipolytic enzyme inhibitor (Orlistat) is known as a fat absorption inhibitory drug. It inhibits the action of pancreatic lipase and inhibits the absorption of fat-soluble vitamin and absorption of fat-soluble vitamin. .

Drugs that inhibit appetite usually act on neurotransmitters (catecholamines) that are present in the brain, reducing appetite. However, dexpenfluramine and phenfluramine have side effects of neurotoxicity, valvular heart disease, and sibutramine has side effects that increase heart rate and blood pressure.

On the other hand, many efforts have been made to prevent or treat obesity by using lactic acid bacteria which have been regarded as safe microorganisms. Lactic acid bacteria have been reported to exhibit effects such as maintenance of intestinal flora, improvement of intestinal flora, antidiabetic and anti-hyperlipidemic effect, inhibition of carcinogenesis, suppression of colitis, and nonspecific activity of host immune system. Among them, the Lactobacillus sp. Strain is a major member of normal microbial communities in the intestinal tract of the human body and has long been known to be important for maintaining healthy digestive organs and vaginal environment and has been approved by the US Public Health Service guidelines ), All of the Lactobacillus strains currently deposited with the American Type Culture Collection (ATCC) are classified as "Bio-safty Level 1", which is recognized as having no known potential risk of causing diseases in humans or animals .

As an anti-obesity effect mechanism of Lactobacillus strains, promotion of adiponectin secretion, promotion of energy consumption by sympathetic nerve stimulation, and induction of satiety have been known so far. However, there is little research on how Lactobacillus strains regulate the expression of fat metabolism-related genes in 3T3-L1 adipocytes.

Thus, the present inventors have found that Lactobacillus plantarum KY1032 inhibits 3T3-L1 adipocyte differentiation ultimately by inhibiting mRNA and protein expression of genes involved in induction of 3T3-L1 adipocyte differentiation, Thereby completing the invention.

Disclosure of the Invention The present invention has been conceived to solve the above-mentioned problems. It is an object of the present invention to provide Lactobacillus plantarum KY1032, which has an effect of inhibiting adipocyte differentiation induction gene expression and ultimately inhibiting adipocyte differentiation, , A fermented milk, a beverage, and a health functional food.

In order to achieve the above object, the present invention provides a method for producing 3T3-L1 adipocytes, which comprises expressing PPARγ2, C / EBPα (CCAAT / enhancer binding protein-alpha), FAS (fatty acid synthase and FABP4 (fatty acid binding protein 4), thereby ultimately providing Lactobacillus plantarum KY1032 having 3T3-L1 adipogenic differentiation inhibitory activity.

Hereinafter, the present invention will be described in detail.

In order to isolate the strain according to the present invention, kimchi prepared in a domestic home was added to an MRS liquid medium containing 0.02% sodium azide and cultured at 37 ° C for 24 hours, , The culture was taken and plated on a medium containing 0.02% sodium azide and cultured at 37% for 48 hours. A strain having the inhibitory effect on the differentiation of 3T3-L1 adipocytes was isolated from the thus formed herpes.

The isolates were cultured on MRS agar plates at 37 ° C for 48 hours. The size and morphology of the isolated strains were observed by light microscopy (SAMWON, CSB-FEI) through Gram stain (Cowan, 1974) Biochemical characteristics of a strain were classified and identified according to Bergey's Manual of Systematic Bacteriology and Bergey's Manual of Determinative Bacteriology after Cowan and Steel (1984) and Macfaddin (1984) methods.

Therefore, the characteristics of the novel lactic acid bacteria according to the present invention are as follows.

1) Form of bacteria: Bacillus

2) Colony color: Milk light circle shape

3) Optimum growth temperature: 37 ℃

4) Motility: None

5) Gram staining: positive

6) Catalase: negative

7) Oxygen effect: Beta anaerobic

8) Fermentation experiment and identification

Table 1 shows the results of sugar fermentation experiments using API 50CHL kit from Biomerieux.

