KR20220102891A - Lactobacillus johnsonii JNU3402 strain and food comprising the same - Google Patents

Abstract

The present invention relates to a novel Lactobacillus johnsonii JNU3402 strain, and more particularly, the Lactobacillus johnny JNU3402 strain, accession number KCCM12892P, exhibits excellent acid resistance and bile resistance. The strain is the strain having a nucleotide sequence encoding 16S rRNA represented by SEQ ID NO: 1.

Description

Lactobacillus johnsonii JNU3402 strain and food containing the same {Lactobacillus johnsonii JNU3402 strain and food comprising the same}

The present invention relates to a Lactobacillus johnny JNU3402 strain and food containing the same.

In the early 1980s, probiotic products began to be used industrially in Korea. In the early days of industrial use, the effect was recognized for abnormal fermentation in the intestine, diarrhea, indigestion and constipation, and it has been used for the human body. .

Lactic acid bacteria, which are widely used as probiotics, are taxonomically Gram-positive, catalase-negative, non-spore-forming, and non-cytochrome as an electron transporter. The shape of lactic acid bacteria is spherical or rod-shaped, has resistance to oxygen and low pH, and ferments carbohydrates to produce lactic acid as a final metabolite. do.

Korean Patent No. 1868517

An object of the present invention is to provide a Lactobacillus johnsny strain.

An object of the present invention is to provide a food containing the Lactobacillus johnsny strain.

1. Lactobacillus johnsonii ( Lactobacillus johnsonii ) JNU3402 (Accession No. KCCM12892P) strain.

2. The strain according to 1 above, wherein the strain has a nucleotide sequence encoding 16S rRNA shown in SEQ ID NO: 1.

3. The strain according to the above 1, characterized in that it has acid resistance and bile resistance.

4. Lactobacillus johnsonii ( Lactobacillus johnsonii ) Food containing JNU3402 (Accession No. KCCM12892P) strain or the culture filtrate of the strain as an active ingredient.

5. The food according to 4 above, characterized in that the strain, the culture solution of the strain, or the culture filtrate of the strain has acid resistance and bile resistance.

6. In the above 4, the food is ice cream, milk, soy milk, yogurt, cheese, meat, sausage, bread, chocolate, candy, confectionery, pizza, ramen, gum, soup, beverage, tea, drink, alcoholic beverage, and At least one selected from the group consisting of lactic acid bacteria preparations in stick packaging, food.

The present invention Lactobacillus Johnsney JNU3402 strain, the culture solution of the strain or the culture filtrate of the strain has excellent acid resistance and bile resistance, and is not killed when passing through the stomach and can reach the intestine. In addition, the Lactobacillus johnsny JNU3402 strain, the culture solution of the strain, or the culture filtrate of the strain temporarily form colonies in the intestinal mucosa, thereby inhibiting the growth of pathogenic bacteria in the intestine and stabilizing the intestinal permeability.

1 shows a phylogenetic diagram showing the phylogenetic classification of Lactobacillus johnsney JNU3402.
Figure 2 shows the results of confirming the intestinal adhesion of Lactobacillus Johnsney JNU3402.
3 shows the results of confirming that there is no difference in the amount of food intake depending on the type of food or the administration of Lactobacillus johnsny JNU3402 dead cells.
Figure 4 shows the weight loss effect according to the administration of Lactobacillus johnsny JNU3402 dead cells.
5 shows the results of reducing the weight of adipose tissue and liver tissue according to the administration of Lactobacillus johnsny JNU3402 dead cells.
6 shows the results of reducing the triglyceride content in blood and tissues according to the administration of Lactobacillus johnsny JNU3402 dead cells.
7 shows a result of a decrease in blood sugar according to the administration of Lactobacillus johnsny JNU3402 dead cells.
8 shows the results of the decrease in the insulin content in the blood according to the administration of the Lactobacillus johnsny JNU3402 dead cells.
9 shows the result of reducing the lipid accumulation of 3T3-L1 adipocytes according to the treatment of the Lactobacillus Johnsney JNU3402 dead cell culture medium from which the Lactobacillus johnsny JNU3402 dead cells were removed.
10 shows that the expression of genes involved in lipid accumulation in epididymal adipose tissue (eWAT) and inguinal white fat (iWAT) is decreased according to the administration of Lactobacillus johnsny JNU3402 dead cells, and genes and fat cells involved in fatty acid beta oxidation. The expression of genes involved in browning shows the course of increased expression.
11 shows that the expression of genes involved in lipid accumulation in 3T3-L1 adipocytes decreased, and genes involved in fatty acid beta oxidation and genes involved in fat cell browning according to the treatment of Lactobacillus johnny JNU3402 dead cells conditioned medium. It shows the result of increased expression.
12 shows the results of increased transcriptional activity of PPARγ and increased activity of UCP1 promoter and PPARγ promoter according to the treatment of Lactobacillus johnsny JNU3402 dead cells conditioned medium.
13 shows the results confirming that the expression of genes related to energy metabolism by the Lactobacillus johnsny JNU3402 dead cell conditioned medium is regulated through the PPARγ pathway.
14 shows the results of increased mitochondrial DNA copy number and CS activity in adipose tissue or adipocytes according to the treatment of Lactobacillus johnsny JNU3402 dead cells or a conditioned medium thereof.
15 shows the result of increasing the rectal temperature of mice by the treatment of Lactobacillus johnsny JNU3402 dead cells.

