KR101545551B1 - The composition containing combination of 7 probiotics which have efficacy preventing from insulin resistance which cause type 2 diabetes mellitus as a effector component - Google Patents

Abstract

The present invention Bifidobacterium ronggeom (Bifidobacterium longum) HY8005, Bifidobacterium Infante Tees (Bifidobacterium infantis) HY8401, Bifidobacterium lactis (Bifidobacterium lactis) HY8101, Bifidobacterium breve (Bifidobacterium breve) HY8201, The present invention relates to a composition comprising seven kinds of lactic acid bacterial strain having an insulin resistance improving effect consisting of Lactobacillus gasseri HY7023, Lactobacillus acidophilus HY7037 and Lactobacillus casei HY7212 as active ingredients A food composition, a fermented milk, a beverage, a health functional food, and the like, which have an effect of preventing or improving diabetes through improvement of insulin resistance.

Description

[0001] The present invention relates to a composition comprising seven kinds of lactic acid bacterial strains having an insulin resistance improving effect as an active ingredient. [0002] The present invention relates to a composition containing 7 kinds of probiotics,

The present invention relates to a composition containing a lactic acid complex strains of seven species having the insulin resistance-improving effect as an active ingredient, and more particularly Bifidobacterium ronggeom (Bifidobacterium longum) HY8005, Bifidobacterium Infante tooth (Bifidobacterium infantis ) HY8401, Bifidobacterium lactis HY8101, Bifidobacterium breve HY8201, Lactobacillus gasseri HY7023, Lactobacillus acidophilus HY7037, and Lactobacillus acidophilus And a composition comprising seven kinds of lactic acid bacterial strain having an insulin resistance improving effect consisting of Lactobacillus casei HY7212 as an active ingredient.

Diabetes mellitus is a typical chronic disease characterized by hyperglycemia, which is caused by a deficiency of insulin hormone or insulin resistance produced in the beta cells of the pancreas, and both of these defects. Such diabetes can be divided into insulin dependent diabetes mellitus and non insulin dependent diabetes mellitus which occurs in insulin resistance and insulin secretory damage.

On the other hand, insulin resistance, which is known to be a major cause of non-insulin-dependent diabetes, is a phenomenon in which insulin moves to target tissues through blood, but does not cause a reaction in target tissues. It is a symptom of loss of fat homeostasis. Therefore, those who show insulin resistance have a high blood sugar level and a high level of insulin in their blood. If these symptoms continue, glucose absorption does not occur in peripheral tissues such as adipose tissue or muscle tissue, and glucose production continues to occur in the liver. These symptoms continue to persist insulin resistance and obesity, fatty liver, diabetes, and so on. In recent years, many researches have been made to solve the cause of such insulin resistance and to improve the insulin resistance.

Tumor necrosis factor-α (TNF-α) is a multipurpose cytokine involved in immune regulation, death of apoptotic cells and lysis of tumor cells. Recent studies have shown that TNF-α induces insulin resistance by inhibiting insulin signal transduction. TNF-α promotes protein phosphorylation of IRS-1 (Ser). This results in the inhibition of protein phosphorylation of insulin-mediated IRS-1 (Tyr), which in turn affects the signal pathway driven by insulin and ultimately affects GLUT4, which feeds glucose . These TNF-α are expressed in adipose tissue and muscle tissue, and are also known to be expressed in animal models involved in obesity and insulin resistance. On the other hand, PPAR-γ and PGC-1α have been shown to be low in the muscle and adipose tissues of obesity and diabetic animal models through many existing papers, and they are also implicated in insulin sensitivity, and these have also been shown to be strongly related to insulin resistance.

On the other hand, lactic acid bacterium, which exists in many fermented foods such as man's cabbage, kimchi and cheese, and milk and milk, plays a role of decomposing fibrous and complex proteins into important nutrients while symbiosis with the human digestive system. To inhibit the growth of harmful bacteria such as E. coli and Chlostridum sp., And to improve diarrhea and constipation, as well as to play a role in vitamin synthesis and blood cholesterol lowering. Especially lactic acid bacteria can bind strongly to the mucous membrane and epithelial cells of the intestines, which helps a lot of the action.

The present inventors have found a total of seven kinds of lactic acid complex strains in age 7 days fetus fecal a Lactobacillus (Lactobacilus sp.) And Bifidobacterium (Bifidobacterium sp.) Isolated from the previous having the improved insulin resistance and glucose control efficacy Thereby completing the present invention.

The present invention Bifidobacterium ronggeom (Bifidobacterium longum) HY8005, Bifidobacterium Infante Tees (Bifidobacterium infantis) HY8401, Bifidobacterium lactis (Bifidobacterium lactis) HY8101, Bifidobacterium breve (Bifidobacterium breve) HY8201, The present invention provides a method for preventing or preventing diabetes by improving insulin resistance by containing a complex strain of Lactobacillus gasseri HY7023, Lactobacillus acidophilus HY7037 and Lactobacillus casei HY7212 as an active ingredient, A pharmaceutical composition for therapeutic use, a food composition for preventing diabetes, a fermented milk for preventing diabetes, a functional beverage for preventing diabetes, and a health functional food for the prevention of diabetes.

In order to achieve the above object, the present invention provides Bifidobacterium ronggeom (Bifidobacterium longum) HY8005, Bifidobacterium Infante tooth (Bifidobacterium infantis) HY8401, Bifidobacterium lactis (Bifidobacterium lactis) HY8101, Bifidobacterium The present invention relates to a method for producing a lactic acid bacterium which contains as an active ingredient a lactic acid bactericidal strain consisting of Bifidobacterium breve HY8201, Lactobacillus gasseri HY7023, Lactobacillus acidophilus HY7037 and Lactobacillus casei HY7212 The present invention provides a pharmaceutical composition for preventing or treating diabetes by improving insulin resistance, a food composition for preventing diabetes, a fermented milk for preventing diabetes, a functional beverage for diabetes prevention, and a diabetic health functional food.

In particular, the pharmaceutical composition for preventing or treating diabetes through improvement of insulin resistance comprising 7 kinds of the lactic acid bacterial strain according to the present invention as an active ingredient is characterized in that GLUT4 (glucose transporter type 4) gene, PPAR-gamma (Peroxisome proliferator-activated receptor gene and PGC-1α (Peroxisome proliferator-activated receptor gamma coactivator 1-alpha) gene to increase insulin resistance by increasing glucose uptake in muscle cells.

Particularly, the pharmaceutical composition for preventing or treating diabetes by improving the insulin resistance by containing the seven kinds of the lactic acid bacterial strain of the present invention as an active ingredient is a composition for preventing or treating diabetes mellitus in a diabetic animal model induced by STZ (Streptozotocin) , Insulin resistance by promoting glucose uptake in the liver and muscle tissue and glycogen synthesis by increasing the amount of glucose in the blood due to dietary intake and abnormalities of glycated hemoglobin (HbA1c) and decreased insulin secretion. .

Hereinafter, the present invention will be described in detail.

Bifidobacterium < RTI ID = 0.0 > longum ) HY8005 (Accession No .: KCTC 12514BP), Bifidobacterium infantis) HY8401 (accession number: KCTC 12419BP), Bifidobacterium lactis (Bifidobacterium lactis ) HY8101 (Accession No .: KCTC 12418BP), Bifidobacterium breve ) HY8201 (Accession No .: KCTC 12515BP), Lactobacillus gasseri) HY7023 (Accession No: KCTC 12512BP), Lactobacillus ash FIG pillar's (Lactobacillus acidophilus) HY7037 (Accession No: KCTC 12409BP) and Lactobacillus casei (Lactobacillus casei) HY7212 (Accession No: KCTC 12513BP) is the age of 7 days Were isolated from the feces of newborn infants and identified as a single strain.

Regarding the availability of glucose in muscle cells, GLUT4 is closely involved in the insulin receptor (receptor) in muscle cells and the insulin secreted by beta pancreatic cells. Sugars like glucose in the blood are used in various organs such as adipocytes and muscle tissues. In the case of muscle cells, the action of insulin causes an insulin signal due to the action of insulin receptor. Finally, GLUT4 expression and translocation in muscle cells increase glucose in the blood into the muscle, which makes it possible to synthesize and use glycogen and the like. However, when a fatty acid such as TNF-α or palmitic acid acts in this process, an abnormality in the insulin signaling mechanism results in an abnormality in the expression and translocation of GLUT4, resulting in the inflow of glucose This does not work well.

In the present invention, L6 skeletal muscle cells, which are muscle cells of normal rat, or L6 skeletal muscle cells, which are rat muscle cells induced by insulin resistance by TNF- Seven kinds of lactic acid bacteria (Bifidobacterium longum sword HY8005, Bifidobacterium infantis HY8401, Bifidobacterium lactis HY8101, Bifidobacterium breve HY8201, Lactobacillus ashdo filler LYU-Bacillus gasseri HY7023, Lactobacillus casei HY7212) were selected.

In addition, when blood insulin reaches muscles and adipocytes, factors that contribute to insulin sensitivity such as PPAR-γ and PGC-1α are increased. When insulin resistance is induced, their expression decreases and expression of GLUT4 and translocation translocation.

In the present invention, seven types of lactic acid bacteria (Bifidobacterium sp.) Which increase the expression of PPAR-γ and PGC-1α using L6 skeletal muscle cells, which are the muscle cells of the rat induced by insulin resistance by TNF- Bifidobacterium lepthitis HY8101, Bifidobacterium breve HY8201, Lactobacillus ashidophilus HY7037, Lactobacillus gasseri HY7023, Lactobacillus casei HY7212) were screened Respectively.

On the other hand, in STZ (Streptozotocin) -injected animal models, STZ causes pancreatic beta cells to be destroyed and normal insulin secretion is inhibited. This leads to insulin-dependent diabetes mellitus, which leads to increased diabetes mellitus and increases the amount of glucose in the blood as well as increased glycated hemoglobin (HbA1c) due to increased glucose. STZ (Streptozotocin) causes a sudden decrease in body weight and, in contrast, a decrease in body weight results in polyuria and multiforme.

In the present invention, the concentration of glucose and glycosylated hemoglobin (HbA1c) increased due to STZ (Streptozotocin) is decreased, and the abnormal weight, tissue weight, 7 kinds of lactic acid bacteria (Bifidobacterium longum HY8005, Bifidobacterium infantis HY8401, Bifidobacterium lactis HY8101, Bifidobacterium breve HY8201, Lactobacillus acidophilus HY7037, Lactobacillus gasseri HY7023, Lactobacillus casei HY7212).

