KR101266328B1 - Lactobacillus gasseri HY7021 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, by inhibiting the mRNA and protein expression of genes involved in 3T3-L1 adipocyte differentiation, ultimately Lactobacillus gasseri ( Lactobacillus gasseri ) HY7021 having the effect of inhibiting 3T3-L1 adipocyte differentiation and containing it as an active ingredient Pharmaceutical compositions, fermented milk, beverages, functional foods, Lactobacillus gasseri according to the invention ( Lactobacillus gasseri ) HY7021 is very effective in inhibiting 3T3-L1 fat cell differentiation, Lactobacillus gasseri ( Lactobacillus gasseri ) HY7021 Functional foods and pharmaceutical compositions that affect fat metabolism can be provided.

Description

Lactobacillus gasseri HY7021 having inhibitory activity against adipocyte-specific gene expression and adipocyte differentiation, and product containing specifically as an effective factor}

The present invention, Lactobacillus gasseri ( Lactobacillus gasseri ) having the effect of inhibiting the expression of genes involved in the induction of adipocyte differentiation ( Lactobacillus gasseri ) HY7021 and pharmaceutical compositions containing the same as an active ingredient, fermented milk, beverages, health functional foods More specifically, peroxisome proliferator activated receptor gamma 2 (PPARγ2), C / EBPα (CCAAT / enhancer binding protein-alpha), and FAS (fatty acid synthase), which are representative genes involved in inducing 3T3-L1 adipocyte differentiation , Lactobacillus gasseri HY7021 having the inhibitory effect of 3T3-L1 adipocyte differentiation by inhibiting the expression of mRNA and protein of FABP4 (fatty acid binding protein 4) and a pharmaceutical composition containing the same as an active ingredient, fermented milk , Beverages and dietary supplements.

3T3-L1 cells are mouse embryonic fibroblasts and are a popular cell line for biological studies of adipose tissue. Treatment of insulin-like hormones with 3T3-L1 mouse embryonic fibroblasts activates the fat synthesis signaling pathway, increasing intracellular fat mass and altering the shape of the cells. This process of changing the intracellular signaling pathway and the shape of the cells is called differentiation, and differentiation of 3T3-L1 cells into 'maturation 3T3-L1 pre-adipocyte', differentiation The completed 3T3-L1 cells are called 'mature 3T3-L1 adipocytes'.

Researches to search for substances that inhibit the differentiation of these 3T3-L1 cells are in progress in each country. For example, epigallocatechin gallate (Obesity res. 2005; 13: 982-90), a green tea polyphenol component, resveratrol (Phytother Res. 2008; 22: 1367-71), a red wine component, and ajoene (Phytother Res. 2009), a garlic component. ; 23: 513-8) and the like have been reported to have the effect of inhibiting the differentiation of 3T3-L1 cells. Substances that inhibit the differentiation of 3T3-L1 cells can be considered to have anti-obesity effects, so they are very useful in the fields of food and medicine.

Obesity is still known as a chronic disease that is caused by a combination of factors that are still unclear. Obesity is a cause of hypertension, diabetes, cardiovascular disease, gallstones, osteoarthritis, sleep apnea, respiratory disorders, endometrium, prostate, breast or colon cancer.

Treatments for obesity are largely diet-exercise therapy, surgical therapy and drug therapy. Diet-exercise therapy is a therapeutic method through low calorie-low fat intake and physical activity that consumes oxygen, and it is recognized that it is difficult to see the public effect because it must be carried out repeatedly and continuously with patience. Surgical therapy is a method of physically removing body fat through surgical surgery, which has the advantage of being effective in a short period of time, but it is limited in use due to the need for surgery, insufficiency of effect, and cost It is becoming. Drug therapy is a method to treat or prevent obesity using agents that cause decreased appetite or inhibit fat absorption. The drugs for preventing and treating obesity developed to date have various physiological mechanisms.

Pancreatic lipase inhibitors (Orlistat) are known as inhibitors of fat absorption, which inhibits the action of pancreatic lipase and thus inhibits the absorption of drugs or fat-soluble vitamins, which may cause breast cancer. .

Appetizers that inhibit appetite mainly affect neurotransmitters (catecholamines) present in the brain, which reduces appetite. However, dexfenfluramine and fenfluramine have side effects of neurotoxicity and valve heart disease, and sibutramine has side effects of increasing heart rate and blood pressure.