The present invention provides a novel Lactobacillus johnsonii ( Lactobacillus johnsonii ) JNU3402 strain.

Lactobacillus johnsonii ( Lactobacillus johnsonii ) JNU3402 strain has a 16S rRNA base sequence set forth in SEQ ID NO: 1.

The Lactobacillus johnsney JNU3402 strain was deposited with the Korea Microorganism Conservation Center and registered with the accession number KCCM12892P.

Lactobacillus johnsney JNU3402 strain may be isolated from infant feces.

Lactobacillus johnsney JNU3402 strain exhibits excellent acid resistance and bile resistance (bile acid resistance). Specifically, the Lactobacillus Johnsny JNU3402 strain of the present invention does not die even after 24 hours or 48 hours after 0.5% (w/v) oxgall treatment, and exhibits an excellent proliferation effect.

Lactobacillus johnsney JNU3402 strain has excellent resistance to acid resistance and bile resistance, so it is not killed when passing through the stomach and can reach the intestine.

Lactobacillus Johnsney JNU3402 strain can temporarily form colonies in the intestinal mucosa to inhibit the growth of pathogenic bacteria and stabilize intestinal permeability.

Lactobacillus johnsney JNU3402 strain may exhibit excellent intestinal adhesion.

In addition to the above effects, Lactobacillus johnsny JNU3402 dead cells killed by heat treatment also show weight loss, adipose tissue and liver tissue weight reduction effects, and a decrease in triglyceride content in tissues and blood, and decrease in blood glucose and insulin content. effect and inhibiting the accumulation of lipids in adipocytes. In addition, Lactobacillus johnsny JNU3402 dead cells killed by heat treatment also have the effect of inhibiting the expression of genes involved in lipid accumulation (eg, FAS, ACC, SREBP1c), genes involved in fatty acid beta oxidation (eg, ACOX, CPT1, PGC1α) ) and the effect of increasing the expression of genes involved in adipocyte browning (eg, UCP1, PPARγ, Cidea), increasing the mitochondrial DNA copy number and citrate synthase activity, and increasing the temperature of the rectum.

In addition, the present invention provides a composition comprising the Lactobacillus johnsny JNU3402 strain or a culture filtrate of the strain as an active ingredient.

The composition of the present invention may include the Lactobacillus johnsney JNU3402 strain. For example, the composition of the present invention may include a culture solution containing the Lactobacillus johnny JNU3402 strain, a concentrated culture solution, or a dried culture solution.

The culture medium or the culture filtrate may contain various antibacterial organic acids and non-proteinaceous antibacterial substances produced by the strain.

The composition of the present invention may include a culture filtrate from which the Lactobacillus johnsny JNU3402 strain is removed. For example, the composition of the present invention may include a culture filtrate of the Lactobacillus johnsny JNU3402 strain, a concentrated culture filtrate, or a dried product of the culture filtrate.

The culture filtrate from which the Lactobacillus johnsny JNU3402 strain is removed may be a filtrate from which the strain is removed from the cultured solution for the Lactobacillus johnsney strain.

A composition comprising the Lactobacillus johnsny JNU3402 strain or the culture filtrate of the strain as an active ingredient may be food.

The food is ice cream, milk, soy milk, yogurt, cheese, meat, sausage, bread, chocolate, candy, confectionery, pizza, ramen, gum, soup, beverage, tea, drink, alcoholic beverage, and lactic acid bacteria preparation in stick packaging It may be at least one selected from the group.

The food may be a health functional food for improving or preventing intestinal health.

When the food is a beverage, it may contain various flavoring agents or natural carbohydrates as an additional component like a conventional beverage. The natural carbohydrate may be, for example, glucose, fructose, maltose, sucrose, dextrin, cyclodextrin, xylitol, sorbitol and erythritol.

The food may contain food additives. For example, food additives include nutrients, vitamins, minerals (electrolytes), synthetic flavoring agents and flavoring agents such as natural flavoring agents, coloring agents and thickening agents (cheese, chocolate, etc.), pectic acid and its salts, alginic acid and its salts, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohols, and carbonating agents used in carbonated beverages.