On the other hand, Bifidobacterium ronggeom (Bifidobacterium longum) HY8005, Bifidobacterium Infante tooth (Bifidobacterium infantis) HY8401, Bifidobacterium lactis (Bifidobacterium lactis) HY8101, Bifidobacterium breve (Bifidobacterium breve) of the present invention A method for producing an insulin resistance-resistant insulin resistance product, which comprises, as an active ingredient, a complex of a lactic acid bacterium consisting of HY8201, Lactobacillus gasseri HY7023, Lactobacillus acidophilus HY7037 and Lactobacillus casei HY7212 The pharmaceutical composition for the prevention or treatment of diabetes through the improvement can be used alone or in formulations such as tablets, capsules and the like according to a method commonly used in pharmaceuticals together with excipients used for pharmaceuticals.

For 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 the route of administration, the age, sex and weight of the patient, the health condition, and the 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 for long-term administration for preventive purposes.

Further, Bifidobacterium ronggeom (Bifidobacterium longum) HY8005, Bifidobacterium Infante tooth (Bifidobacterium infantis) HY8401, Bifidobacterium lactis (Bifidobacterium lactis) HY8101, Bifidobacterium breve (Bifidobacterium breve) of the present invention A method for producing an insulin resistance-resistant insulin resistance product, which comprises, as an active ingredient, a complex of a lactic acid bacterium consisting of HY8201, Lactobacillus gasseri HY7023, Lactobacillus acidophilus HY7037 and Lactobacillus casei HY7212 The diabetic food composition for improving diabetes can be used as food, food additive, beverage, beverage additive, fermented milk, health functional food, and the like. When used as a food, a food additive, a beverage, a beverage additive, or a health functional food, it is possible to use various foods, fermented milk, meat, beverage, chocolate, snack, confectionery, pizza, ramen, other noodles, gums, ice cream, But may be, but not limited to, a combination, a mainstream and other health functional food.

In particular, Bifidobacterium ronggeom (Bifidobacterium longum) HY8005, Bifidobacterium Infante Tees (Bifidobacterium infantis) HY8401, Bifidobacterium lactis (Bifidobacterium lactis) HY8101, Bifidobacterium breve (Bifidobacterium breve) HY8201, Lactobacillus The present invention relates to a method for improving insulin resistance, which comprises, as an active ingredient, a complex of a lactic acid bacterium comprising Lactobacillus gasseri HY7023, Lactobacillus acidophilus HY7037 and Lactobacillus casei HY7212 The fermented milk for diabetes prevention is prepared by uniformly mixing the 7 kinds of the above-mentioned lactic acid bacterial strain, the culture liquid of lactic acid bacteria, the ultrasonic disruption liquid of lactic acid bacteria and the mixed fruit syrup at a certain ratio at 150 bar, do.

In particular, Bifidobacterium ronggeom (Bifidobacterium longum) HY8005, Bifidobacterium Infante Tees (Bifidobacterium infantis) HY8401, Bifidobacterium lactis (Bifidobacterium lactis) HY8101, Bifidobacterium breve (Bifidobacterium breve) HY8201, Lactobacillus The present invention relates to a method for improving insulin resistance, which comprises, as an active ingredient, a complex of a lactic acid bacterium comprising Lactobacillus gasseri HY7023, Lactobacillus acidophilus HY7037 and Lactobacillus casei HY7212 The functional beverage for the prevention of diabetes was prepared by mixing mixed juice syrup, 7 types of lactic acid bacteria complex and water at a constant ratio, homogenizing at 150 bar, cooling to below 10 ° C, packaging in a small bottle container such as glass bottles and plastic bottles, .

In particular, Bifidobacterium ronggeom (Bifidobacterium longum) HY8005, Bifidobacterium Infante Tees (Bifidobacterium infantis) HY8401, Bifidobacterium lactis (Bifidobacterium lactis) HY8101, Bifidobacterium breve (Bifidobacterium breve) HY8201, Lactobacillus The present invention relates to a method for improving insulin resistance, which comprises, as an active ingredient, a complex of a lactic acid bacterium comprising Lactobacillus gasseri HY7023, Lactobacillus acidophilus HY7037 and Lactobacillus casei HY7212 The diabetic health functional food contains 7 kinds of the lactic acid bacteria combined strains as well as vitamin B ₁ , B ₂ , B ₅ , B ₆ , E and acetic acid ester, nicotinic acid amide and oligosaccharide as nutritional supplement components And other food additives may be added.

As described above, the present invention provides a pharmaceutical composition, food composition, fermented milk, beverage, health functional food, and the like that prevent or ameliorate the metabolic syndrome including seven kinds of lactic acid bacterial strain having an effect of improving insulin resistance as an active ingredient And the like.

FIG. 1 is a graph showing glucose uptake by normal L6 muscle cell treatment of 7 kinds of lactic acid bacteria complex strains. FIG.
FIG. 2 is a graph showing glucose uptake by L6 muscle cell treatment induced by insulin resistance of seven kinds of lactic acid bacteria combined strains.
FIG. 3 is a graph showing changes in expression of GLUT4 gene involved in glucose uptake by L6 muscle cell treatment induced by insulin resistance in seven kinds of lactic acid bacteria complex strains.
Fig. 4 is a graph showing changes in PPAR-y gene expression involved in insulin sensitivity by L6 muscle cell treatment induced by insulin resistance of seven kinds of lactic acid bacteria combined strains.
FIG. 5 is a graph showing changes in PGC-1 alpha gene expression involved in insulin sensitivity by L6 muscle cell treatment induced by insulin resistance of seven complex strains of lactic acid bacteria.
6 is a graph showing changes in body weight of mice in which diabetes was induced by STZ of seven kinds of lactic acid bacteria complex strains.
FIG. 7 is a graph showing changes in dietary intake of mice in which diabetes was induced by STZ of seven kinds of lactic acid bacteria combined strains.
8 is a graph showing liver and epididymal fat weights of mice induced by diabetes mellitus by STZ of seven kinds of lactic acid bacteria complex strains.
FIG. 9 is a graph showing fasting glucose values of mice in which diabetes was induced by STZ of 7 kinds of lactic acid bacteria combined strains.
FIG. 10 is a graph showing the glycated hemoglobin (HbA1c) levels of mice induced by diabetes mellitus by STZ of seven kinds of lactic acid bacteria complex strains.
11 is a graph showing blood glucose changes with time of experimental animals after oral administration of glucose to mice in which diabetes was induced by STZ of 7 kinds of lactic acid bacteria complex strains.
FIG. 12 is a graph showing the synthesis of glycogen in muscle tissue of seven kinds of lactic acid bacteria complex strains, and analysis of glucose uptake and insulin sensitivity related genes.
FIG. 13 is a graph showing the synthesis of glycogen in hepatic tissues and the analysis of gene related to glucose uptake synthesis, glucose uptake and insulin sensitivity-related genes of seven kinds of lactic acid bacteria complex strains.

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 >

Isolation and identification of seven strains of lactic acid bacteria

1-1. Bifidobacterium Infante Tees (Bifidobacterium infantis) Separation and Identification of HY8401

In order to isolate the new strain according to the present invention, the feces collected from the neonate 7 days before birth were collected, and 0.1 g of the collected neonatal feces was decanted into the anaerobic dilution, and the mixture was diluted with a BL solid medium containing 1.5% agar After culturing for 48 hours under anaerobic condition at 37 ° C, colony was taken as a loop and inoculated on fresh BL solid medium and purified. The purely isolated colonies were inoculated into BL liquid medium and cultured at 37 占 폚 for 24 hours.

The isolated strains were observed by microscopic observation through Crystal Violet stain. DNA was isolated and identified by Bifidobacterial 16S rRNA analysis (Im3, Im26). Blast search (http: //blast.ncbi. nlm.nih.gov/Blast.cgi?PAGE_

TYPE = BlastSearch & BLAST_SPEC = MicrobialGenomes) are shown in Table 1 below.

As shown in Table 1 above, the 16S rRNA strain of the new strain of the present invention was identified as Bifidobacterium infantisBifidobacterium longum subsp. infantis) With the highest homology with ATCC 15697 and Bifidobacterium infantis (Bifidobacterium infantis) Showed the highest molecular phylogenetic relationship with the Bifidobacterium infantisBifidobacterium infantis) HY8401, and deposited it on June 10, 2013 with the Korea Gene Expression Recovering Gene Bank (Accession No .: KCTC 12419BP).

The characteristics of Bifidobacterium infantis HY8401 according to the present invention are as follows.

1) Types of bacteria

The characteristics of the bacteria when cultured under anaerobic conditions for 2 days at 37 ° C in Biel (BL) agar plate medium

① Cell shape: amorphous hepatoblastic

② Mobility: None

③ Spore forming ability: None

④ Gram staining: positive

2) Physiological properties

① Growth temperature: Growth possible Growth temperature 15 ~ 42 ℃

            Optimum growth temperature 36 ~ 38 ℃

② Growth pH: Growable Growth pH 4.5 ~ 8.0

           Optimum pH 6.5 ~ 7.0

③ Effect on oxygen: anaerobic

3) Catalase: -

4) Whether gas is formed: -

5) Growth at 15 ℃: -

6) Growth at 45 ° C: +

7) Indole production: -

8) Lactic acid production: +

9) Fermentation experiments and identification using API 20 A kit from Biomerieux

1-2. Bifidobacterium lactis (Bifidobacterium lactis) Separation and Identification of HY8101

In order to isolate the new strain according to the present invention, the feces collected from the neonate 7 days before birth were collected, and 0.1 g of the collected neonatal feces was decanted into the anaerobic dilution, and the mixture was diluted with a BL solid medium containing 1.5% agar After culturing at 37 ° C in anaerobic condition for 48 hours, colony was taken as a loop and inoculated into fresh BL solid medium and purified. The purely isolated colonies were inoculated into BL liquid medium and cultured at 37 占 폚 for 24 hours.

The isolated strains were observed by microscopic observation through Crystal Violet stain. DNA was isolated and identified by Bifidobacterial 16S rRNA analysis (Im3, Im26). Blast search (http: //blast.ncbi. nlm.nih.gov/Blast.cgi?PAGE_

TYPE = BlastSearch & BLAST_SPEC = MicrobialGenomes) are shown in Table 2 below.