On the other hand, efforts to prevent or treat obesity have also been made using lactic acid bacteria, which have been considered safe microorganisms. Lactobacillus has been reported to show the effects of maintaining normal intestinal flora, improving intestinal flora, antidiabetic and antihyperlipidemic effects, inhibiting carcinogenesis, inhibiting colitis, and nonspecific activity of the host's immune system. Among them, the strain Lactobacillus is a major member of the normal microbial community in the intestine of the human body, which has long been known to be important in maintaining a healthy digestive system and the vaginal environment, and the US Public Health Service guidelines. ), All of the Lactobacillus strains currently deposited with the American Strain Deposit Agency (ATCC) are classified as 'Bio-safty Level 1', which is not known for any potential risk of causing disease in humans or animals. .

The mechanism of anti-obesity of Lactobacillus strains is known to promote adiponectin secretion, to promote energy consumption by sympathetic stimulation, to induce satiety. However, studies on how the Lactobacillus strain regulates the expression of fat metabolism related genes in 3T3-L1 adipocytes are insufficient.

The present inventors have discovered that Lactobacillus gasseri HY7021 inhibits mRNA and protein expression of genes involved in inducing 3T3-L1 adipocyte differentiation, thereby ultimately inhibiting 3T3-L1 adipocyte differentiation. To complete.

The present invention has been made to solve the above problems, Lactobacillus gasseri ( Lactobacillus gasseri ) HY7021 having the effect of ultimately inhibiting adipocyte differentiation by inhibiting adipocyte differentiation induction gene expression and containing it as an active ingredient To provide a pharmaceutical composition, fermented milk, beverages, health functional food.

In order to achieve the above object, the present invention is a representative gene involved in inducing 3T3-L1 adipocyte differentiation, PPARγ2 (peroxisome proliferator activated receptor gamma 2), C / EBPα (CCAAT / enhancer binding protein-alpha), FAS (fatty) It is characterized by providing Lactobacillus gasseri HY7021, which ultimately inhibits 3T3-L1 adipocyte differentiation by inhibiting the expression of acid synthase), mRNA and protein expression of FABP4 (fatty acid binding protein 4).

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 3T3-L1 adipocyte differentiation inhibitory effect was isolated from the colonies thus formed.

Observation of the isolated strains was incubated for 48 hours at 37 ℃ in MRS agar plate medium and the size and shape of the isolated strains were observed by light microscopy (SAMWON, CSB-FEI) through Gram staining (Cowan, 1974). The 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 the characteristics of the strains were investigated according to the methods of Cowan, Steel (1984) and Macfaddin (1984). Catalase test was used to observe the presence of oxygen after adding 3% hydrogen peroxide. Oxidase test was carried out by adding 1% dimethyl-p-phenylenediamine gydrochloride to observe discoloration.

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

1) Types of bacteria

The characteristics of the bacteria when cultured on an MRS agar plate medium at 37 ° C for 2 days

① Cell Type: Bacillus

② Mobility: None

③ Sporulation ability: None

④Gram staining: positive

2) Physiological properties

① Growth temperature: Growth possible Growth temperature: 20 ~ 45 ℃

Optimum growth temperature: 36 ~ 38 ℃

② Growth pH: Growth possible Growth pH: 5.0 ~ 7.5

Optimum pH: 6.0-6.5

③ Effects on oxygen: anaerobic anaerobic

3) Catalase:-

4) Whether gas is formed:-

5) Growth at 15 ℃:-

6) Growth at 45 ℃: +

7) Indole Production:-

8) lactic acid production: +

9) sugar fermentation experiment and identification

The results of the sugar fermentation experiment using the API 50CHL kit of Biomerieux are shown in Table 1 and FIG. 1.

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 -

10) 16s rRNA Identification

To identify the strain of the present invention, 16s rRNA gene was PCR. At this time, the forward primer base sequence was 5'-AGAGTTTGATCCTGGCTCAG-3 ', the reverse primer base sequence was used 5'-GGTTACCTTGTTACGACTT-3'. 16s rRNA gene was identified the strain of the present invention on the basis of the nucleotide sequence as Lactobacillus biasing Li (Lactobacillus gasseri), the inventors ended Li (Lactobacillus this Lactobacillus gasseri ) was named HY7021 and was deposited with Korea Biotechnology Research Institute (Accession No .: KCTC 11722BP), an international depository institution, on July 5, 2010.