The present invention Lactobacillus johnny JNU3402 strain or its culture filtrate can exhibit excellent acid resistance, bile resistance, cholesterol lowering effect, obesity suppression effect, etc., and has excellent stability due to strong resistance to various environmental factors. Accordingly, the Lactobacillus johnsny JNU3402 strain of the present invention, its culture medium, or its culture filtrate can be widely used in pharmaceutical compositions, health functional foods, foods, and the like.

Hereinafter, the configuration and effect of the present invention will be described in more detail by way of examples. However, the following examples are provided for illustrative purposes only to aid understanding of the present invention, and the scope and scope of the present invention are not limited thereto.

One. Lactobacillus johnsonii Isolation and Identification of JNU3402

Lactic acid bacteria were isolated using feces from infants within 2 months of age as samples. Specifically, the sample was smeared on MRS agar medium (Difco, USA) containing 0.004% of bromocresol purple by a streak plate method, followed by anaerobic conditions (5 to 15% carbon dioxide). C, and incubated for 48 hours. Among the cultured strains, strains showing yellow colonies were isolated, and excellent strains were selected by examining Gram staining, catalase and oxidase tests, gas formation, sugar utilization, and the like. The selected strain was named Lactobacillus johnsonii JNU3402.

The morphological and physiological characteristics of Lactobacillus johnsonii JNU3402 in the medium are shown in Table 1 below.

cell shape bacilli (rod) motility doesn't exist sporulation doesn't exist Gram dye positivity catalase voice Growth temperature and time 37, 20-48 hours oxygen demand facultative anaerobic

In addition, 16S rDNA sequencing was performed to identify the selected strain, and the result is shown in SEQ ID NO: 1. The nucleotide sequence analysis was commissioned by Bionics, and the sequencing results were confirmed through NCBI blast search. As a result of sequencing, strain JNU3402 of the present invention showed 99% sequence homology with 16S rDNA of Lactobacillus johnsny, and it was confirmed that the isolated strain JNU3402 was Lactobacillus johnsny. The present inventors deposited the isolated strain as described above with the accession number KCCM12892P at the Korea Microorganism Conservation Center, an international depository, on December 09, 2020.

2. Lactobacillus johnsonii Evaluation of acid resistance and bile resistance of JNU3402

The Lactobacillus johnsonii JNU3402 (Accession No. KCCM12892P) strain was subcultured in MRS broth for 18 hours, followed by centrifugation (2,000 rpm, 20 minutes), followed by repeated washing with sterile physiological saline three times and used for this evaluation.

For acid resistance evaluation, a 0.05 M sodium phosphate buffer was prepared and pepsin (Sigma Co., USA) was added to 1,000 unit/mL in MRS broth adjusted to 2.0 and 2.5 pH with HCl, respectively, and used as a medium for measuring acid resistance. did. The initial number of bacteria was suspended in 0.05M sodium phosphate buffer to about 10 ⁷ cfu/ml, and then inoculated into the medium. After culturing in a shaking incubator at 37°C for 2 hours, the number of colonies was counted to evaluate acid resistance. As a result of acid resistance evaluation, the Lactobacillus johnny JNU3402 strain of the present invention was confirmed to have excellent acid resistance by maintaining a level of 10 ⁷ even when treated at pH 2.5 for 1 hour (see Table 2).

pH Number of viable cells (CFU/mL) initial number of bacteria 4.3 x 10 ⁷ Control (pH 7.0) Bacteria count after 1 hour 4.7 x 10 ⁷ Bacteria count after 2 hours 3.4 x 10 ⁷ pH 2.5 treatment group Bacteria count after 1 hour 2.79 x 10 ⁷ Bacteria count after 2 hours 8.1 x 10 ⁶ pH 2.0 treatment group Bacteria count after 1 hour 1.42 x 10 ⁶ Bacteria count after 2 hours 5.6 x 10 ⁶

In addition, for the evaluation of bile resistance, a filter-sterilized oxgall (Difco, USA) solution was added to the MRS medium so that the oxgall content was 0.5% (w/v). The same lactic acid bacteria suspension as used in acid resistance evaluation was inoculated into the medium and cultured at 37 °C for 24 hours, followed by evaluation of the number of viable cells. As a result of the evaluation, it was confirmed that growth was possible even at a concentration of 0.5% oxgall and exhibited a high growth rate. Specifically, it was confirmed that the number of bacteria when 24 hours had elapsed after inoculation was 200% or more higher than that at the time of initial inoculation (see Table 3).

Number of viable cells after 0.5% oxgall treatment
(CFU/mL) Initial Inoculation Bacteria 1.62 x 10 ⁶ Bacteria count after 24 hours 4.1 x 10 ⁶ Bacteria count after 48 hours 5.8 x 10 ⁷

3. Lactobacillus johnsonii Evaluation of intestinal adhesion of JNU3402

Intestinal epithelial cells (HT-29) forming a monolayer were washed 5 times with PBS buffer, and RPMI 1640 medium without antibiotics was added. The JNU3402 strain was suspended in RPMI to a concentration of 1 to 2 X 10 ¹⁰ CFU/mL, then inoculated into a well-plate, and incubated at 37° C. for 2 hours in the presence of 5% CO ₂ . After the culture was completed, washing was performed three times using PBS buffer while stirring at 200 rpm for 3 minutes for removal and washing of non-adherent lactic acid bacteria. After washing was completed, 0.2% Trypsin-EDTA was dispensed to remove the attached cells, and the number of viable cells was measured by plated on MRS solid medium by continuous dilution using peptone water and cultured at 37 for 24 hours.