0-24 per Whether or not to use Per 25-49 Whether or not to use 0 Control - 25 esculin + 1 glycerol - 26 Living + 2 Erythritol - 27 Cellobios + 3 D arabinose - 28 Maltose + 4 L arabinose + 29 lactose + 5 ribos + 30 Melibiose + 6 D xylose - 31 sucrose + 7 L xylose - 32 Trehalose + 8 Adonitor - 33 inulin - 9 beta methyl-D-xyloside - 34 Melitos + 10 galactose + 35 raffinos + 11 glucose + 36 starches - 12 fructose + 37 glycogen - 13 Manno + 38 xylitol - 14 sorbos - 39 Gentiobios + 15 Lambos + 40 D Toranos + 16 Dorsitol - 41 License Source - 17 Synoshitol - 42 D Tagatos - 18 Mannitol + 43 D Fuchos - 19 sorbitol + 44 L Fuchos - 20 [alpha] -methyl-D-mannoside + 45 D arabitol + 21? -Methyl-D-glucoside + 46 L arabitol - 22 N-acetyl-glucosamine + 47 Gluconate + 23 Amigalline + 48 2-keto-gluconate - 24 Arbutin + 49 5-keto-gluconate -

Based on the morphological, physiological and growth characteristics of the above-mentioned bacteria, the strain of the present invention was treated with Lactobacillus plantarum KY1032 (Accession Number: Korean Microorganism Conservation Center, deposit date: October 2, 2002, : KCCM-10430). This strain has already been patented in Korean Patent No. 0464642 (Lactobacillus plantarum kewaii 1032).

On the other hand, the pharmaceutical composition containing Lactobacillus plantarum KY1032 having an adipocyte differentiation inhibiting effect by inhibiting the expression of a gene involved in induction of adipocyte differentiation induction of the present invention as an active ingredient can be used alone or in combination with pharmaceutical excipients And may be used in the form of tablets, capsules, and the like according to a method commonly used in pharmaceuticals.

In the case of humans, a typical daily dose may range from 1 to 30 mg / kg body weight, and may be administered in single or divided doses. However, the actual dosage should be determined in light of various relevant factors such as route of administration, age, sex, weight, health status and severity of the disease.

Of course, since the pharmaceutical composition of the present invention has little toxicity and side effects, it can be safely used even when taken for a long time.

In addition, the fermented milk containing Lactobacillus plantarum KY1032 as an active ingredient having inhibitory effect on adipocyte differentiation inhibition by inhibiting the expression of a gene involved in induction of adipocyte differentiation can be obtained by culturing the lactic acid bacteria culture solution, Lactobacillus plantarum KY1032 and mixed juice syrup They are homogenized at a constant ratio of 150 bar, cooled to below 10 ° C and packaged in a container.

In addition, the beverage containing Lactobacillus plantarum KY1032 as an active ingredient having inhibitory effect on adipocyte differentiation inhibition by inhibiting expression of a gene involved in induction of adipocyte differentiation can be obtained by mixing mixed fruit syrup, Lactobacillus plantarum KY1032, , Homogenized at 150 bar, cooled to below 10 ° C and packaged in glass bottles, plastic bottles, etc.

In addition, the health functional food containing Lactobacillus plantarum KY1032, which has an effect of inhibiting the differentiation of adipocytes by inhibiting the expression of a gene involved in the induction of adipocyte differentiation, as an active ingredient includes the aforementioned Lactobacillus plantarum KY1032 Vitamin B1, B2, B5, B6, E and acetic acid ester, nicotinamide, oligosaccharide, etc. may be added as nutritional supplement components, and other food additives may be added.

Lactobacillus plantarum KY1032 according to the present invention inhibits the expression of a gene involved in the induction of adipocyte differentiation, and thus has an excellent effect of inhibiting adipocyte differentiation. Therefore, it is preferable to use Lactobacillus plantarum KY1032 Functional foods and pharmaceutical compositions that affect metabolism, and the like.