As can be seen from the above Table 2, it was confirmed that the 16S rRNA strain of the new strain of the present invention was 100% identical to Bifidobacterium lactis . As a result, the inventors found that the Bifidobacterium lactis ( Bifidobacterium lactis ) HY8101, and deposited on June 10, 2013 with the Korean Genealogy Saving Gene Bank (Accession No .: KCTC 12418BP).

The characteristics of Bifidobacterium lactis HY8101 according to the present invention are as follows.

1) Types of bacteria

The characteristics of the bacteria when cultured under anaerobic conditions for 2 days at 37 ° C in Biel (BL) agar plate medium

① Cell shape: amorphous hepatoblastic

② Mobility: None

③ Spore forming ability: None

④ Gram staining: positive

2) Physiological properties

① Growth temperature: Growth possible Growth temperature 15 ~ 42 ℃

            Optimum growth temperature 36 ~ 38 ℃

② Growth pH: Growable Growth pH 4.5 ~ 8.0

           Optimum pH 6.5 ~ 7.0

③ Effect on oxygen: anaerobic

3) Catalase: -

4) Whether gas is formed: -

5) Growth at 15 ℃: -

6) Growth at 45 ° C: +

7) Indole production: -

8) Lactic acid production: +

9) Fermentation experiments and identification using API 20 A kit from Biomerieux

1-3. Lactobacillus Ash's also a filler (Lactobacillus acidophilus) Isolation and Identification of HY7037

In order to isolate the new strain according to the present invention, the feces collected from the neonate 7 days before birth were collected, and 0.1 g of the feces of the collected neonate was decanted into the exhalation dilution to obtain an MRS ), Cultured at 37 ° C for 48 hours, colony was taken as a loop and inoculated on fresh MRS solid medium and purified. The purely isolated colonies were inoculated into the MRS liquid medium and cultured at 37 DEG C for 24 hours.

Table 5 shows the results of sugar fermentation experiments using an API 50 CH kit from Biomerieux to identify the newly isolated strains.

As can be seen in the following Table 5, it was confirmed that the new strain of the present invention had 100% agreement with the sugar fermentation test of Lactobacillus acidophilus as a result of sugar fermentation of the present strain, and the inventors confirmed that the Lactobacillus ash It was named Lactobacillus acidophilus HY7037, and deposited on May 9, 2013 with the Korean Life Performance Reduction Gene Bank (Accession No .: KCTC 12409BP).

The Lactobacillus acidophilus ( Lactobacillus < RTI ID = 0.0 > acidophilus ) HY7037 has the following characteristics.

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 shape: Bacillus

② Mobility: None

③ Spore forming ability: None

④ Gram staining: positive

2) Morphology

When cultured on MRS agar plate medium at 37 ° C for 2 days,

① Shape: Circular

② Bump: convex

③ Surface: Rough

3) Physiological properties

① Growth temperature: Growth possible Growth temperature 15 ~ 40 ℃

            Optimum growth temperature 37 ℃

② Growth pH: Growable growth pH 5.0 ~ 7.5

           Optimum pH 6.0 to 6.5

③ Effect on oxygen: Tumor anaerobic

4) Catalase: -

5) Whether gas is formed: -

6) Growth at 15 ℃: -

7) Growth at 45 ° C: +

8) Indole production: -

9) Lactic acid production: +

10) Fermentation experiments and identification with API 50 CH kit from Biomerieux

1-4. Bifidobacterium Ronggeom (Bifidobacterium longum) Isolation and Identification of HY8005

In order to isolate the new strain according to the present invention, the feces collected from the neonate 7 days before birth were collected, and 0.1 g of the collected neonatal feces was decanted into the anaerobic dilution, and the mixture was diluted with a BL solid medium containing 1.5% agar After culturing at 37 ° C in anaerobic condition for 48 hours, colony was taken as a loop and inoculated into fresh BL solid medium and purified. The purely isolated colonies were inoculated into BL liquid medium and cultured at 37 占 폚 for 24 hours.

The isolated strains were observed through a microscope through Crystal Violet staining. DNA was isolated and identified by Bifidobacteria 16S rRNA analysis (Im3, Im26) and blast search (http: //blast.ncbi.nlm .nih.gov / Blast.cgi? PAGE_

TYPE = BlastSearch & BLAST_SPEC = MicrobialGenomes) are shown in Table 3 below.

As can be seen from Table 3, are identified 16S rRNA strains result of identification of the novel strain of the present invention that the bipyridinium Te matches Solarium ronggeom (Bifidobacterium longum) and 100% gaming present inventors it Bifidobacterium ronggeom (Bifidobacterium longum ) HY8005 and donated to Korea Life Performance Salvage Gene Bank (Accession No .: KCTC 12514BP) on November 5, 2013.

The characteristics of Bifidobacterium longum HY8005 according to the present invention are as follows.

1) Types of bacteria

The characteristics of the bacteria when cultured under anaerobic conditions for 2 days at 37 ° C in Biel (BL) agar plate medium

① Cell shape: amorphous hepatoblastic

② Mobility: None

③ Spore forming ability: None

④ Gram staining: positive

2) Physiological properties

① Growth temperature: Growth possible Growth temperature 15 ~ 42 ℃

            Optimum growth temperature 36 ~ 38 ℃

② Growth pH: Growable Growth pH 4.5 ~ 8.0

           Optimal pH 6.0 ~ 7.0

③ Effect on oxygen: Absolute anaerobic

3) Catalase: -

4) Whether gas is formed: -

5) Growth at 15 ℃: -

6) Growth at 45 ° C: +

7) Indole production: -

8) Lactic acid production: +

1-5. Bifidobacterium breve (Bifidobacterium breve) Separation and Identification of HY8201

In order to isolate the new strain according to the present invention, the feces collected from the neonate 7 days before birth were collected, and 0.1 g of the collected neonatal feces was decanted into the anaerobic dilution, and the mixture was diluted with a BL solid medium containing 1.5% agar After culturing at 37 ° C in anaerobic condition for 48 hours, colony was taken as a loop and inoculated into fresh BL solid medium and purified. The purely isolated colonies were inoculated into BL liquid medium and cultured at 37 占 폚 for 24 hours.

The isolated strains were observed through a microscope through Crystal Violet staining. DNA was isolated and identified by Bifidobacteria 16S rRNA analysis (Im3, Im26) and blast search (http: //blast.ncbi.nlm .nih.gov / Blast.cgi? PAGE_

TYPE = BlastSearch & BLAST_SPEC = MicrobialGenomes) are shown in Table 4 below.

As can be seen from the above Table 4, the 16S rRNA strain of the new strain of the present invention was found to be 100% identical to Bifidobacterium breve , and the present inventors confirmed this strain as Bifidobacterium breve ( Bifidobacterium breve ) HY8201, and donated to KCT 12515BP, Korea, on November 5, 2013.

The characteristics of Bifidobacterium breve HY8201 according to the present invention are as follows.

1) Types of bacteria

The characteristics of the bacteria when cultured under anaerobic conditions for 2 days at 37 ° C in Biel (BL) agar plate medium

① Cell shape: amorphous hepatoblastic

② Mobility: None

③ Spore forming ability: None

④ Gram staining: positive

2) Physiological properties

① Growth temperature: Growth possible Growth temperature 15 ~ 42 ℃

            Optimum growth temperature 36 ~ 38 ℃

② Growth pH: Growable Growth pH 4.5 ~ 8.0

           Optimal pH 6.0 ~ 7.0

③ Effect on oxygen: Absolute anaerobic

3) Catalase: -

4) Whether gas is formed: -

5) Growth at 15 ℃: -

6) Growth at 45 ° C: +

7) Indole production: -

8) Lactic acid production: +

9) Fermentation experiments and identification with API 50 CH kit from Biomerieux

1-6. Lactobacillus biasing Li (Lactobacillus gasseri) Isolation and Identification of HY7023

In order to isolate the new strain according to the present invention, the feces collected from the neonate 7 days before birth were collected, and 0.1 g of the feces of the collected neonate was decanted into the exhalation dilution to obtain an MRS ), Cultured at 37 ° C for 48 hours, colony was taken as a loop and inoculated on fresh MRS solid medium and purified. The purely isolated colonies were inoculated into the MRS liquid medium and cultured at 37 DEG C for 24 hours.

The DNA of the isolated isolate was isolated and subjected to blast search by homologous identification through 16S rRNA analysis (27F, 1492R) (http://blast.ncbi.nlm.nih.gov/Blast.cgi

? PAGE_TYPE = BlastSearch & BLAST_SPEC = MicrobialGenomes) are shown in Table 5 below.

As can be seen from Table 5, is found to 16S rRNA matching strain identification result rock Fig Bacillus biasing Li (Lactobacillus gasseri), and 100% of the novel strain of the present invention the inventors Lee (Lactobacillus this lock also Bacillus in addition gasseri ) HY7023 and donated to Korea Life Performance Salvage Gene Bank (Accession No .: KCTC 12512BP) on November 5, 2013.

The characteristics of Lactobacillus gasseri HY7023 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 shape: Bacillus

② Mobility: None

③ Spore forming ability: None

④ Gram staining: positive

2) Physiological properties

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

  Optimum growth temperature: 36 ~ 38 ℃

② Growth pH: Growable growth pH 5.0 ~ 7.5

  Optimum pH: 6.0 to 6.5

③ Effect on oxygen: Tumor anaerobic

3) Catalase: -

4) Whether gas is formed: -

5) Growth at 15 ℃: -

6) Growth at 45 ° C: +

7) Indole production: -

8) Lactic acid production: +

9) Fermentation experiments and identification with API 50 CH kit from Biomerieux

1-7. Lactobacillus Casei (Lactobacillus casei) Isolation and Identification of HY7212

In order to isolate the new strain according to the present invention, the feces collected from the neonate 7 days before birth were collected, and 0.1 g of the feces of the collected neonate was decanted into the exhalation dilution to obtain an MRS ), Cultured at 37 ° C for 48 hours, colony was taken as a loop and inoculated on fresh MRS solid medium and purified. The purely isolated colonies were inoculated into the MRS liquid medium and cultured at 37 DEG C for 24 hours.