As an experimental control, Lactobacillus GG with high intestinal adhesion activity was used. As a result of the experiment, strain JNU3402 showed intestinal epithelial cell adhesion activity similar to that of Lactobacillus GG. However, Lactobacillus plantarum isolated from kimchi showed low intestinal adhesion (see FIG. 2).

4. Lactobacillus johnsonii Preparation of JNU3402 dead cells and their culture filtrate

One) Lactobacillus johnsonii JNU3402 dead cell (NV-LJ3402) production

Lactobacillus johnsonii JNU3402 (Accession No. KCCM12892P) strain was inoculated into MRS broth (BD, Difco Laboratories, Detroit, MI, USA) and cultured at 37° C. for 24 hours. The culture medium was adjusted to OD 600 = 1.0 x 10 ⁸ cfu/mL. . The adjusted culture was killed by heating at 80° C. for 15 minutes. It was confirmed that bacterial colonies were killed using MRS agar plates (BD, Difco Laboratories, Detroit, MI, USA).

2) Lactobacillus johnsonii Dead cell culture filtrate that does not contain JNU3402 dead cells (Lactobacillus johnsny JNU3402 dead cell conditioned medium, NV-LJ3402-CM) manufacturing

After the Lactobacillus johnsny JNU3402 strain was killed by heating the above-adjusted culture solution at 80 ° C. for 15 minutes, centrifuged at a speed of 4,000 g for 15 minutes to separate only the supernatant without dead cells, Lactobacillus johnsny JNU3402 Co., Ltd. A cell conditioned medium was prepared.

5. Lactobacillus johnsonii Weight change analysis by JNU3402 dead cells

21 6-week-old C57BL/6J experimental rats (weight 19-20 g, Central Animal Research Center, Daejeon, Korea) were acclimatized for 1 week and then normal diet (Normal diet, ND) from 7 weeks (16% of total calories from fat, LabDiet, St. Louis, MO, USA) or high-fat diet (HFD) (45% of total calories from fat, Research Diets Inc., New Brunswick, NJ, USA) to induce obesity (see Table 4).

Thereafter, 200 μl of Lactobacillus johnsny JNU3402 dead cells or PBS was orally administered to mice every day for 14 weeks. During 14 weeks of administering Lactobacillus johnsny JNU3402 dead cells or PBS, the dietary intake of each control group and experimental group was measured 3 times a week at 0, 8, 10, and 14 weeks. The dietary intake of the control group and the experimental group did not show a significant difference (Fig. 3, ND: control group 1, HFD: control group 2, HFD+NV-LJ3402: experimental group 1). As a result of measuring the body weight of the control group and the experimental group once every 2 weeks, Compared to Control 1, which is the regular diet group, the weight of Control 2, the high-fat diet group, increased by 49%. On the other hand, the body weight of Experimental Group 1, a group that ingested a high-fat diet and orally administered Lactobacillus johnsny JNU3402 dead cells, was reduced by about 10% compared to Control 2, which ingested a high-fat diet (Fig. 4, ND: Control 1, HFD: control group 2, HFD+NV-LJ3402: experimental group 1).

6. Lactobacillus johnsonii Changes in fat and liver tissue weight by JNU3402 dead cells

The weights of liver, groin white fat (iWAT), epididymal white fat (eWAT), and brown adipose tissue (BAT) in experimental group 1 to which Lactobacillus Johnsney JNU3402 dead cells were orally administered were 16, respectively, compared to control group 2, which was a high-fat diet group. %, 29%, 26%, and 30% reduction was confirmed (FIG. 5, ND: control group 1, HFD: control group 2, HFD+NV-LJ3402: experimental group 1).

7. Lactobacillus johnsonii Changes in triglycerides in tissues and blood by JNU3402 dead cells

To investigate the effect of Lactobacillus Johnsney JNU3402 dead cells on triglyceride content in blood and tissues, triglycerides were measured in each tissue using a TG quantification kit (SCG Biomax, Seoul, Korea). As a result, the triglyceride content was reduced by 49% in the blood of Experimental Group 1, which is the group orally administered with Lactobacillus Johnsney JNU3402 dead cells, compared to Control 2, which is the high-fat diet group. In addition, as a result of measuring the triglyceride content in the liver and adipose tissue, the triglyceride content was 38% and 23%, respectively, in Experimental Group 1, the group that was orally administered with Lactobacillus Johnsney JNU3402 dead cells, compared to Control 2, the high-fat diet group. was decreased (FIG. 6, ND: control group 1, HFD: control group 2, HFD+NV-LJ3402: experimental group 1).