FIG. 1 is a graph showing the results of measuring the inhibitory effect of 3T3-L1 cell fat accumulation by Lactobacillus plantarum KY1032 by the AdipoRed assay method. FIG.
FIG. 2 is a graph showing the results of observing the inhibitory effect of 3T3-L1 cell fat accumulation by Lactobacillus plantarum KY1032 by Oil-red-O staining method.
FIG. 3 is a graph showing relative mRNA expression levels of PPARγ2, C / EBPα, FAS and FABP4 genes in 3T3-L1 cells using a real-time PCR method.
FIG. 4 is a graph of protein expression levels of PPARγ2, C / EBPα, FAS and FABP4 genes in 3T3-L1 cells using a Western blotting method.

Hereinafter, the present invention will be described in more detail with reference to examples. However, the following embodiments are not intended to limit the scope of the present invention, and ordinary variations by those skilled in the art within the scope of the technical idea of the present invention are possible.

≪ Example 1 >

Manufacture of freeze-dried powder containing Lactobacillus planta KY1032

The Lactobacillus plantarum KY1032 of the present invention is prepared by preparing Proteose peptone # 3, Yeast Extract, Beef Extract, and glucose-containing liquid medium for foodstuffs, culturing the mixture at 37 ° C for about 16 hours, centrifuging the culture, Washed with physiological saline and then dispersed in sterile oil. And then lyophilized again to obtain about 10 ¹¹ cfu bacterium per gram of freeze-dried powder (g). The lyophilized powder was used as an inhibitor of adipocyte differentiation inducing gene expression and adipocyte differentiation inhibition.

Lactobacillus plantarum KY1032 of the present invention may be provided in the form of a lyophilized powder or a culture as described above.

≪ Example 2 >

Production of a pharmaceutical composition containing Lactobacillus plantarum KY1032 as an active ingredient

The preparation examples of the pharmaceutical composition containing Lactobacillus plantarum KY1032 of the present invention as an active ingredient will be described, but the present invention is not intended to be limited thereto but is specifically described.

Manufacture of tablets

100 mg of lyophilized powder containing Lactobacillus plantarum KY1032 of Example 1, 100 mg of corn starch, 100 mg of lactose and 97 mg of polyvinylpyrrolidone were homogeneously mixed and granulated by a wet granulation method to obtain magnesium stearate 2 Was added to the mixture, and the resulting mixture was compressed to 400 mg per tablet.

Preparation of capsules

100 mg of lyophilized powder containing Lactobacillus plantarum KY1032 of Example 1, 100 mg of corn starch, 100 mg of lactose, and 2 mg of magnesium stearate were thoroughly mixed, and the resulting mixture was added to hard gelatine capsules To prepare a capsules.

≪ Example 3 >

Production of fermented milk containing Lactobacillus planta KY1032 as an active ingredient

A method for producing fermented milk comprising the lactic acid bacteria culture solution and the lactobacillus plantarum KY1032 of the present invention and the mixed fruit juice syrup is as follows.

The lactic acid bacteria culture solution was prepared by stirring 95.36 wt% of crude oil and 4.6 wt% of skim milk powder (or mixed powdered milk) and measuring specific gravity at 15 ° C from 1.0473 to 1.0475, a titratable acidity of 0.200 to 0.220%, pH of 6.65 to 6.70, of Brix (Brix ^o) were mixed such that the degree of 16.3 ~ 16.5%. After mixing, the mixture was heat-treated with UHT (sterilized at 135 ° C. for 2 seconds), cooled to 40 ° C., added with 0.02% by weight of Streptococcus thermophilus and Lactolytic enzyme (Valley laboratory, USA) The total number of lactic acid bacteria in the medium was 1.0 × 10 ⁹ cfu / ml or more, the titratable acidity was 0.89 to 0.91%, and the pH was 4.55 to 4.65.