On the other hand, the pharmaceutical composition containing Lactobacillus gaseri HY7021 having the effect of inhibiting adipocyte differentiation by inhibiting the expression of genes involved in the induction of adipocyte differentiation of the present invention as an active ingredient is an excipient used alone or pharmaceutically. It can be used by formulating together in the form of tablets, capsules and the like according to the method commonly used pharmaceutically.

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 gaseri HY7021, which has the effect of inhibiting adipocyte differentiation by inhibiting the expression of genes involved in the induction of adipocyte differentiation, as an active ingredient, has a certain ratio of lactic acid bacteria culture medium, Lactobacillus gaseri HY7021 and mixed fruit syrup. After homogeneous at 150bar in combination with the cooling to 10 ℃ or less and prepared by packaging in a container.

In addition, a beverage containing Lactobacillus fern HY7021 as an active ingredient, which inhibits the expression of a gene involved in inducing adipocyte differentiation, as an active ingredient, has a ratio of mixed juice syrup, Lactobacillus fern HY7021 and water at a constant ratio. The mixture is homogenized at 150 bar, cooled to 10 ° C. or lower, and then packaged in a small packaging container such as a glass bottle or a plastic bottle.

In addition, a dietary supplement containing Lactobacillus gasteria HY7021 as an active ingredient, which inhibits the expression of genes involved in inducing adipocyte differentiation, as an active ingredient, is supplemented with nutrition other than the Lactobacillus gasteria HY7021. As ingredients, vitamins B1, B2, B5, B6, E and acetate esters, nicotinic acid amides, oligosaccharides and the like may be added, and other food additives may be added.

Lactobacillus gasseri according to the present invention ( Lactobacillus gasseri ) HY7021 by inhibiting the expression of genes involved in the induction of adipocyte differentiation is very excellent in suppressing the effect of adipocyte differentiation, so Lactobacillus gasseri ( Lactobacillus gasseri ) for fat metabolism using HY7021 It can be used as a functional food, pharmaceutical composition and the like to affect.

1 is a diagram showing the results of a sugar fermentation experiment of Lactobacillus gaseri HY7021 using api 50 CH kit of Biomerieux.
Figure 2 is a graph showing the results of measuring the inhibitory effect of 3T3-L1 cell fat accumulation by Lactobacillus gaseri HY7021 by the AdipoRed assay method.
Figure 3 is a diagram showing the results observed by the Oil-red-O staining method for inhibiting the accumulation of 3T3-L1 cell fat by Lactobacillus gaseri HY7021.
Figure 4 is a graph of the relative mRNA expression of PPARγ2, C / EBPa, FAS, FABP4 gene in 3T3-L1 cells using real-time PCR method.
5 is a graph illustrating the protein expression levels of PPARγ2, C / EBPa, FAS and FABP4 genes in 3T3-L1 cells using Western blotting.

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

≪ Example 1 >

Preparation of lyophilized powders, including Lactobacillus gasteria HY7021

The Lactobacillus gasteria HY7021 of the present invention is a liquid medium containing Proteose peptone # 3 for food ingredients, Yeast Extract, Beef Extract, and glucose, and cultured at 37 ° C. for about 16 hours, followed by centrifugation of the culture solution and sterilization. Washed with brine and then dispersed in sterile oil. Freeze-drying again yielded about 10 ¹¹ cfu bacteria per gram (g) of lyophilized powder. This lyophilized powder was used as a material for inhibiting adipocyte differentiation inducing gene expression and adipocyte differentiation.

Meanwhile, the Lactobacillus fern HY7021 of the present invention may be provided in the form of a lyophilized powder or culture.

<Example 2>

Preparation of a pharmaceutical composition containing Lactobacillus gaseri HY7021 as an active ingredient

Although the formulation example of the pharmaceutical composition containing the Lactobacillus gaseri HY7021 of the present invention as an active ingredient will be described, the present invention is not intended to limit this, only intended to explain in detail.

Manufacture of tablets

Lyophilized powder 100 mg, Lactobacillus 100 g, 100 mg of corn starch, 100 mg of lactose, and 97 mg of polyvinylpyrrolidone were homogeneously mixed, granulated by wet granulation, and magnesium stearate 2 mg. After adding and mixing, one tablet was compressed to 400 mg.

Preparation of capsules

After completely mixing the lyophilized powder 100mg, corn starch 100mg, lactose 100mg, magnesium stearate 2mg, including Lactobacillus gaseous HY7021 of Example 1, and filled into hard gelatin capsules according to the conventional method for preparing capsules To prepare a capsule.