8. Lactobacillus johnsonii Changes in blood glucose and insulin by JNU3402 dead cells

To investigate the effect of Lactobacillus Johnsney JNU3402 dead cells on the increase in blood glucose and insulin due to a high-fat diet, blood glucose levels were measured using a blood glucose meter, and insulin ELISA kit (ALPCO, Windham, NH, USA) ) was used to measure blood insulin levels. In order to measure fasting blood glucose, rats in the control group and the experimental group were maintained in a fasting state for 12 hours. Compared to Control 2, which is the group that received a high-fat diet, fasting blood glucose and postprandial blood glucose were decreased by 43% and 27%, respectively, in the rats of Experimental Group 1, which is the group orally administered with Lactobacillus johnsny JNU3402 dead cells (Fig. 7, ND: Control 1, HFD: Control 2, HFD+NV-LJ3402: Experimental Group 1). In addition, the blood insulin content was found to be 51% lower in the rats of Experimental Example 1, which is the group orally administered with Lactobacillus johnsny JNU3402 dead cells, compared to the rats in Control 2, which is the group that ate a high-fat diet (FIG. 8, ND: control group) 1, HFD: control group 2, HFD+NV-LJ3402: experimental group 1).

9. Lactobacillus johnsonii JNU3402 dead cells Changes in lipid accumulation in 3T3-L1 adipocytes according to treatment with conditioned medium

Oil-Red O staining was performed on 3T3-L1 adipocytes to confirm the effect of Lactobacillus johnsny JNU3402 dead cell culture conditioned medium on lipid accumulation in adipocytes. 3T3-L1 adipocytes when treated with Lactobacillus johnsny JNU3402 dead cells conditioned medium (NV-LJ3402-CM) for 24, 36, 48 hours compared to the condition (con) treated using heated MRS broth as a control medium of lipid accumulation was decreased by 22%, 46%, and 61%, respectively (FIG. 9). Statistical differences in lipid accumulation in adipocytes were analyzed using Students' t-test.

10. Lactobacillus johnsonii Gene expression regulation by JNU3402 dead cells or conditioned medium thereof

One) Lactobacillus johnsonii Gene expression regulation by JNU3402 dead cells

RT-qPCR was performed in the epididymis and groin white fat of mice to investigate whether or not Lactobacillus johnsny JNU3402 dead cells were orally administered to high-fat diet mice to determine whether the expression of genes involved in lipid accumulation and energy metabolism in adipose tissue was regulated. carried out.

Specifically, total RNA was isolated from 3T3-L1 cells and adipose tissue (WAT) using Trizol reagent (Invitrogen, Waltham, MA, USA), and Moloney Murine Leukemia Virus (M-MLV) Reverse Transcriptase (Promega, Madison WIS). , USA) was used to synthesize cDNA from 1 g of total RNA. RT-qPCR was performed by a known method. The results were normalized to 36B4 mRNA expression. The relative quantification of PCR products was calculated as the difference in Ct values between the target gene and the 36B4 gene using the 2 ^- ΔΔCt method. Table 5 below shows the primer sequences for PCR.

gene Sense primer (5' → 3') Antisense primer (5' → 3') fatty acid synthase (FAS) AGATCCTGGAACGAGAACACGAT
(SEQ ID NO: 2) GAGACGTGTCACTCCTGGACTTG
(SEQ ID NO: 3) acetyl-CoA carboxylase (ACC) GTATGTTCGAAGGGCTTACATTG
(SEQ ID NO: 4) TGGGCAGCATGAACTGAAATT
(SEQ ID NO: 5) sterol regulatory element-binding protein-1c (SREBP1c) ACTGTGACCTCACAGGTCCA
(SEQ ID NO: 6) GGCAGTTTGTCTGTGTCCACA
(SEQ ID NO: 7) acetyl-CoA oxidase (ACOX) TCGAGGCTTGGAAACCACTG
(SEQ ID NO: 8) TCGAGTGATGAGCTGAGCC
(SEQ ID NO: 9) carnitine palmitoyltransferase 1 (CPT1) ACTCCTGGAAGAAGAAGTTC
(SEQ ID NO: 10) TAGGGTCCGATTGATCTTTG
(SEQ ID NO: 11) peroxisome proliferator-activated receptor gamma coactivator 1-α (PGC1α) GAGACTTTGGAGGGCCAGCA
(SEQ ID NO: 12) CGCCATCCCTTAGTTCACTGG
(SEQ ID NO: 13) uncoupling protein 1 (UCP1) GGAGGTGTGGCAGTGTTC
(SEQ ID NO: 14) TCTGTGGTGGCTATAACTCTG
(SEQ ID NO: 15) PPARγ GAAGACCACTCGCATTCCTT
(SEQ ID NO: 16) GAAGGTTCTTCATGAGGCCTG
(SEQ ID NO: 17) Cidea ATCACAACTGGCCTGGTTACG
(SEQ ID NO: 18) TACTACCCGGTGTCCATTTCT
(SEQ ID NO: 19) 36B4 AGATGCAGCAGATCCGCAT
(SEQ ID NO: 20) ATATGAGGCAGCAGTTTCTCCAG
(SEQ ID NO: 21) D-loop AATCTACCATCCTCCGTG
(SEQ ID NO: 22) GACTAATGATTCTTCACCGT
(SEQ ID NO: 23) GAPDH GTTGTCTCCTGCGACTTCA
(SEQ ID NO: 24) GGTGGTCCAGGGTTTCTTA
(SEQ ID NO: 25)