Next, the mixed fruit juice syrup was prepared by mixing 13 wt% of liquid fructose, 5 wt% of white sugar, 10.9 wt% of mixed juice concentrate 56Brix ^o , 1.0 wt% of pectin, 0.1 wt% of fresh fruit mix essence and 70 wt% of purified water at 30-35 캜 Mixed with stirring, heat-treated with UHT (sterilized at 135 ° C for 2 seconds), and cooled.

Then, a mixture of 69.5% by weight of the lactic acid bacteria culture solution and 0.1% by weight of freeze-dried powder containing Lactobacillus planta KY1032 of Example 1 and 30.4% by weight of the mixed fruit juice syrup were homogenized at 150 bar, A fermented milk containing Lactobacillus plantarum KY1032 as an active ingredient having inhibitory effect on adipocyte differentiation was produced by inhibiting the expression of a gene involved in induction of adipocyte differentiation.

<Example 4>

Production of functional beverage containing Lactobacillus plantarum KY1032 as an active ingredient

A method for producing a functional beverage comprising the lactobacillus plantarum KY1032 of the present invention and a mixed fruit syrup is as follows.

First, a mixed juice syrup was 13% by weight of liquid fructose, white sugar, 2.5 wt%, brown sugar 2.5% by weight, mixed juice concentrate 56Brix ^o 10.9% by weight of pectin, 1.0% by weight, fresh 0.1% by weight fruit mix essence and purified water 70% The mixture was stirred at 30 to 35 ° C, mixed, and heat-treated by UHT (sterilized at 135 ° C for 2 seconds) and cooled.

Then, 30.4% by weight of the mixed juice syrup prepared by the above method, 0.1% by weight of freeze-dried powder containing the lactobacillus planta KY1032 of Example 1 and 69.5% by weight of the remaining purified water were combined and homogenized at 150 bar, After cooling, it is packaged in small containers such as glass bottles and PET bottles to inhibit the expression of genes involved in the induction of adipocyte differentiation, thereby producing a functional beverage containing Lactobacillus plantarum KY1032 as an active ingredient having an effect of inhibiting adipocyte differentiation Respectively.

&Lt; Example 5 >

Production of health functional food containing Lactobacillus planta KY1032 as active ingredient

(Vitamin B1, B2, B5, B6, E and acetic acid ester, nicotinic acid amide) and oligosaccharide were added to 0.1 wt% of the lyophilized powder containing the lactobacillus planta KY1032 of Example 1, 10 parts by weight based on 100 parts by weight of the freeze-dried powder containing Bacillus planta KY1032 were added and mixed in a high-speed rotary mixer. To 100 parts by weight of the mixture, 10 parts by weight of sterilized purified water was added and mixed to form granules having a diameter of 1 to 2 mm. The molded granules were dried in a vacuum drier at 40 to 50 DEG C and then passed through 12 to 14 mesh to prepare uniform granules. The granules thus prepared were extruded in suitable amounts to be purified or powdered or filled into hard capsules to prepare hard capsule products.

&Lt; Test Example 1 >

3T3-L1 induces differentiation of adipocytes and secures Lactobacillus plantarum KY1032 cell extract

1-1. Induction of 3T3-L1 adipocyte differentiation

, 37℃이다.
3T3-L1 mouse embryonic fibroblasts purchased from the American Type Culture Collection (ATCC) were cultured in Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal bovine serum (FBS). The procedure for differentiating 3T3-L1 mouse embryonic fibroblasts into adipocytes is as follows. After 2 days from the time point when confluent growth of 3T3-L1 mouse embryonic fibroblast (day 0), the culture medium was cultured in DMEM medium (differentiation medium) containing 10% FBS, 1 μg / ml insulin, 0.5 mM isobutylmethylxanthine (IBMX) and 1 μM dexamethasone Exchange culture. Two days later, the cells were cultured in DMEM medium supplemented with 10% FBS and 1 μg / ml insulin. After 2 days (day 4), the medium was replaced with a medium containing only 10% FBS. Four days later, on day 8, more than 90% of 3T3-L1 cells were completely differentiated into adipocytes. 3T3-L1 mouse embryonic fibroblasts were termed maturing 3T3-L1 pre-adipocyte (maturing 3T3-L1 pre-adipocyte) until complete differentiation into adipocytes (day 0 ~ day 8) (after day 8) is termed mature 3T3-L1 adipocyte (mature 3T3-L1 adipocyte). Cell culture conditions were 5% CO

, And 37 ° C.