The DNA of the isolated isolate was isolated and subjected to blast search by homologous identification through 16S rRNA analysis (27F, 1492R) (http://blast.ncbi.nlm.nih.gov/Blast.cgi

? PAGE_TYPE = BlastSearch & BLAST_SPEC = MicrobialGenomes) are shown in Table 6 below.

As can be seen from Table 6 above, the 16S rRNA strain of the new strain of the present invention was identified to be 100% identical to Lactobacillus casei , and the present inventors confirmed that the Lactobacillus casei HY7212, and donated to Korea Biotechnology Saving Gene Bank (Accession No .: KCTC 2513BP) on November 5, 2013.

The characteristics of Lactobacillus casei HY7212 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 shape: Bacillus

② Mobility: None

③ Spore forming ability: None

④ Gram staining: positive

2) Morphology

When cultured on MRS agar plate medium at 37 ° C for 2 days,

① Shape: Circular

② Bump: convex

③ Surface: Smooth

3) Physiological properties

① Growth temperature: Growth possible Growth temperature 15 ~ 40 ℃

            Optimum growth temperature 37 ℃

② Growth pH: Growable growth pH 5.0 ~ 7.5

           Optimum pH 6.0 to 6.5

③ Effect on oxygen: Tumor anaerobic

4) Catalase: -

5) Whether gas is formed: -

6) Growth at 15 ℃: -

7) Growth at 45 ° C: +

8) Indole production: -

9) Lactic acid production: +

10) Fermentation experiments and identification with API 50 CH kit from Biomerieux

≪ Example 2 >

Manufacture of freeze-dried powder containing 7 kinds of lactic acid bacteria complex strains

2-1. Bifidobacterium Infante Tees (Bifidobacterium infantis) freeze dried powder containing HY8401

Bifidobacterium infantis HY8401 2 × 10 ⁹ cfu / ml bacterium of the present invention was inoculated into BL liquid medium and batch cultured at 37 ° C. for 24 hours under anaerobic conditions in an incubator. The supernatant was removed by centrifugation at 4000 rpm for 15 minutes, washed with phosphate buffered saline (PBS), and centrifuged at 4000 rpm for 15 minutes to remove the supernatant. The collected cells were mixed with a skim milk powder as a preservative at a weight ratio of 1: 1 and lyophilized to prepare a powder.

2-2. Bifidobacterium lactis (Bifidobacterium lactis) freeze-dried powder production, including HY8101

BP Gaming invention Te Solarium lactis (Bifidobacterium lactis ) HY8101 2 × 10 ⁹ cfu / ml were inoculated into BL liquid medium and batch cultured at 37 ° C. for 24 hours under anaerobic conditions in an incubator. The supernatant was removed by centrifugation at 4000 rpm for 15 minutes, washed with phosphate buffered saline (PBS), and centrifuged at 4000 rpm for 15 minutes to remove the supernatant. The collected cells were mixed with a skim milk powder as a preservative at a weight ratio of 1: 1 and lyophilized to prepare a powder.

2-3. Lactobacillus Ash's also a filler (Lactobacillus freeze-dried powder production, including acidophilus) HY7037

Lactobacillus acidophilus HY7037 of the present invention 2 x 10 ⁹ cfu / ml bacterium was inoculated into MRS broth and batch cultured in an incubator at 37 ° C for 24 hours. The supernatant was removed by centrifugation at 4000 rpm for 15 minutes, washed with phosphate buffered saline (PBS), and centrifuged at 4000 rpm for 15 minutes to remove the supernatant. The collected cells were mixed with a skim milk powder as a preservative at a weight ratio of 1: 1 and lyophilized to prepare a powder.

2-4. Ronggeom Bifidobacterium (Bifidobacterium longum) freeze-dried powder production, including HY8005

Bifidobacterium longum HY8005 of the present invention Bifidobacterium longum HY8005 2 x 10 ⁹ cfu / ml bacterium was inoculated into BL liquid medium and cultured in an incubator under anaerobic conditions at 37 ° C for 24 hours. The supernatant was removed by centrifugation at 4000 rpm for 15 minutes, washed with phosphate buffered saline (PBS), and centrifuged at 4000 rpm for 15 minutes to remove the supernatant. The collected cells were mixed with a skim milk powder as a preservative at a weight ratio of 1: 1 and lyophilized to prepare a powder.

2-5. Bifidobacterium breve (Bifidobacterium breve) freeze-dried powder production, including HY8201

Bifidobacterium breve HY8201 of the present invention Bifidobacteria of 2 X 10 ⁹ cfu / ml were inoculated into BL liquid medium and batch cultured at 37 ° C under anaerobic conditions for 24 hours in an incubator. The supernatant was removed by centrifugation at 4000 rpm for 15 minutes, washed with phosphate buffered saline (PBS), and centrifuged at 4000 rpm for 15 minutes to remove the supernatant. The collected cells were mixed with a skim milk powder as a preservative at a weight ratio of 1: 1 and lyophilized to prepare a powder.

2-6. Lactobacillus biasing Li (Lactobacillus gasseri), including lyophilized powder prepared HY7023

Lactobacillus gasseri HY7023 2 × 10 ⁹ cfu / ml of the present invention was inoculated into MRS broth and batch cultured in an incubator at 37 ° C. for 24 hours. The supernatant was removed by centrifugation at 4000 rpm for 15 minutes, washed with phosphate buffered saline (PBS), and centrifuged at 4000 rpm for 15 minutes to remove the supernatant. The collected cells were mixed with a skim milk powder as a preservative at a weight ratio of 1: 1 and lyophilized to prepare a powder.

2-7. Lactobacillus casei (Lactobacillus casei) freeze-dried powder prepared including HY7212

Lactobacillus casei HY7212 of the present invention 2 x 10 ⁹ cfu / ml bacterium was inoculated into MRS broth and batch cultured in an incubator at 37 ° C for 24 hours. The supernatant was removed by centrifugation at 4000 rpm for 15 minutes, washed with phosphate buffered saline (PBS), and centrifuged at 4000 rpm for 15 minutes to remove the supernatant. The collected cells were mixed with a skim milk powder as a preservative at a weight ratio of 1: 1 and lyophilized to prepare a powder.

2-8. Preparation of freeze-dried powder containing 7 kinds of lactic acid bacteria complex strains

The lyophilized powders containing the strains of each of Examples 2-1 to 2-7 were mixed at the same weight ratios (1: 1: 1: 1: 1: 1: 1) And the total number of bacteria was 1 X 10 ¹¹ cfu / g.

Meanwhile, the seven strains of the lactic acid bacteria of the present invention can be provided in the form of a lyophilized powder or a culture as described above.

≪ Example 3 >

Preparation of a pharmaceutical composition containing seven kinds of lactic acid bacteria complex strains as effective ingredients

Manufacture of liquid agent

100 mg of the lyophilized powder containing seven complex strains of lactic acid bacteria of Example 2-8, 10 g of isomerized sugar, and 5 g of mannitol were dissolved in purified water in accordance with the usual liquid preparation method, dissolved in lemon flavor Purified water was added to adjust the total volume to 100 ml, and the solution was filled in a brown bottle and sterilized to prepare a liquid preparation.

Preparation of capsules

100 mg of corn starch, 100 mg of lactose and 2 mg of magnesium stearate were thoroughly mixed with 100 mg of the lyophilized powder containing the seven kinds of the lactic acid bacterial strain of Example 2-8, and the mixture was filled into hard gelatin capsules according to the preparation method of capsules, To prepare a capsule preparation.

<Example 4>

Production of fermented milk containing 7 kinds of lactic acid bacteria complex strains as active ingredients

A method for producing a fermented milk containing seven kinds of the lactic acid bacteria complex strain of the present invention as an active ingredient is as follows.

The lactic acid bacteria culture solution was prepared by stirring 95.36% by weight of crude oil and 4.6% by weight of skim milk powder (or mixed powdered milk) with a specific gravity of 1.0473 to 1.0475 at 15 ° C, a titratable acidity of 0.200 to 0.220%, a pH of 6.55 to 6.70, Of Brix ⁰ was mixed to be about 16.3 to 16.5%. After the mixture was subjected to UHT heat treatment (sterilization at 135 캜 for 2 seconds) and cooling to 40 캜, Lactobacillus acidophilus HY7037, Lactobacillus casei HY7212 and Lactobacillus casei HY7212, After adding 0.02% by weight of HY8005 ( Bifidobacterium longum HY8005) and lyophilizing enzyme (Valley laboratory, USA) for 6 hours, the total number of lactic acid bacteria in the BCP medium was 1.0 × 10 ⁹ cfu / 0.91%, and a pH of 4.55 to 4.65. Then, the mixed juice syrup was prepared by mixing 13 wt% of liquid fructose, 5 wt% of white sugar, 10.9 wt% of mixed juice concentrate 56Brix ⁰ , 1.0 wt% of pectin, 0.1 wt% of fresh fruit mix essence and 70 wt% of purified water at 30-35 째 C 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 culture liquid of lactic acid bacteria, 0.1% by weight of lyophilized powder containing seven kinds of the lactic acid bacteria of Example 2-8 and 30.4% by weight of the mixed fruit syrup was homogenized at 150 bar, Followed by cooling to prepare fermented milk containing seven kinds of lactic acid bacteria complexes as active ingredients.

&Lt; Example 5 >

Preparation of vegetable juice containing 7 kinds of lactic acid bacteria complex strains as active ingredients

A method for producing vegetable juice containing the seven kinds of the lactic acid bacterial strain of the present invention as an active ingredient is as follows.

Citric acid juice 1 wt.%, Spinach juice 1 wt.%, Lettuce juice 2 wt.%, Celery juice 1 wt.%, Anhydrous citric acid 0.2 wt.%, 0.1% by weight of the lyophilized powder containing the seven kinds of lactic acid bacterial strains of Examples 2-8 were homogeneously mixed in a mixing machine and sterilized at 85 to 97 ° C using a sterilizer to prepare 7 kinds of lactic acid bacteria complex Vegetable juices were prepared.

&Lt; Example 6 >

Production of functional beverage containing 7 kinds of lactic acid bacteria complex strains as active ingredients

A method for producing a functional beverage containing the seven strains of the lactic acid bacteria of the present invention as an active ingredient 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 ⁰ 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 fruit juice syrup prepared by the above method, 0.1% by weight of the lyophilized powder containing the seven kinds of the lactic acid bacteria of the Example 2-8 and 69.5% by weight of the remaining purified water were combined and homogenized at 150 bar. ℃ and packaged in small containers such as glass bottles and PET bottles to produce functional beverages containing seven kinds of lactic acid bacteria complexes as active ingredients.