As a result, compared to Control 2, which is a high-fat diet group, fatty acid synthase (FAS), acetyl, which are genes involved in lipid accumulation, in the epididymal adipose tissue (eWAT) of Experimental Group 1, which is the group orally administered with Lactobacillus Johnsney JNU3402 dead cells, compared to Control 2, which is the high-fat diet group. The expression of -CoA carboxylase (ACC) and sterol regulatory element-binding protein-1c (SREBP1c) was 40%, 32%, and 51% lower, respectively. Conversely, the expression of acetyl-CoA oxidase (ACOX), carnitine palmitoyltransferase 1 (CPT1), and peroxisome proliferator-activated receptor gamma coactivator 1-α (PGC1α) genes involved in fatty acid beta oxidation was higher than in Control 2, a high-fat diet group. Lactobacillus johnsny JNU3402 dead cells showed 1.8 times, 1.9 times, and 2.3 times higher values in Experimental Group 1, which is the group orally administered (FIG. 10, HFD: Control 2, HFD+NV-LJ3402: Experimental Group 1).

In addition, compared with Control 2, which is a high-fat diet group, uncoupling protein 1 (UCP1), a gene involved in adipocyte browning in the groin white fat (iWAT) of Experimental Group 1, which is a group orally administered with Lactobacillus johnsny JNU3402 dead cells, Expressions of PPARγ and Cidea were 2.6-fold, 4.5-fold, and 2.2-fold higher, respectively (FIG. 10, HFD: control group 2, HFD+NV-LJ3402: experimental group 1).

2) Controlling gene expression by treatment with dead cells conditioned medium

Similar to the results of expression control of genes involved in lipid accumulation and energy metabolism in adipose tissue by the above-mentioned JNU3402 dead cells, 3T3-L1 adipocytes were treated with Lactobacillus Johnsney JNU3402 dead cells conditioned medium (NV-LJ3402-CM). Compared to the condition without (con), the expression of FAS, ACC, and SREBP1c, which are genes involved in lipid accumulation, was 50% and 51%, respectively, when Lactobacillus johnsny JNU3402 dead cell conditioned medium (NV-LJ3402-CM) was treated. , decreased by 48%, and the expression of ACOX, CPT1, and PGC1α, genes involved in fatty acid beta oxidation, increased by 3.4-fold, 2-fold, and 2-fold. In addition, the expression of UCP1, PPARγ, Cidea, which are genes involved in adipocyte browning, increased 2.3-fold, 1.7-fold, and 1.9-fold ( FIG. 11 ). Statistical differences in gene expression were analyzed using Students’ t-test.

11. Lactobacillus johnsonii Increased transcriptional activity of PPARγ by JNU3402 dead cell conditioned medium

PPARγ is a major regulator of lipid metabolism in adipose tissue and plays a role in regulating the activity and expression of genes involved in energy metabolism. To investigate the effect of Lactobacillus johnsny JNU3402 dead cells conditioned medium on PPARγ transcriptional activity, a reporter gene assay was performed in HEK293T cells.

As a result, when a reporter containing only the PPARγ response element (PPRE) was treated with the Lactobacillus johnsny JNU3402 dead cells conditioned medium, compared to the condition not treated with the Lactobacillus johnsny JNU3402 dead cells conditioned medium (PPARγ in FIG. 12), PPARγ of the transcriptional activity was increased by about 2.3 times, and the activities of the UCP1 promoter and PPARγ promoter, which are target genes, were increased by 2 times and 2.2 times, respectively (FIG. 12).