<Example 3>

Preparation of Fermented Milk Containing Lactobacillus Gaseri HY7021 as an Active Ingredient

Method for producing a fermented milk consisting of the lactic acid bacteria culture medium, Lactobacillus gaseri HY7021 and mixed juice syrup of the present invention 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, it was UHT heat-treated (sterilized at 135 ° C for 2 seconds) and cooled to 40 ° C. Then, Streptococcus thermophilus and Lactobacillus (Valley laboratory, USA) were each added 0.02% by weight and incubated for 6 hours to incubate BCP. Total lactic acid bacteria in the medium was 1.0 × 10 ⁹ cfu / ㎖ or more, the titratable acidity was 0.89 ~ 0.91%, pH was prepared to be 4.55 ~ 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, 69.5% by weight of the lactic acid bacteria culture medium, 0.1% by weight of the lyophilized powder including Lactobacillus gaseri HY7021 of Example 1 and 30.4% by weight of the mixed fruit juice syrup were homogenized at 150 bar and then cooled to 10 ° C or less. By inhibiting the expression of genes involved in cell differentiation, fermented milk containing Lactobacillus gaseri HY7021 having the effect of inhibiting adipocyte differentiation was prepared as an active ingredient.

<Example 4>

Preparation of Functional Drink Containing Lactobacillus Gaseri HY7021 as an Active Ingredient

Method for producing a functional beverage consisting of Lactobacillus gastrius HY7021 and mixed juice syrup of the present invention 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.

And after mixing the homogeneous at 150 bar and 30.4% by weight of the mixed fruit juice syrup prepared in the above method and 0.1% by weight of the lyophilized powder including the Lactobacillus gaseri HY7021 and 69.5% by weight of the remaining purified water and cooled to 10 ℃ or less. Then, by packaging the same in a small package container such as glass bottles, plastic bottles to suppress the expression of genes involved in the induction of adipocyte differentiation, a functional beverage containing Lactobacillus gaseri HY7021 having the effect of inhibiting adipocyte differentiation was prepared as an active ingredient.

<Example 5>

Preparation of Health Functional Foods Containing Lactobacillus Gaseri HY7021 as an Active Ingredient

Lactobacillus HY7021 of Example 1 in the freeze-dried powder containing 0.1% by weight of nutritional supplements (vitamins B1, B2, B5, B6, E and acetate esters, nicotinic acid amide) and oligosaccharides To 100 parts by weight of the freeze-dried powder, including petroleum HY7021 was added to 10 parts by weight 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 prepared as described above were extruded by appropriate amounts into tablets or powders or filled into hard capsules to produce hard capsule products.

&Lt; Test Example 1 >

Induction of 3T3-L1 Adipocyte Differentiation and Acquisition of Lactobacillus Casey HY7021 Cell Extract

1-1. Induces 3T3-L1 Adipocyte Differentiation

, 37℃이다.
3T3-L1 cells 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 cells into adipocytes is listed as follows. Two days after 3T3-L1 cells grew confluent (day 0), the culture medium was exchanged with DMEM medium (differentiation medium) containing 10% FBS, 1 μg / ml insulin, 0.5 mM isobutylmethylxanthine (IBMX) and 1 μM dexamethasone. Incubated. After 2 days (day 2) and incubated with DMEM medium containing 10% FBS and 1μg / ㎖ insulin. Two days later (day 4), the culture was replaced with a medium containing only 10% FBS. Four days later (day 8), more than 90% of 3T3-L1 cells are fully differentiated into adipocytes. Before 3T3-L1 cells fully differentiate into adipocytes (day 0 to day 8), they are termed maturing 3T3-L1 pre-adipocytes and are fully differentiated into adipocytes (day 8 and later), the mature 3T3-L1 adipocytes (mature 3T3-L1 adipocytes). Cell culture conditions were 5% CO

, 37 ° C.

1-2. Acquisition of Cell Extracts of Lactobacillus Gasteria HY7021

cfu/ml 이 되도록 PBS에 resuspension한 후 sonication하여 균체를 lysis 하였다. 이를 원심분리한 후 상층액을 락토바실러스 가세리 HY7021 세포 추출물로 시험에 사용하였다.
Lactobacillus fern HY7021 was incubated in MRS medium at 37 ° C. for 24 hours. The cells were centrifuged and then rinsed with Phosphate Buffered Saline (PBS) to remove the remaining media components. Lactobacillus gaseri HY7021 cells were lyophilized and powdered, followed by serial dilution. Cell concentration is 10

The cells were resuspensioned in PBS to cfu / ml and then sonicated to lyse the cells. After centrifugation, the supernatant was used for testing as a Lactobacillus fern HY7021 cell extract.