&Lt; Example 7 >

Production of Health Functional Foods Containing 7 Lactic Acid Bacterial Strain as Active Ingredient

(Vitamins B ₁ , B ₂ , B ₅ , B ₆ , E and acetic acid esters, nicotinic acid amide) and oligosaccharides were added to 0.1% by weight of the lyophilized powder containing the seven kinds of lactic acid bacterial strains of Example 2-8 Was added in an amount of 10 parts by weight based on 100 parts by weight of the lyophilized powder containing the seven kinds of lactic acid bacterial strains of Example 2-8, and the mixture was mixed in a high-speed rotary mixer. 10 parts by weight of sterilized purified water was added to the mixture, mixed and molded into 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 >

Investigation of the effect of 7 kinds of lactic acid bacteria on glucose absorption

As for the utilization of sugar cells in muscle cells, insulin secreted from pancreatic β-cells and insulin receptor in muscle cells, GLUT4 (glucose transporter type 4), are closely related. Sugars such as glucose in the blood are used in various organs such as adipocytes and muscle tissue. In the case of muscle cells, insulin action causes insulin signal due to the action of insulin receptor Operation. Finally, the expression of GLUT4 and the increase in translocation in muscle cells allows glucose in the blood to enter the muscle, allowing it to be synthesized and used with glycogen and the like.

However, when fatty acids such as TNF-α and palmitic acid act in this process, an abnormality in the insulin signaling mechanism results in an abnormal expression of GLUT4 and translocation, resulting in poor blood glucose inflow .

Therefore, in order to ascertain the increase of blood glucose uptake in the case where the blood glucose uptake is decreased due to the intracellular TNF-a of the seven kinds of the lactic acid bacteria of the present invention, glucose uptake assay is performed through the glucose uptake assay, Respectively.

1-1. Muscle cell culture

L6 cell myoblast, a skeletal muscle cell derived from rat, was purchased from the Korean Cell Line Bank (cellbank.snu.ac.kr) and used for the animal cells used in this test. Cell culture was carried out in a medium containing 90% DMEM medium (Gibco #) and 10% FBS (Gibco # 16000) for maintenance of normal cells, and subculture was performed twice a week at a ratio of 1: 8.

1-2. Obtain cell extracts of 7 kinds of lactic acid bacteria complex strains

① Bifidobacterium Infante Tees (Bifidobacterium infantis) HY8401 cell extracts

BP Gaming invention Te Solarium Infante Tees (Bifidobacterium infantis) were inoculated in the BL HY8401 liquid medium and anaerobic incubation for 20 hours at 37 ℃. The culture was centrifuged at 3,000 rpm at 4 ° C for 10 minutes to obtain cells, which were then washed three times with sterilized PBS buffer. Then, the cells were suspended in a sterilized PBS buffer solution, and the absorbance was measured. The OD600 was adjusted to 1.0 and the suspension was sterilized at 100 DEG C for 20 minutes. Bifidobacterium infantis HY8401 thus killed was centrifuged at 3,000 rpm at 4 ° C for 10 minutes to obtain cells, which were then suspended in 1 ml of sterilized PBS buffer to obtain Bifidobacterium infantis ( Bifidobacterium infantis ) HY8401 strain suspension was prepared and a sample was prepared.

② Bifidobacterium Lactis ( Bifidobacterium lactis ) HY8101 cell extract

Bifidobacterium lactis HY8101 of the present invention was cultured in BL medium (Difco) under anaerobic conditions at 37 DEG C for 24 hours. The cells were centrifuged, washed with PBS 2-3 times, and the remaining medium was removed. The cells were lyophilized and pulverized, and then the number of bacteria was measured by serial dilution. The cells were resuspended in PBS to a concentration of 10 ¹⁰ cfu / ml and sonicated to lysis the cells. After centrifugation, the supernatant was used for the test as a lactic acid bacterium cell extract.

③ Lactobacillus Ash's also a filler (Lactobacillus acidophilus ) HY7037 cell extract

Lactobacillus acidophilus HY7037 of the present invention was cultured in MRS medium (Difco) at 37 占 폚 for 24 hours. The cells were centrifuged, washed with PBS 2-3 times, and the remaining medium was removed. The cells were lyophilized and pulverized, and then the number of bacteria was measured by serial dilution. The cells were resuspended in PBS to a concentration of 10 ¹⁰ cfu / ml and sonicated to lysis the cells. After centrifugation, the supernatant was used for the test as a lactic acid bacterium cell extract.

④ ronggeom Bifidobacterium (Bifidobacterium longum) HY8005 cell extracts

Bifidobacterium longum HY8005 of the present invention was inoculated into BL liquid medium and anaerobically cultured at 37 占 폚 for 20 hours. The culture was centrifuged at 3,000 rpm at 4 ° C for 10 minutes to obtain cells, which were then washed three times with sterilized PBS buffer. Then, the cells were suspended in a sterilized PBS buffer solution, and the absorbance was measured. The OD600 was adjusted to 1.0 and the suspension was sterilized at 100 DEG C for 20 minutes. The thus-killed Bifidobacterium longum HY8005 was centrifuged at 3,000 rpm at 4 DEG C for 10 minutes to obtain cells, which were then suspended in 1 mL of sterilized PBS buffer to obtain Bifidobacterium longum HY8005 A suspension was prepared and a sample was prepared.

⑤ Bifidobacterium breve (Bifidobacterium breve) HY8201 cell extracts

BP gaming of the present invention, Te Solarium breve (Bifidobacterium breve ) HY8201 were inoculated into BL liquid medium and anaerobically cultured at 37 ° C for 20 hours. The culture was centrifuged at 3,000 rpm at 4 ° C for 10 minutes to obtain cells, which were then washed three times with sterilized PBS buffer. Then, the cells were suspended in a sterilized PBS buffer solution, and the absorbance was measured. The OD600 was adjusted to 1.0 and the suspension was sterilized at 100 DEG C for 20 minutes. Thus killing the Bifidobacterium breve (Bifidobacterium breve) the HY8201 bacteria 3,000rpm, after obtaining the cells were separated by 10 minutes centrifugation at 4 ℃ and suspended in sterile PBS buffer 1㎖ Bifidobacterium breve (Bifidobacterium breve ) HY8201 strain was prepared and a sample was prepared.

⑥ Lactobacillus joined Lee (Lactobacillus gasseri) HY7023 cell extracts

Lactobacillus of the present invention in addition Lee (Lactobacillus gasseri ) HY7023 were cultured in MRS medium (Difco) at 37 DEG C for 24 hours. The cells were centrifuged, washed with PBS 2-3 times, and the remaining medium was removed. The cells were lyophilized and pulverized, and then the number of bacteria was measured by serial dilution. The cells were resuspended in PBS to a concentration of 10 ¹⁰ cfu / ml and sonicated to lysis the cells. After centrifugation, the supernatant was used for the test as a lactic acid bacterium cell extract.

⑦ Lactobacillus casei (Lactobacillus casei) HY7212 cell extracts

Lactobacillus casei HY7212 of the present invention was cultured in MRS medium (Difco) at 37 DEG C for 24 hours. The cells were centrifuged, washed with PBS 2-3 times, and the remaining medium was removed. The cells were lyophilized and pulverized, and then the number of bacteria was measured by serial dilution. The cells were resuspended in PBS to a concentration of 10 ¹⁰ cfu / ml and sonicated to lysis the cells. After centrifugation, the supernatant was used for the test as a lactic acid bacterium cell extract.

⑧ Cell extracts of 7 kinds of lactic acid bacteria complex strains

Each of the lactic acid bacterial cell extracts of (1) to (7) was mixed at the same weight ratios (1: 1: 1: 1: 1: 1: 1) and used as a cell extract of the seven lactic acid bacterial strains of the present invention.

1-3. Differentiation of muscle cells

The L6 skeletal muscle cells were subcultured in a 96-well plate at 5 × 10 ⁵ cells / well and cultured for 3 to 4 days in DMEM medium supplemented with 2% FBS.

1-4. Measurement of glucose uptake in L6 skeletal muscle cells by treatment with 7 kinds of lactic acid bacteria complex strains

(1) Each of the seven Lactobacillus strains of Example 1 was treated with 10 ⁶ cfu / well for 24 hours in the differentiated L6 skeletal muscle cells of Test Example 1-3, then the culture medium was removed, and Cayman 2-NBDG (1:70), insulin (500 μM), and the seven kinds of lactic acid bacterial strain extracts of Test Example 1-2 were added to the glucose included in the kit of Chemical Co., Lt; / RTI &gt; for 30 minutes.

200 [mu] l of each well was washed with an assay buffer in a kit of Cayman Chemical Co. for 5 minutes, and then 100 [mu] l of assay buffer was added to the same plate. Using a plate reader, Excitation / emission = 485/535 nm fluorescence measurement.

(2) Each Bifidobacterium ronggeom (Bifidobacterium longum) HY8005, Bifidobacterium Infante tooth (Bifidobacterium infantis) HY8401, Bifidobacterium lactis (Bifidobacterium lactis), instead of lactic acid complex strains of seven of the above (1) HY8101, Bifidobacterium breve HY8201, Lactobacillus ( Lactobacillus &lt; RTI ID = 0.0 &gt; acidophilus HY7037, Lactobacillus gasseri HY7023, Lactobacillus &lt; RTI ID = 0.0 &gt; casei ) Test was carried out by the same test method as in (1) except that HY7212 was used.

The results are shown in Fig.