In order to investigate whether the increase in the expression of the target gene of PPARγ by the Lactobacillus Johnsney JNU3402 dead cell conditioned medium occurs through the increase in PPARγ activity, 3T3-L1 adipocytes were treated with Lactobacillus Johnsney JNU3402 dead cell conditioned medium together with the PPARγ antagonist GW9662. was treated to examine the expression of the PPARγ target gene. As a result, when the Lactobacillus Johnsney JNU3402 dead cell conditioned medium was treated (FIG. 13 NV LJ3402-CM) compared to the case where the Lactobacillus Johnsney JNU3402 dead cell conditioned medium was not treated (FIG. 13 con), PPARγ, UCP1, ACOX Expression was increased by about 2.2-fold, 2.1-fold, and 3.1-fold, respectively ( FIG. 13 ). On the other hand, in the condition (GW9662 + NV LJ3402-CM of FIG. 13) treated with Lactobacillus johnsny JNU3402 dead cell conditioned medium with GW9662, a PPARγ antagonist, the expression of all three genes was reduced (FIG. 13). According to the above results, it is considered that the expression of genes related to energy metabolism by the Lactobacillus johnsny JNU3402 dead cell conditioned medium is regulated through the PPARγ pathway.

12. Lactobacillus johnsonii Changes in mitochondrial content and activity by JNU3402 dead cells or their conditioned medium

Energy metabolism in adipocytes occurs mainly in mitochondria, and thermogenesis in mitochondria is associated with increased UCP1 expression. To measure mitochondrial copy number and activity by Lactobacillus johnsny JNU3402 dead cells in adipose tissue and 3T3-L1 adipocytes, mitochondrial DNA content was measured using qPCR, and citrate synthase (CS) activity assay kit (BioVision, Milpitas) , CA, USA) was used to measure mitochondrial activity. As a result, the mitochondrial DNA copy number in the groin and epididymal adipose tissue of the experimental mice increased 1.6 times and 1.3 times in Experimental Group 1, the group treated with Lactobacillus johnsny JNU3402 dead cells, respectively, compared to Control 2, the high-fat diet group. The activity was increased 2.9-fold and 2.2-fold, respectively (FIG. 14, HFD: control group 2, HFD+NV-LJ3402: experimental group 1).

In addition, as a result of treating 3T3-L1 adipocytes with Lactobacillus johnsny JNU3402 dead cells conditioned medium (FIG. 14 NV-LJ3402-CM), the mitochondrial DNA copy number was increased by 1.9 times compared to the control group (FIG. 14 con), and CS activity was increased by 4.3 times (Fig. 14).

13. Lactobacillus johnsonii Increased rectal temperature by JNU3402 dead cells

The rectal temperature of the mice was measured to determine whether a thermogenic response was induced due to the increase in mitochondrial function and UCP1 expression by Lactobacillus johnsny JNU3402 dead cells. Control 2, a high-fat diet rat group, and Experimental Group 1, a rat group orally administered with Lactobacillus johnsny JNU3402 dead cells, were exposed to 4 ° C. It was confirmed that the rectal temperature of the mice orally administered Lactobacillus johnsny JNU3402 was higher than the rectal temperature of the mice on the high-fat diet (FIG. 15, HFD: control group 2, HFD+NV-LJ3402: experimental group 1).