1-2. Securing cell extract of Lactobacillus planta KY1032

cfu/㎖ 이 되도록 PBS에 resuspension한 후 sonication하여 균체를 lysis 하였다. 이를 원심분리한 후 상층액을 락토바실러스 플란타룸 KY1032 세포 추출물로 시험에 사용하였다.
Lactobacillus plantarum KY1032 was cultured in MRS medium at 37 DEG C for 24 hours. The cells were centrifuged and rinsed with phosphate buffered saline (PBS) to remove the remaining medium. Lactobacillus plantarum KY1032 cells were lyophilized and pulverized and serial dilution was performed to measure the number of bacteria. When the concentration of the cells is 10

cfu / ml, and then lysed by sonication. After centrifugation, the supernatant was used for the test with Lactobacillus plantarum KY1032 cell extract.

&Lt; Test Example 2 &

Measurement of 3T3-L1 adipocyte differentiation inhibitory ability by Lactobacillus plantarum KY1032

2-1. 3T3-L1 AdipoRed assay

3T3-L1 cells cultured on a 96-well plate were treated with 1% (v / vo) of the Lactobacillus plantarum KY1032 cell extract of Test Example 1-2 at the time of treatment of the differentiation medium (day 0) . In addition, Lactobacillus plantarum KY1032 cell extracts were diluted to 1/10, 1/100, and 1/1000, respectively, and then treated with 1% (v / vo). The maturation of the 3T3-L1 pre-adipocyte maturation on day 6 (day 6) was rinsed with PBS to remove media components and treated with 200 μl PBS and 5 μl AdipoRed Assay Reagent (Lonza) . AdipoRed reagents are very useful for measuring the amount of fat in a cell because they enter the fat mass and display fluorescence. After incubation at room temperature for 10 minutes, the fluorescence was measured with a fluorescence detector (excitation 485 nm, emission 572 nm).

The results are shown in Fig.

1 is a group treated with PBS, B is a group treated with 1/1000 diluted Lactobacillus plantarum KY1032 cell extract, C is a group treated with 1/100 diluted Lactobacillus plantarum KY1032 cell extract, D is a group treated with Lactobacillus The group treated with 1/10 dilution of Plantarum KY1032 cell extract, E is the group treated with Lactobacillus plantarum KY1032 cell extract, and the relative fat accumulation of each of B, C, D and E against A is shown.

As can be seen in FIG. 1, the degree of fat accumulation of 3T3-L1 cells was decreased by about 4% for B, about 10% for D, about 33% for D and about 36% for E, As a result, it was concluded that as the concentration of the cell extract of Lactobacillus plantarum KY1032 of the present invention is increased, the amount of accumulated fat is further reduced as 3T3-L1 cells are differentiated.

2-2. 3T3-L1 Oil-red-O staining

OH 1.5㎖과 70% ethanol 98.5㎖ 혼합 용액에 10번 담근 후 증류수에 10번 담근다. 성숙 중인 3T3-L1 지방전구세포(maturing 3T3-L1 pre-adipocyte)에 축적되어 있던 지방이 이 과정에서 적색으로 염색되었고 이를 현미경(AxioObser Z1, Carl Zeiss)으로 관찰하였다.3T3-L1 cells cultured on cover glass were treated with 1% (v / vo) of Lactobacillus plantarum KY1032 cell extract of Test Example 1-2 at the time of treatment of the differentiation medium (day 0). Maturing 3T3-L1 preadipocyte cells, which were grown 6 days later (day 6), were rinsed with PBS and fixed with 10% formalin for 30 minutes. The oil-red-O reagent was dissolved in isopropanol at a concentration of 3 g / L, filtered and mixed with distilled water at a ratio of 6: 4, and the cells were treated for 1 hour. Cells were rinsed with distilled water, treated with hematoxylin, reacted for 5 minutes, and rinsed again with distilled water. Next, immerse in 2% glacial acetic acid 10 times and rinse with distilled water. Next, 30% NH