&Lt; Test Example 2 &

Determination of 3T3-L1 Adipocyte Differentiation Ability by Lactobacillus Gaseri HY7021

2-1. 3T3-L1 AdipoRed assay

At the time of treatment of the differentiation medium to the undifferentiated 3T3-L1 cells cultured in a 96-well plate (day 0), the Lactobacillus gasteria HY7021 cell extract of Test Example 1-2 was treated with 1% (vo / vo) together. . Additionally, the Lactobacillus gaseri HY7021 cell extract was diluted to 1/10, 1/100, and 1/1000, respectively, and then treated with 1% (vo / vo). After 6 days (day 6), the mature 3T3-L1 pre-adipocytes (maturing 3T3-L1 pre-adipocytes) were rinsed with PBS to remove the media components and treated with 200μl PBS and 5μl AdipoRed Assay Reagent (Lonza). . AdipoRed reagents are very useful for measuring the amount of fat in a cell because they penetrate the fat mass and fluoresce. After reacting at room temperature for 10 minutes, fluorescence was measured by a fluorescence detector (excitation 485 nm, emission 572 nm).

The results are shown in Fig.

Figure 2 A is a group treated with PBS, B is a group of 1/1000 dilution of Lactobacillus gaseous HY7021 cell extract, C is a group treated with 1/100 dilution of Lactobacillus gaseous HY7021 cell extract, D is a Lactobacillus gasseri HY7021 cell extract 1/10 dilution group, E is a group treated with Lactobacillus gasi HY7021 cell extract as it is, showing the relative fat accumulation of each of B, C, D, E for A.

As can be seen in Figure 2, compared to A, B is about 5%, C is about 13%, D is about 34%, E is about 40% reduced fat accumulation. It was concluded that the higher the concentration of the cell extract of the Lactobacillus gasteria HY7021 of the present invention, the more the amount of fat accumulated in the maturing 3T3-L1 pre-adipocytes was reduced.

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.At the time of treatment of the differentiation medium to the 3T3-L1 cells cultured in the cover glass (day 0), the Lactobacillus fern HY7021 cell extract of Test Example 1-2 was treated with 1% (vo / vo) together. After 6 days (day 6), the resulting mature 3T3-L1 pre-adipocytes (maturing 3T3-L1 pre-adipocytes) were rinsed with PBS and fixed with 10% formalin for 30 minutes. Oil-red-O reagent was dissolved in isopropanol at a concentration of 3g / L, filtered, mixed with distilled water and a ratio of 6: 4, and treated with cells for 1 hour. The cells were rinsed with distilled water and then treated with hematoxylin for 5 minutes and rinsed again with distilled water. Next, immersed in 2% glacial acetic acid 10 times and rinsed with distilled water. Next 30% NH

Dip 10 times in a mixture of 1.5ml OH and 98.5ml 70% ethanol, and then immerse in distilled water 10 times. Fat accumulated in maturing 3T3-L1 pre-adipocytes was stained red in this process and observed under a microscope (AxioObser Z1, Carl Zeiss).

The results are shown in Fig.

3A is maturing 3T3-L1 pre-adipocyte cultured in differentiation medium, B is maturing 3T3-L1 cultured in differentiation medium containing Lactobacillus fern HY7021 cell extract As fat precursor cells (maturing 3T3-L1 pre-adipocyte), Oil-red-O stained fat was observed under a microscope.

As can be seen in Figure 3, in the case of A treated with differentiation medium to induce differentiation into adipocytes, a significant amount of fat accumulated. However, in case of B, the accumulation of fat in maturing 3T3-L1 pre-adipocytes was significantly inhibited compared to A by Lactobacillus gasi HY7021 cell extract contained in differentiation medium. Could.