&Quot; Negative control group &quot; represents a group in which none of the differentiated L6 skeletal muscle cells was treated,

The 'positive control group' represents the group treated with only insulin (500 μM) to differentiated L6 skeletal muscle cells,

'HY8005' is a system in which differentiated L6 skeletal muscle cells are supplemented with insulin (500 μM) and Bifidobacterium longum ) HY8005 treated group,

'HY8401' represents the insulin (500μM) and Bifidobacterium Infante tooth group treated with HY8401 (Bifidobacterium infantis) in the differentiated L6 skeletal muscle cells,

'HY8101' represents the group treated with insulin (500 μM) and Bifidobacterium lactis HY8101 in differentiated L6 skeletal muscle cells,

'HY8201' represents the group treated with insulin (500 μM) and Bifidobacterium breve HY8201 in differentiated L6 skeletal muscle cells,

'HY7023' represents a group treated with insulin (500 μM) and Lactobacillus gasseri HY7023 to differentiated L6 skeletal muscle cells,

'HY7037' represents a group treated with insulin (500 μM) and Lactobacillus acidophilus HY7037 in differentiated L6 skeletal muscle cells,

'HY7212' insulin (500μM) and Lactobacillus casei (Lactobacillus in the differentiated L6 skeletal muscle cells casei ) HY7212 treated group,

'Birth-7' represents the group treated with insulin (500 μM) and 7 kinds of lactic acid bacteria complexes to differentiated L6 skeletal muscle cells.

As can be seen from FIG. 1, when the seven kinds of lactic acid bacteria were treated with the combination of the above-mentioned lactic acid bacteria alone, the increase in blood glucose uptake in the normal L6 skeletal muscle cells in which insulin resistance was not induced I could confirm.

1-5. Measurement of glucose uptake in L6 skeletal muscle cells induced by insulin resistance induced by TNF-α by the combination of seven strains of lactic acid bacteria

(1) Each of the seven lactic acid bacterial strains of Example 1 was treated with 10 ⁶ cfu / well for 24 hours in the differentiated L6 skeletal muscle cells of Test Example 1-3, and then TNF-α (100 ng / ml) Was treated with 200 [mu] L per well for 6 hours to induce insulin resistance.

After the culture medium was removed, the fluorescent substance-treated 2-NBDG (1:70), insulin (500 μM), and the seven kinds of the test samples described in Test Example 1-2 were added to the glucose included in the kit of Cayman Chemical Co. Lactic acid bacterial cell extracts were mixed together and treated with 100 μl each for 30 minutes.

200 [mu] l of each well was washed with an assay buffer in a kit of Cayman Chemical Co. for 5 minutes, and then 100 [mu] l of assay buffer was added to the same plate. Using a plate reader, Excitation / emission = 485/535 nm fluorescence measurement.

(2) Each Bifidobacterium ronggeom (Bifidobacterium longum) HY8005, Bifidobacterium Infante tooth (Bifidobacterium infantis) HY8401, Bifidobacterium lactis (Bifidobacterium lactis), instead of lactic acid complex strains of seven of the above (1) Except for using HY8101, Bifidobacterium breve HY8201, Lactobacillus acidophilus HY7037, Lactobacillus gasseri HY7023, and Lactobacillus casei HY7212. Was tested by the same test method as in (1) above.

The results are shown in Fig.

&Quot; Negative control group &quot; represents a group in which none of the differentiated L6 skeletal muscle cells was treated,

The 'positive control group' represents the group treated with only insulin (500 μM) to differentiated L6 skeletal muscle cells,

Induction group represents the group treated with only insulin (500 μM) and TNF-α (100 ng / ml) in differentiated L6 skeletal muscle cells,

'HY8005' represents the group treated with insulin (500 μM), TNF-α (100 ng / ml) and Bifidobacterium longum HY8005 in differentiated L6 skeletal muscle cells,

'HY8401' represents the insulin (500μM) and TNF-α (100ng / ㎖) and Bifidobacterium Infante tooth group treated with HY8401 (Bifidobacterium infantis) in the differentiated L6 skeletal muscle cells,

'HY8101' represents a group treated with insulin (500 μM), TNF-α (100 ng / ml) and Bifidobacterium lactis HY8101 to differentiated L6 skeletal muscle cells,

'HY8201' insulin (500μM) and TNF-α (100ng / ㎖) and Bifidobacterium breve (Bifidobacterium in the differentiated L6 skeletal muscle cells breve ) HY8201 treated group,

'HY7023' represents a group treated with insulin (500 μM), TNF-α (100 ng / ml) and Lactobacillus gasseri HY7023 to differentiated L6 skeletal muscle cells,

'HY7037' represents a group treated with insulin (500 μM), TNF-α (100 ng / ml) and Lactobacillus acidophilus HY7037 to differentiated L6 skeletal muscle cells,

'HY7212' insulin (500μM) and TNF-α (100ng / ㎖) and Lactobacillus casei (Lactobacillus in the differentiated L6 skeletal muscle cells casei ) HY7212 treated group,

'Birth-7' represents the group treated with insulin (500 μM) and 7 kinds of lactic acid bacteria complexes to differentiated L6 skeletal muscle cells.

As can be seen in FIG. 2, when seven kinds of lactic acid bacteria were treated with the combination of the respective lactic acid bacteria alone, the glucose uptake in L6 skeletal muscle cells induced by insulin resistance by TNF- .

&Lt; Test Example 2 &

Measurement of expression levels of GLUT4, PPAR-γ, and PGC-1α in L6 skeletal muscle cells by treatment with 7 different strains of lactic acid bacteria

Myocardial glucose uptake is caused by a protein called GLUT4. In particular, the expression of peroxisome proliferator-activated receptor gamma (PPAR-γ) and peroxisome proliferator-activated receptor gamma coactivator 1-alpha (GLP-1α) It affects the translocation. Therefore, in order to confirm the increase of glucose uptake by GLUT4, PPAR-γ and PGC-1α gene expression of Lactobacillus multiforme of the present invention in L6 skeletal muscle cells induced by insulin resistance by TNF-α, GLUT4 , PPAR-γ and PGC-1α genes were measured.

2-1. Differentiation of muscle cells

L6 skeletal muscle cells of Test Example 1-1 were subcultured on a 6-well plate at 1 × 10 ⁶ cells / well and cultured for 3 to 4 days using DMEM medium containing 2% FBS.

2-2. Measurement of GLUT4 gene expression in L6 skeletal muscle cells induced by insulin resistance induced by TNF-α by 7 kinds of Lactobacillus complexes

(1) The differentiated muscle cells of Test Example 2-1 were treated with each of the ^seven lactic acid bacterial strains of Example 1 at 10 ⁷ cfu / well for 24 hours, and then the following day, TNF-α (100 ng / and treated with 2 ml per well for 6 hours to induce insulin resistance.

After the culture medium was removed, insulin (500 μM) and the seven kinds of lactic acid bacterial strain extracts of Test Example 1-2 were mixed together and treated in 2 ml portions for 30 minutes.

RNA from muscle cells was obtained from Qiagen's RNA prep. Kit (RNeasy mini kit, Qiagen 74106). CDNA was synthesized from the separated RNA using Quantit reverse transcription kit (Qiagen, Valencia, Calif.). RNA expression was quantitated using Taqman probe and Real-time PCR (applied biosystems, 7500 real time PCR) shown in Table 7 below. GAPDH (Glyceraldehyde 3-phosphate dehydrogenase) was used as an internal control.

(2) Each Bifidobacterium ronggeom (Bifidobacterium longum) HY8005, Bifidobacterium Infante tooth (Bifidobacterium infantis) HY8401, Bifidobacterium lactis (Bifidobacterium lactis), instead of lactic acid complex strains of seven of the above (1) Except for using HY8101, Bifidobacterium breve HY8201, Lactobacillus acidophilus HY7037, Lactobacillus gasseri HY7023, and Lactobacillus casei HY7212. Was tested by the same test method as in (1) above.

The results are shown in Fig.

&Quot; Negative control group &quot; represents a group in which none of the differentiated L6 skeletal muscle cells was treated,

The 'positive control group' represents the group treated with only insulin (500 μM) to differentiated L6 skeletal muscle cells,

Induction group represents the group treated with only insulin (500 μM) and TNF-α (100 ng / ml) in differentiated L6 skeletal muscle cells,

'HY8005' represents the group treated with insulin (500 μM), TNF-α (100 ng / ml) and Bifidobacterium longum HY8005 in differentiated L6 skeletal muscle cells,

'HY8401' represents the insulin (500μM) and TNF-α (100ng / ㎖) and Bifidobacterium Infante tooth group treated with HY8401 (Bifidobacterium infantis) in the differentiated L6 skeletal muscle cells,

'HY8101' represents a group treated with insulin (500 μM), TNF-α (100 ng / ml) and Bifidobacterium lactis HY8101 to differentiated L6 skeletal muscle cells,

'HY8201' represents a group treated with insulin (500 μM), TNF-α (100 ng / ml) and Bifidobacterium breve HY8201 in differentiated L6 skeletal muscle cells,

'HY7023' expresses insulin (500 μM), TNF-α (100 ng / ml) and Lactobacillus gasseri gasseri ) HY7023,

'HY7037' expresses insulin (500 μM), TNF-α (100 ng / ml) and Lactobacillus ( Lactobacillus) acidophilus HY7037,

'HY7212' insulin (500μM) and TNF-α (100ng / ㎖) and Lactobacillus casei (Lactobacillus in the differentiated L6 skeletal muscle cells casei ) HY7212 treated group,

'Birth-7' represents the group treated with insulin (500 μM) and 7 kinds of lactic acid bacteria complexes to differentiated L6 skeletal muscle cells.

As can be seen in FIG. 3, GLUT4 gene expression, which is involved in glucose uptake by insulin, is increased in the normal muscle cells of the positive control group, whereas in the induction group, TNF-α is involved in glucose uptake due to normal muscle cells GLUT4 gene expression was reduced.

In addition, the expression of GLUT4 gene was decreased in each of the individual strains, but the expression of GLUT4 gene was increased in the seven strains of the lactic acid bacteria of the present invention.

Thus, it was confirmed that the seven strains of the lactic acid bacteria of the present invention increased the expression amount of GLUT4 reduced by TNF-α, thereby increasing blood glucose absorption.

gene Taqman probe Rat GAPDH Rn01775763_g1 Rat solute carrier family 2 (facilitated glucose transporter), member 4, GLUT-4 Rn00562597_m1 Rat PPAR-γ Rn00440945_m1 Rat peroxisome proliferative activated receptor, gamma, coactivator 1 alpha Rn00580241_m1

2-3. Measurement of PPAR-γ gene expression level in L6 skeletal muscle cells induced by insulin resistance induced by TNF-α by 7 kinds of Lactobacillus complexes

The expression level of the PPAR-gamma gene was measured by the same test method as in Test Example 2-2.

The results are shown in Fig.