OH and 98.5 ml of 70% ethanol for 10 times, and soak them in distilled water 10 times. The fat accumulated in the maturing 3T3-L1 pre-adipocyte was stained red in this process and observed with a microscope (AxioObser Z1, Carl Zeiss).

The results are shown in Fig.

FIG. 2A shows a 3T3-L1 cell with undifferentiated 3T3-L1 cells, B shows a maturing 3T3-L1 pre-adipocyte cultured in a differentiation medium, and C shows an eruption containing Lactobacillus plantarum KY1032 cell extract (3T3-L1 pre-adipocyte) cultured in medium was observed by microscopic observation of Oil-red-O stained fat.

As can be seen from FIG. 2, in the case of A, there was almost no fat because it was not differentiated into adipocytes. In case of B, a large amount of fat was accumulated because the differentiation medium was treated to induce differentiation into adipocytes. However, in the case of C, it was confirmed that the degree of fat accumulation in 3T3-L1 cells was significantly inhibited by the Lactobacillus plantarum KY1032 cell extract contained in the differentiation medium as compared with B.

&Lt; Test Example 3 >

A significant difference in the expression of obesity-related biomarker genes in 3T3-L1 cells by Lactobacillus plantarum KY1032

3-1. Real-time PCR analysis

Representative genes involved in the differentiation of 3T3-L1 cells into adipocytes include PPARγ2, C / EBPα, FAS, and FABP4. Real-time PCR was performed to confirm that the 3T3-L1 adipocyte differentiation inhibitory effect of Lactobacillus flutarium KY1032 of Test Example 2 was suppressed by suppressing mRNA expression of the gene.

3T3-L1 cells cultured on a 6-well plate were treated with 1% (v / vo) of the Lactobacillus plantarum KY1032 cell extract of Test Example 1-2 at the time of treatment of the differentiation medium (day 0) . RNA was extracted from the mature 3T3-L1 preadipocyte (maturing 3T3-L1 pre-adipocyte) generated 6 days later (day 6) and analyzed by High Capacity RNA-to-cDNA kit (Applied Biosystems, 4387406) Complementary DNA (hereinafter referred to as "cDNA") for RNA was obtained. Real-time RCR (Applied Biosystems, 7500 Real time PCR) was performed using the purchased Taqman probes (purchased from Applied Biosystems) and the cDNAs as shown in Table 2 below.

The results are shown in Fig.

FIG. 3A shows the maturation of 3T3-L1 preadipocyte cultured in the differentiation medium and B shows the mature 3T3-L1 pre-adipocyte cultured in the differentiation medium containing the Lactobacillus plantarum KY1032 cell extract. L1 preadipocyte (maturing 3T3-L1 pre-adipocyte), and C is undifferentiated 3T3-L1 cell, indicating the relative gene mRNA expression of B and C for A, respectively.

GAPDH mRNA expression in Table 2 was used as an internal control.

As can be seen from FIG. 3, mRNA expression levels of C / EBPα, FABP4, FAS, and PPARγ2 genes involved in 3T3-L1 adipogenic differentiation were significantly increased in A compared to C. PPARγ2 and C / EBPα induce lipid differentiation of 3T3-L1 cells as transcription factors, while FAS and FABP4 increase unsaturated fatty acid synthesis and fatty acid uptake. However, in the case of B, mRNA expression levels of PPARγ2, C / EBPα, FAS and FABP4 were inhibited by 28%, 23%, 30%, and 36%, respectively, These results suggest that inhibition of mRNA expression of the gene inducing adipocyte differentiation by Lactobacillus plantarum KY1032 inhibits 3T3-L1 adipocyte differentiation.