<Test Example 3>

Determination of Significant Differences in Obesity-Related Biomarker Gene Expression in 3T3-L1 Cells by Lactobacillus Gaseri HY7021

3-1. Real-Time PCR Analysis

Representative genes involved in the process of differentiating 3T3-L1 cells into adipocytes include PPARγ2, C / EBPa, FAS, FABP4, and the like. Real-time PCR was performed to confirm whether the inhibitory effect of 3T3-L1 adipocyte differentiation of Lactobacillus gasery HY7021 of Test Example 2 is indicated by suppressing mRNA expression of the gene.

At the time of treatment of the differentiation medium to the 3T3-L1 cells cultured in 6-well plate (day 0), the Lactobacillus gasery HY7021 cell extract of Test Example 1-2 was treated with 1% (vo / vo) together. After 6 days (day 6), RNA was extracted from the maturing 3T3-L1 pre-adipocytes, which were produced using a 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 Taqman probes (purchased from Applied Biosystems, Inc.) and the cDNA purchased as shown in Table 2 below.

The results are shown in FIG.

4A is maturing 3T3-L1 pre-adipocyte cultured in differentiation medium, B is maturing 3T3-L1 cultured in differentiation medium containing Lactobacillus fern HY7021 cell extract Adipocytes (maturing 3T3-L1 pre-adipocyte), C, are undifferentiated 3T3-L1 cells, indicating the relative gene mRNA levels of B and C, respectively, relative to A.

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

As can be seen in Figure 4, it was confirmed that the mRNA expression of PPARγ2, C / EBPa, FAS, FABP4 genes involved in the process of 3T3-L1 adipocyte differentiation was significantly increased in A compared to C. PPARγ2 and C / EBPa induce adipose differentiation of 3T3-L1 cells as transcription factors and FAS and FABP4 increase unsaturated fatty acid synthesis and fatty acid influx. However, in the case of B, the mRNA expression levels of PPARγ2, C / EBPa, FAS, and FABP4 were inhibited by 30%, 36%, 23%, and 32%, respectively, compared to A. These results showed that 3T3-L1 adipocyte differentiation was inhibited as a result of inhibiting mRNA expression of a gene inducing adipocyte differentiation by Lactobacillus acetyl HY7021.

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

3-2. Western blotting analysis

In addition to the fact that the mRNA expression of the 3-1 fat cell differentiation-related genes is inhibited by the Lactobacillus gasery HY7021 of the present invention, to confirm whether the protein expression of these genes is also inhibited by the Lactobacillus gasery HY7021 of the present invention. Western blotting analysis was performed.

At the time of treatment of the differentiation medium to the 3T3-L1 cells cultured in 6-well plate (day 0), the Lactobacillus gasery HY7021 cell extract of Test Example 1-2 was treated with 1% (vo / vo) together. After extracting the protein from the maturation 3T3-L1 pre-adipocyte (maturation 3T3-L1 pre-adipocyte) produced after 6 days (day 6), the extracted proteins were electrophoresed on acrylamide gel. Proteins separated according to molecular weight on the gel were transferred to a nitrocellulose film, and the antibodies purchased as shown in Table 3 were reacted using Western blotting automation equipment (Benchpro 4100, Invitrogen).

The results are shown in Fig.

Fig. 5A is maturation cultured in differentiation medium containing undifferentiated 3T3-L1 cells, B is maturing 3T3-L1 pre-adipocyte, C is Lactobacillus fern HY7021 cell extract As 3T3-L1 pre-adipocytes, the protein expression level can be determined through the band area.

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

As can be seen in Figure 5, it was confirmed that the protein expression of PPARγ2, C / EBPa, FAS, FABP4 genes involved in the process of 3T3-L1 adipocyte differentiation was significantly increased in B compared to A. However, in the case of C, the amount of protein expression of PPARγ2, C / EBPa, FAS and FABP4 was all significantly inhibited compared to B. These results showed that 3T3-L1 adipocyte differentiation was inhibited as a result of inhibition of protein expression of genes that induce adipocyte differentiation by Lactobacillus sp. HY7021.

Antibody name product no anti-PPARγ2 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 Biotechnology Research Institute KCTC11722BP 20100705

Claims (6)

delete Peroxisome proliferator activated receptor gamma 2 (PPARγ2), CCAAT / enhancer binding protein-alpha (C / EBPα), fatty acid synthase (FAS) and fatty acid binding protein 4 (FABP4), genes involved in inducing 3T3-L1 adipocyte differentiation Lactobacillus gasseri HY7021 (Accession No .: KCTC 11722BP), which has the effect of inhibiting the expression of 3T3-L1 adipocyte differentiation. delete delete delete delete

Images