&Quot; Negative control group &quot; represents a group in which none of the differentiated L6 skeletal muscle cells was treated,

The 'positive control group' represents the group treated with only insulin (500 μM) to differentiated L6 skeletal muscle cells,

Induction group represents the group treated with only insulin (500 μM) and TNF-α (100 ng / ml) in differentiated L6 skeletal muscle cells,

'HY8005' represents the group treated with insulin (500 μM), TNF-α (100 ng / ml) and Bifidobacterium longum HY8005 in differentiated L6 skeletal muscle cells,

'HY8401' expresses insulin (500 μM), TNF-α (100 ng / ml) and Bifidobacterium represents a group treated with infantis) HY8401,

'HY8101' represents a group treated with insulin (500 μM), TNF-α (100 ng / ml) and Bifidobacterium lactis HY8101 to differentiated L6 skeletal muscle cells,

'HY8201' represents a group treated with insulin (500 μM), TNF-α (100 ng / ml) and Bifidobacterium breve HY8201 in differentiated L6 skeletal muscle cells,

'HY7023' represents a group treated with insulin (500 μM), TNF-α (100 ng / ml) and Lactobacillus gasseri HY7023 to differentiated L6 skeletal muscle cells,

'HY7037' represents a group treated with insulin (500 μM), TNF-α (100 ng / ml) and Lactobacillus acidophilus HY7037 to differentiated L6 skeletal muscle cells,

'HY7212' insulin in differentiated L6 skeletal muscle cells (500μM) and TNF-α (100ng / ㎖) and Lactobacillus casei (Lactobacillus casei ) HY7212 treated group,

'Birth-7' represents the group treated with insulin (500 μM) and 7 kinds of lactic acid bacteria complexes to differentiated L6 skeletal muscle cells.

As can be seen in FIG. 4, the expression of PPAR-γ gene, which is involved in insulin sensitivity and glucose uptake by insulin, is increased in the normal muscle cells of the positive control group. In contrast, in the induction group, PPAR-γ gene expression, which is involved in sensitivity and glucose uptake, was reduced. In addition, the expression of PPAR-γ gene was decreased in each of the individual strains except for Bifidobacterium lactis HY8101 and Lactobacillus acidophilus HY7037, but the expression of the seven lactic acid bacteria of the present invention The complex strain showed an increase in the gene expression of PPAR-γ. Thus, it was confirmed that the seven strains of the lactic acid bacteria of the present invention increased the expression of PPAR-γ reduced by TNF-α, thereby increasing blood glucose uptake.

2-4. Measurements of PGC-1α gene expression in L6 skeletal muscle cells induced by insulin resistance induced by TNF-α by the combination of seven strains of lactic acid bacteria

The expression level of PGC-1 alpha gene was measured by the same test method as in Test Example 2-2.

The results are shown in Fig.

&Quot; Negative control group &quot; represents a group in which none of the differentiated L6 skeletal muscle cells was treated,

The 'positive control group' represents the group treated with only insulin (500 μM) to differentiated L6 skeletal muscle cells,

Induction group represents the group treated with only insulin (500 μM) and TNF-α (100 ng / ml) in differentiated L6 skeletal muscle cells,

'HY8005' was obtained by culturing the differentiated L6 skeletal muscle cells with insulin (500 μM), TNF-α (100 ng / ml) and Bifidobacterium longum ) HY8005 treated group,

'HY8401' represents the insulin (500μM) and TNF-α (100ng / ㎖) and Bifidobacterium Infante tooth group treated with HY8401 (Bifidobacterium infantis) in the differentiated L6 skeletal muscle cells,

'HY8101' represents a group treated with insulin (500 μM), TNF-α (100 ng / ml) and Bifidobacterium lactis HY8101 to differentiated L6 skeletal muscle cells,

'HY8201' represents a group treated with insulin (500 μM), TNF-α (100 ng / ml) and Bifidobacterium breve HY8201 in differentiated L6 skeletal muscle cells,

'HY7023' represents a group treated with insulin (500 μM), TNF-α (100 ng / ml) and Lactobacillus gasseri HY7023 to differentiated L6 skeletal muscle cells,

'HY7037' represents a group treated with insulin (500 μM), TNF-α (100 ng / ml) and Lactobacillus acidophilus HY7037 to differentiated L6 skeletal muscle cells,

'HY7212' insulin (500μM) and TNF-α (100ng / ㎖) and Lactobacillus casei (Lactobacillus in the differentiated L6 skeletal muscle cells casei ) HY7212 treated group,

'Birth-7' represents the group treated with insulin (500 μM) and 7 kinds of lactic acid bacteria complexes to differentiated L6 skeletal muscle cells.

As can be seen from FIG. 5, in normal muscle cells as a positive control group, expression of PGC-1 alpha gene, which is involved in insulin sensitivity and glucose uptake, was increased by insulin, whereas in the induction group, Sensitivity and PGC-1α gene expression involved in glucose absorption were reduced. Further, Bifidobacterium Infante tooth (Bifidobacterium infantis) HY8401, Bifidobacterium lactis (Bifidobacterium lactis) HY8101 and Lactobacillus each strain other than the Li (Lactobacillus gasseri) HY7023 biasing increases the gene expression of the PGC-1α However, it was found that the gene expression of PGC-1α was significantly increased in the seven strains of the lactic acid bacteria of the present invention. Therefore, it was confirmed that the seven strains of the lactic acid bacteria of the present invention increased the expression of PGC-1α reduced by TNF-α, thereby increasing blood glucose absorption.

As shown in the test results of Test Example 2 above, the seven strains of the lactic acid bacteria of the present invention increased the expression of GLUT4, PPAR-γ and PGC-1α reduced by TNF-α, .

&Lt; Test Example 3 >

Assessment of Improvement of Blood Glucose Control by Seven Lactic Acid Bacterial Diet in Diabetic Animal Model Induced by STZ (Streptozotocin) Treatment

When STZ is administered intraperitoneally to C57BL / 6J normal male at 4 weeks of age, the beta cells of the pancreas are destroyed and insulin secretion is not performed well. Insulin secretion does not work well, resulting in abnormal weight loss, polysomnia, and polyuria. Abnormal glucose metabolism leads to an increase in blood glucose and HbA1c. Therefore, the improvement effect of the seven kinds of lactic acid bacterial strains in the diabetic animal model induced by STZ (Streptozotocin) treatment of the seven kinds of the lactic acid bacterial strain of the present invention was evaluated.

3-1. C57BL / 6J mouse preparation and dietary preparation

Animal experiments were conducted to confirm the improvement of blood glucose control efficacy due to the combination of seven strains of lactic acid bacteria in a diabetic animal model induced by treatment with STZ (Streptozotocin) according to the present invention. Experimental animals were purchased from Ronbio Co., Ltd., C57BL / 6J mice, and 4-week-old male rats were used for 1 week after being fed with Chow diet. Table 3 shows the composition ratios of the feeds administered to each test group. The basic diet was AIN-76A from Research diets.

3-2. Pancreatic beta cell destruction due to intraperitoneal administration of STZ

The experimental mice of Test Example 3-1 were intraperitoneally administered at a concentration of 200 μl / ml in all remaining rats (n = 30) STZ (150 mg / kg) except for the normal group, and the beta cells of the pancreas were destroyed. Five days after the start of the peritoneal administration, the blood was collected from the tail blood, and the plasma was separated from the blood by measuring the glucose in the blood. Only the experimental mice having a concentration of 250 mg / dl or more were selected, and 6 mice per test group were administered to administer the diet of the above-mentioned Test Example 3-1.

3-3. Weight and Dietary Intake Measurements

The body weight (FIG. 6) and the feed intake (FIG. 7) were measured once a week by administering the experimental diet for a total of 3 weeks.

The results are shown in Fig. 6 and Fig.

The 'normal' group represents the group treated with STZ (Streptozotocin) in the experimental group of Table 8,

'Induction group' refers to the group treated with STZ (Streptozotocin) -treated mice in Table 8

&Quot; Birth-7 &quot; represents a group treated with STZ (Streptozotocin) -treated mice in Table 8.

As can be seen from FIG. 6, when the 7 kinds of lactic acid bacterial strains of the present invention were subjected to dietary administration for 3 weeks, it was confirmed that the body weight decreased by STZ was significantly increased compared to the induction group there was.

As can be seen from FIG. 7, when the 7 kinds of lactic acid bacterial strains of the present invention were subjected to dietary administration for 3 weeks, the pattern of polysaccharide increased by STZ was significantly And the dietary intake was decreased.

3-4. Weight measurement for each organization

After a total of 3 weeks of experimental diets, the mice were sacrificed and the weight of liver and epididymal fat was measured through the scales.

The results are shown in Fig.

The 'normal' group represents the group treated with STZ (Streptozotocin) in the experimental group of Table 8,

'Induction group' refers to the group treated with STZ (Streptozotocin) -treated mice in Table 8

&Quot; Birth-7 &quot; represents a group treated with STZ (Streptozotocin) -treated mice in Table 8.

As can be seen from FIG. 8, it was confirmed that the weight of epididymal fat was significantly increased by the seven kinds of the lactic acid bacteria complex strain of the present invention compared to the induction group.

However, there was no significant difference in liver weight between the inducible group and normal group and Birth-7.

3-5. Fasting blood glucose measurement

Fasting blood glucose was measured by fasting for 6 hours at intervals of once a week for a total of 3 weeks.

The results are shown in Fig.

The 'normal' group represents the group treated with STZ (Streptozotocin) in the experimental group of Table 8,

'Induction group' refers to the group treated with STZ (Streptozotocin) -treated mice in Table 8

&Quot; Birth-7 &quot; represents a group treated with STZ (Streptozotocin) -treated mice in Table 8.

As can be seen from FIG. 9, when the 7 kinds of the lactic acid bacteria of the present invention were administered by dietary administration for 3 weeks, the fasting blood glucose level increased due to insulin secretion by STZ was significantly And the fasting blood glucose was decreased.

3-6. Glycated hemoglobin (HbA1c) measurement

HbA1c was measured using an HbA1c (Easy A1c) glycated hemoglobin test cartridge, Infopia] with blood collected from animal sacrifice.

The results are shown in Fig.

The 'normal' group represents the group treated with STZ (Streptozotocin) in the experimental group of Table 8,

'Induction group' refers to the group treated with STZ (Streptozotocin) -treated mice in Table 8

&Quot; Birth-7 &quot; represents a group treated with STZ (Streptozotocin) -treated mice in Table 8.