Gene name product no peroxisome proliferator activated receptor gamma 2 (PPARγ2) Mm00440945_m1 CCAAT / enhancer binding protein alpha (C / EBPa) Mm00514283_s1 fatty acid binding protein 4 (FABP4) Mm00445880_m1 fatty acid synthase (FAS) Mm00662319_m1 glyceraldehyde-3-phosphate dehydrogenase (GAPDH) Mm99999915_g1

3-2. Western blot analysis

In addition to the fact that mRNA expression of the adipocyte differentiation-related genes of Test Example 3-1 was inhibited by Lactobacillus plantarum KY1032 of the present invention, protein expression of these genes was also inhibited by Lactobacillus plantarum KY1032 of the present invention Western blotting analysis was performed.

3T3-L1 cells cultured on a 6-well plate were treated with 1% (v / vo) of the Lactobacillus plantarum KY1032 cell extract of Test Example 1-2 at the time of treatment of the differentiation medium (day 0) . Proteins were extracted from mature 3T3-L1 pre-adipocytes generated 6 days after (day 6) and the extracted proteins were electrophoresed on acrylamide gel. Proteins separated according to molecular weight on the gel were transferred to a nitrocellulose film and reacted with antibodies purchased as shown in Table 3 using a Western blotting automated apparatus (Benchpro 4100, Invitrogen).

The results are shown in Fig.

FIG. 4A shows the maturation of 3T3-L1 cells with undifferentiated 3T3-L1 cells, B with maturing 3T3-L1 pre-adipocytes, and C with differentiation maturation in differentiation medium containing Lactobacillus plantarum KY1032 cell extract 3T3-L1 preadipocyte (3T3-L1 pre-adipocyte).

GAPDH mRNA expression in Table 3 was used as an internal control.

As can be seen from FIG. 4, the protein expression levels of PPARγ2, C / EBPα, FAS, and FABP4 genes involved in the 3T3-L1 adipocyte differentiation process were significantly increased in B compared to A. However, in the case of C, protein expression levels of PPARγ2, C / EBPα, FAS and FABP4 were observed to be significantly inhibited compared to B. These results indicate that inhibition of protein expression of the gene inducing adipocyte differentiation by Lactobacillus plantarum KY1032 inhibits 3T3-L1 adipocyte differentiation.

Antibody Name product no anti-PPARgamma2 antibody abcam ab45036 anti-C / EBPa antibody Cell signaling sc-2295 anti-FABP4 antibody Santa cruz sc-18661 anti-FAS antibody Cell signaling 3189 anti-GAPDH antibody Santa cruz sc-137179

Korea Microorganism Conservation Center (overseas) KCCM10430 20021002

Claims (6)

delete delete delete (PPARγ2), C / EBPα (CCAAT / enhancer binding protein-alpha), FAS (fatty acid synthase) and FABP4 (fatty acid binding protein 4), which are genes involved in induction of 3T3- Wherein the cell extract of Lactobacillus plantarum KY1032 (accession number: KCCM-10430) having an inhibitory effect on the differentiation of 3T3-L1 adipocytes is used as an active ingredient.
(PPARγ2), C / EBPα (CCAAT / enhancer binding protein-alpha), FAS (fatty acid synthase) and FABP4 (fatty acid binding protein 4), which are genes involved in induction of 3T3- Wherein the cell extract of Lactobacillus plantarum KY1032 (accession number: KCCM-10430) having an inhibitory effect on the differentiation of 3T3-L1 adipocytes is contained as an active ingredient.
(PPARγ2), C / EBPα (CCAAT / enhancer binding protein-alpha), FAS (fatty acid synthase) and FABP4 (fatty acid binding protein 4), which are genes involved in induction of 3T3- (KCCM-10430) cell extract of Lactobacillus plantarum KY1032 which has an inhibitory effect on 3T3-L1 adipocyte differentiation inhibiting activity by inhibiting the expression of Lactobacillus plantarum KT1032.

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