Korea Microorganism Conservation Center (Overseas) KCCM12892P 20201209

<110> INDUSTRY FOUNDATION OF CHONNAM NATIONAL UNIVERSITY <120> Lactobacillus johnsonii JNU3402 strain and food comprising the same <130> 20P11055 <160> 25 <170> KoPatentIn 3.0 <210> 1 <211> 1443 <212> DNA <213> Artificial Sequence <220> <223> JNU3402 <400> 1 gcagtcgagc gagcttgcct agatgatttt ggtgcttgca ctaaatgaaa ctagatacaa 60 gcgagcggcg gacgggtgag taacacgtgg gtaacctgcc caagagactg ggataacacc 120 tggaaacaga tgctaatacc ggataacaac actagacgca tgtctagagt ttgaaagatg 180 gttctgctat cactcttgga tggacctgcg gtgcattagc tagttggtaa ggtaacggct 240 taccaaggca atgatgcata gccgagttga gagactgatc ggccacattg ggactgagac 300 acggcccaaa ctcctacggg aggcagcagt agggaatctt ccacaatgga cgaaagtctg 360 atggagcaac gccgcgtgag tgaagaaggg tttcggctcg taaagctctg ttggtagtga 420 agaaagatag aggtagtaac tggcctttat ttgacggtaa ttacttagaa agtcacggct 480 aactacgtgc cagcagccgc ggtaatacgt aggtggcaag cgttgtccgg atttattggg 540 cgtaaagcga gtgcaggcgg ttcaataagt ctgatgtgaa agccttcggc tcaaccggag 600 aattgcatca gaaactgttg aacttgagtg cagaagagga gagtggaact ccatgtgtag 660 cggtggaatg cgtagatata tggaagaaca ccagtggcga aggcggctct ctggtctgca 720 actgacgctg aggctcgaaa gcatgggtag cgaacaggat tagataccct ggtagtccat 780 gccgtaaacg atgagtgcta agtgttggga ggtttccgcc tctcagtgct gcagctaacg 840 cattaagcac tccgcctggg gagtacgacc gcaaggttga aactcaaagg aattgagggg 900 ggcccgcaca agcggtggag catgtggttt aattcgaagc aacgcgaaga accttaccag 960 gtcttgacat ccagtgcaaa cctaagagat taggtgttcc cttcggggac gctgagacag 1020 gtggtgcatg gctgtcgtca gctcgtgtcg tgagatgttg ggttaagtcc cgcaacgagc 1080 gcaacccttg tcattagttg ccatcattaa gttgggcact ctaatgagac tgccggtgac 1140 aaaccggagg aaggtgggga tgacgtcaag tcatcatgcc ccttatgacc tgggctacac 1200 acgtgctaca atggacggta caacgagaag cgaacctgcg aaggcaagcg gatctcttaa 1260 agccgttctc agttcggact gtaggctgca actcgcctac acgaagctgg aatcgctagt 1320 aatcgcggat cagcacgccg cggtgaatac gttcccgggc cttgtacaca ccgcccgtca 1380 caccatgaga gtctgtaaca cccaaagccg gtgggataac ctttatagga gtcagccgtc 1440 taa 1443 <210> 2 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> fatty acid synthase sense primer <400> 2 agatcctgga acgagaacac gat 23 <210> 3 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> fatty acid synthase antisense primer <400> 3 gagacgtgtc actcctggac ttg 23 <210> 4 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> acetyl-CoA carboxylase sense primer <400> 4 gtatgttcga agggcttaca ttg 23 <210> 5 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> acetyl-CoA carboxylase antisense primer <400> 5 tgggcagcat gaactgaaat t 21 <210> 6 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> sterol regulatory element-binding protein-1c sense primer <400> 6 actgtgacct cacaggtcca 20 <210> 7 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> sterol regulatory element-binding protein-1c antisense primer <400> 7 ggcagtttgt ctgtgtccac a 21 <210> 8 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> acetyl-CoA oxidase sense primer <400> 8 tcgaggcttg gaaaccactg 20 <210> 9 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> acetyl-CoA oxidase antisense primer <400> 9 tcgagtgatg agctgagcc 19 <210> 10 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> carnitine palmitoyltransferase 1 sense primer <400> 10 actcctggaa gaagaagttc 20 <210> 11 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> carnitine palmitoyltransferase 1 antisense primer <400> 11 tagggtccga ttgatctttg 20 <210> 12 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> peroxisome proliferator-activated receptor gamma coactivator 1-alpha sense primer <400> 12 gagactttgg aggccagca 19 <210> 13 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> peroxisome proliferator-activated receptor gamma coactivator 1-alpha antisense primer <400> 13 cgccatccct tagttcactg g 21 <210> 14 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> uncoupling protein 1 sense primer <400> 14 ggaggtgtgg cagtgttc 18 <210> 15 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> uncoupling protein 1 antisense primer <400> 15 tctgtggtgg ctataactct g 21 <210> 16 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> PPAR gamma sense primer <400> 16 gaagaccact cgcattcctt 20 <210> 17 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> PPAR gamma antisense primer <400> 17 gaaggttctt catgaggcct g 21 <210> 18 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Cidea sense primer <400> 18 atcacaactg gcctggttac g 21 <210> 19 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Cidea antisense primer <400> 19 tactacccgg tgtccatttc t 21 <210> 20 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> 36B4 sense primer <400> 20 agatgcagca gatccgcat 19 <210> 21 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> 36B4 antisense primer <400> 21 atatgaggca gcagtttctc cag 23 <210> 22 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> D-loop sense primer <400> 22 aatctaccat cctccgtg 18 <210> 23 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> D-loop antisense primer <400> 23 gactaatgat tcttcaccgt 20 <210> 24 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> GAPDH sense primer <400> 24 gttgtctcct gcgacttca 19 <210> 25 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> GAPDH antisense primer <400> 25 ggtggtccag ggtttctta 19

Claims (6)

Lactobacillus johnsonii ( Lactobacillus johnsonii ) JNU3402 (Accession No. KCCM12892P) strain.
The method according to claim 1, The strain will have a nucleotide sequence encoding the 16S rRNA shown in SEQ ID NO: 1, the strain.
The strain according to claim 1, characterized in that it has acid resistance and bile resistance.
Lactobacillus johnsonii ( Lactobacillus johnsonii ) Food containing JNU3402 (Accession No. KCCM12892P) strain or the culture filtrate of the strain as an active ingredient.
The food according to claim 4, wherein the strain, the culture solution of the strain, or the culture filtrate of the strain has acid resistance and bile resistance.
The method according to claim 4, wherein the food is ice cream, milk, soy milk, yogurt, cheese, meat, sausage, bread, chocolate, candy, confectionery, pizza, ramen, gum, soup, beverage, tea, drink, alcoholic beverage, and stick packaging At least one selected from the group consisting of lactic acid bacteria preparations, food.

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