As can be seen from FIG. 10, the glycosylated hemoglobin levels increased by increased glucose levels due to insulin secretion by STZ after 3 weeks of dietary administration of the 7 kinds of lactic acid bacteria of the present invention Which was significantly lower than that of the control group.

3-7. Insulin sensitivity measurement

In order to measure the insulin sensitivity of the rats receiving the 3-week experimental diet of the above Test Example 3-2, a glucose tolerance test was performed on an empty stomach. The animals were maintained on fasting for 6 hours and 2 g / kg of glucose was orally administered to each animal. The blood glucose level was measured for 2 hours every 30 minutes.

The results are shown in Fig.

The 'normal' group represents the group treated with STZ (Streptozotocin) in the experimental group of Table 8,

'Induction group' refers to the group treated with STZ (Streptozotocin) -treated mice in Table 8

&Quot; Birth-7 &quot; represents a group treated with STZ (Streptozotocin) -treated mice in Table 8.

As can be seen in FIG. 11, the induction group was found to be slower than the normal group in that the blood sugar level was not lowered to fasting blood glucose level even 120 minutes after the increase of the blood glucose level, whereas the 7 kinds of the lactic acid bacterial strain was AIN-76A (Birth-7) was similar to that of the normal group, and the fasting blood glucose level was low. After a certain period of time (30 minutes after the administration), the fasting blood glucose level was controlled.

These findings suggest that the combination of seven kinds of lactic acid bacteria of the present invention can inhibit the blood glucose level of the present invention when blood glucose is lowered and the blood glucose level is rapidly stabilized even when glucose is consumed such as food consumption, It is possible to consider the use as a descending agent.

3-6. Measurement of gene expression levels in liver and muscle tissue

After a total of 3 weeks of experimental diet, mice were sacrificed and liver and muscle tissues were extracted and placed in RNA later storage solution. RNA from liver and muscle cells was obtained from Qiagen's RNA prep. Kit (RNeasy mini kit, Qiagen 74106). CDNA was synthesized from the separated RNA using Quantit reverse transcription kit (Qiagen, Valencia, Calif.). RNA expression was quantitated using Taqman probe and Real-time PCR (applied biosystems, 7500 real time PCR) in Table 9. GAPDH (Glyceraldehyde 3-phosphate dehydrogenase) was used as an internal control.

Mouse Gsk3? Mm00444911_m1 Mouse Phosphoprotein phosphatase 1 (PP1) Mm00849631_s1 Mouse G6pc Mm00839363_m1 Mouse phosphoenolpyruvate carboxykinase 1 (Pck1) Mm01247058_m1 Mouse GLUT-2 Mm00446224_m1 Mouse GLUT-4 Mm00436615_m1 Mouse peroxisome proliferative activated receptor, gamma, coactivator 1 alpha Mm01208835_m1 Mouse peroxisome proliferator activated receptor gamma 2 Mm00440945_m1 Mouse GAPDH Mm99999915_g1

The results are shown in Fig. 12 and Fig.

The 'normal' group represents the group treated with STZ (Streptozotocin) in the experimental group of Table 8,

'Induction group' refers to the group treated with STZ (Streptozotocin) -treated mice in Table 8

&Quot; Birth-7 &quot; represents a group treated with STZ (Streptozotocin) -treated mice in Table 8.

As can be seen from FIG. 12, the seven strains of the lactic acid bacteria of the present invention significantly lowered the gene expression of the enzyme GSK3? (Glycogen synthase kinase 3 beta) involved in glycogen synthesis in the muscle tissue as compared to the induction group, And GLUT4 were significantly increased compared to the induction group.

As can be seen from FIG. 13, the expression of protein phosphatase 1 (PP1) related to glycogen synthesis in liver tissue was significantly increased compared with the induction group, and GLUT2 related to glucose uptake was significantly It was confirmed that the gene expression was increased.

As can be seen from the test results of Test Example 3 above, the effect of improving the blood glucose level of the seven strains of the lactic acid bacteria of the present invention is that the glucose in the blood is well infused into liver and muscle tissue, And thus the blood glucose level is regulated.

Korea Biotechnology Research Institute KCTC12419BP 20130610 Korea Biotechnology Research Institute KCTC12418BP 20130610 Korea Biotechnology Research Institute KCTC12409BP 20130509 Korea Biotechnology Research Institute KCTC12514BP 20131105 Korea Biotechnology Research Institute KCTC12515BP 20131105 Korea Biotechnology Research Institute KCTC12512BP 20131105 Korea Biotechnology Research Institute KCTC12513BP 20131105

Claims (9)

Bifidobacterium ronggeom (Bifidobacterium longum) HY8005 (accession number: KCTC 12514BP), Bifidobacterium Infante Tees (Bifidobacterium infantis) HY8401 (accession number: KCTC 12419BP), Bifidobacterium lactis (Bifidobacterium lactis) HY8101 (Accession number: KCTC 12418BP), Bifidobacterium breve (Bifidobacterium breve) HY8201 (accession number: KCTC 12515BP), Lactobacillus joined Lee (Lactobacillus gasseri) HY7023 (accession number: KCTC 12512BP), Lactobacillus ash also filler's (Lactobacillus The present invention relates to a method for preventing or treating diabetes by improving insulin resistance, which comprises a complex strain of lactic acid bacteria comprising an acidophilus HY7037 (accession number: KCTC 12409BP) and Lactobacillus casei HY7212 (accession number: KCTC 12513BP) A pharmaceutical composition for therapeutic use.
Bifidobacterium ronggeom (Bifidobacterium longum) HY8005 (accession number: KCTC 12514BP), Bifidobacterium Infante Tees (Bifidobacterium infantis) HY8401 (accession number: KCTC 12419BP), Bifidobacterium lactis (Bifidobacterium lactis) HY8101 (Accession number: KCTC 12418BP), Bifidobacterium breve (Bifidobacterium breve) HY8201 (accession number: KCTC 12515BP), Lactobacillus joined Lee (Lactobacillus gasseri) HY7023 (accession number: KCTC 12512BP), Lactobacillus ash also filler's (Lactobacillus The present invention relates to a method for preventing diabetes by improving insulin resistance, which comprises, as an active ingredient, a complex of a lactic acid bacterium comprising an acidophilus HY7037 (accession number: KCTC 12409BP) and Lactobacillus casei HY7212 (accession number: KCTC 12513BP) Food composition.
Bifidobacterium ronggeom (Bifidobacterium longum) HY8005 (accession number: KCTC 12514BP), Bifidobacterium Infante Tees (Bifidobacterium infantis) HY8401 (accession number: KCTC 12419BP), Bifidobacterium lactis (Bifidobacterium lactis) HY8101 (Accession number: KCTC 12418BP), Bifidobacterium breve (Bifidobacterium breve) HY8201 (accession number: KCTC 12515BP), Lactobacillus joined Lee (Lactobacillus gasseri) HY7023 (accession number: KCTC 12512BP), Lactobacillus ash also filler's (Lactobacillus The present invention relates to a method for preventing diabetes by improving insulin resistance, which comprises, as an active ingredient, a complex of a lactic acid bacterium comprising an acidophilus HY7037 (accession number: KCTC 12409BP) and Lactobacillus casei HY7212 (accession number: KCTC 12513BP) Fermented milk.
Bifidobacterium ronggeom (Bifidobacterium longum) HY8005 (accession number: KCTC 12514BP), Bifidobacterium Infante Tees (Bifidobacterium infantis) HY8401 (accession number: KCTC 12419BP), Bifidobacterium lactis (Bifidobacterium lactis) HY8101 (Accession number: KCTC 12418BP), Bifidobacterium breve (Bifidobacterium breve) HY8201 (accession number: KCTC 12515BP), Lactobacillus joined Lee (Lactobacillus gasseri) HY7023 (accession number: KCTC 12512BP), Lactobacillus ash also filler's (Lactobacillus The present invention relates to a method for preventing diabetes by improving insulin resistance, which comprises, as an active ingredient, a complex of a lactic acid bacterium comprising an acidophilus HY7037 (accession number: KCTC 12409BP) and Lactobacillus casei HY7212 (accession number: KCTC 12513BP) Health functional foods.
Bifidobacterium ronggeom (Bifidobacterium longum) HY8005 (accession number: KCTC 12514BP), Bifidobacterium Infante Tees (Bifidobacterium infantis) HY8401 (accession number: KCTC 12419BP), Bifidobacterium lactis (Bifidobacterium lactis) HY8101 (Accession number: KCTC 12418BP), Bifidobacterium breve (Bifidobacterium breve) HY8201 (accession number: KCTC 12515BP), Lactobacillus joined Lee (Lactobacillus gasseri) HY7023 (accession number: KCTC 12512BP), Lactobacillus ash also filler's (Lactobacillus The present invention relates to a method for preventing diabetes by improving insulin resistance, which comprises, as an active ingredient, a complex of a lactic acid bacterium comprising an acidophilus HY7037 (accession number: KCTC 12409BP) and Lactobacillus casei HY7212 (accession number: KCTC 12513BP) Functional beverage.
The method according to claim 1,
Wherein the improvement in insulin resistance increases the expression of GLUT4 (glucose transporter type 4) gene in muscle cells, thereby increasing glucose uptake in muscle cells.
The method according to claim 1,
The insulin resistance improvement may be caused by glucose uptake by increasing the expression of PPAR-gamma (Peroxisome proliferator-activated receptor gamma) and PGC-1 alpha (Peroxisome proliferator-activated receptor gamma coactivator 1-alpha) Or a pharmaceutically acceptable salt thereof.
The method according to claim 1,
The improvement in insulin resistance is characterized in that blood glucose is regulated by regulating body weight, dietary intake, and abnormal glycemic index (HbA1c) caused by insulin secretion lowering in a diabetic animal model induced by STZ (Streptozotocin) A pharmaceutical composition for therapeutic use.
The method according to claim 1,
The improvement of the insulin resistance may be attributed to an increase in the amount of glucose in the blood due to a decrease in insulin secretion in an animal model of diabetes induced by STZ (Streptozotocin), an increase in glucose uptake and glycogen synthesis in liver and muscle tissue, Wherein the blood glucose level is controlled by enhancing the blood glucose level.

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