KR20150135647A - Lactobacillus brevis JB PML-127 strain having anti-diabetic and anti-hyperlipidemia activity and uses thereof - Google Patents

Abstract

The present invention relates to a Lactobacillus brevis JB PML-127 strain (accession no. KACC91949P) having anti-diabetic and anti-hyperlipidemia activity; to a composition containing the strain or a culture medium of the strain as an active ingredients for anti-diabetes and anti-hyperlipidemia; and to a method for manufacturing a microorganism preparation for anti-diabetes and anti-hyperlipidemia, comprising a step of culturing the strain.

Description

Lactobacillus brevis JB PML-127 strain having anti-diabetic and antihyperlipidemic activity and its use {Lactobacillus brevis JB PML-127 strain having anti-diabetic and anti-hyperlipidemia activity and uses thereof}

The present invention relates to the use of Lactobacillus brevis ( Lactobacillus brevis) the present invention provides a method for producing an antidiabetic or antihyperlipidemic composition comprising an antidiabetic or antihyperlipidemic composition comprising the strain or a culture thereof as an active ingredient and a step of culturing the strain, And a method for manufacturing the same.

Lactic acid bacteria, which produce lactic acid which is beneficial to humans without producing harmful substances such as indole, sucraldehyde, phenol, amine and ammonia, are present in the most abundant density in the intestines of animals. Especially, Holding. The lactic acid is typically a Carnot tumefaciens in (Carnobacterium), Enterococcus genus (Enterococcus), Lactobacillus genus (Lactobacillus), Lactococcus genus (Lactococcus), flow Pocono stock in (Leuconostoc), Phedi O Rhodococcus genus (Pediococcus), the like in Streptococcus (Streptococcus), tetra-gen Rhodococcus genus (Tetragenococcus), Bago Rhodococcus genus (Vagococcus), by sselra in (Weissella) and Bifidobacterium (Bifidobacterium) strains are included. In particular, the strain of the genus Lactobacillus is excellent in safety and is used in various studies. It normally produces a uniform microflora in the intestines of an animal including humans, and enhances immunity, neutralization and synthesis of harmful toxins, increase digestion and absorption of nutrients, It is well known that it represents activity, and it is widely administered to animals as well as humans.

Lactic acid bacteria are widely found in nature and produce carbohydrates by anaerobic use of carbohydrates. The natural environment in which lactic acid bacteria are found is not only present in human or animal intestines but also in various vegetables and fruits and plays an important role in the fermentation process in fermented food such as kimchi, yogurt and German sauerkraut in Korea . These lactic acid bacteria enter the intestines and multiply in the intestinal epithelium, thereby preventing the colonization of harmful microorganisms and secreting antimicrobial substances, thereby suppressing the growth of harmful microorganisms, improving diarrhea and constipation, , Serum cholesterol lowering and the like. In addition, it is generally known that these lactic acid bacteria are directly or indirectly added to foods to improve the storage stability of foods by lactic acid, which is a metabolite thereof, and improve the flavor and texture of foods. The major lactic acid bacteria were Lactobacillus acidophilus , Lactobacillus < RTI ID = 0.0 > casei ), Lactobacillus fermentum fermentum ), Lactobacillus plantarum ( Lactobacillus plantarum ), etc., and these lactic acid bacteria are now widely used as seeds, probiotics and the like of fermented dairy products all over the world.

Diabetes is a disease caused by increasing the concentration of glucose in the blood. α-glucosidase is a digestive enzyme that exists in the brush-border membrane of the small intestine, and acts to hydrolyze the disaccharide or polysaccharide into a monosaccharide that is a state for digestion and absorption of carbohydrates. Absorption of carbohydrates by a-glucosidase in the small intestine is usually done rapidly in the upper small intestine, resulting in a rapid rise in postprandial blood glucose levels. It is known that, in normal people, α-glucosidase such as maltase or sucrase is appropriately suppressed in the small intestine to suppress rapid increase in blood glucose after the meal.

Recently, an increase in blood cholesterol levels due to saturated fatty acids and cholesterol has been recognized as a main cause of atherosclerosis due to excessive intake of animal foods. In order to lower the level of cholesterol in the blood, there are methods such as suppression of cholesterol biosynthesis in the body, and food intake that lowers the level of cholesterol in the blood. For this purpose, attention is paid to development of low cholesterol livestock food and development of functional fermented food that lowers blood cholesterol level . Currently fermented milk and lactic acid bacteria that lower blood cholesterol levels, especially lactobacillus ( Lactobacillus acidophilus have been reported to reduce blood cholesterol levels.

Korean Patent No. 1371648 discloses Lactobacillus brevis which has excellent alcohol decomposing ability, Korean Laid-open Patent Application No. 2013-0068864 discloses Lactobacillus brevis strain producing gamma-amino butyrate, And an Lactobacillus brevis JB PML-127 strain having an antihyperlipidemic activity.

DISCLOSURE OF THE INVENTION The present invention has been made in view of the above-mentioned needs, and it is an object of the present invention to provide Lactobacillus brevis brevis JB PML-127 strain has antidiabetic and anti-hyperlipemic activity, thus completing the present invention.

In order to solve the above problems, the present invention is Lactobacillus brevis (Lactobacillus having anti-diabetic and anti-hyperlipemia activity brevis ) JB PML-127 strain (Accession No. KACC91949P).

The present invention also provides a composition for an anti-diabetic or antihyperlipidemic comprising the strain or a culture thereof as an active ingredient.

In addition, the present invention provides a method for producing a microbial preparation for antidiabetic and antihyperlipidemic comprising the step of culturing the strain.

The inventive Lactobacillus brevis brevis ) JB PML-127 strain has not only cytotoxicity, but also has anti-glucosidase inhibitory activity, intracellular glucose uptake activity, BSH (bile salt hydrolase) activity and cholesterol lowering activity, The antidiabetic or antihyperlipidemic composition containing the Lactobacillus brevis JB PML-127 strain or a culture thereof as an active ingredient can be very usefully used as a food, a food additive or a medicine.

FIG. 1 is a graph comparing cell survival rates after treatment of 293T cells with 0.1%, 0.3%, and 0.5% concentrations of the culture supernatant of Lactobacillus brevis JB PML-127 strain.
Control: No treatment
2 is a graph showing the AGI activity of the culture supernatant of Lactobacillus brevis JB PML-127 strain.
Control: No treatment
FIG. 3 is a graph showing the amount of glucose introduced into muscle cells of the culture supernatant of Lactobacillus brevis JB PML-127 strain.
Control: No treatment
4 is a graph showing BSH (bile salt hydrolase) activity of Lactobacillus brevis JB PML-127 strain.
PC: positive control ( Lactobacillus acidophilus KCTC 3164), NC: negative control ( Escherichia coli ACTC 8739)
5 is a graph showing cholesterol removal rate (%) of Lactobacillus brevis JB PML-127 strain.
Control: no treatment, PC: positive control ( Lactobacillus acidophilus KCTC 3164)

In order to achieve the object of the present invention, the present invention provides a pharmaceutical composition comprising Lactobacillus brevis ( Lactobacillus brevis) brevis JB PML-127 strain.

The lactic acid bacteria was isolated from a traditional Meju In the present invention, it was confirmed that the excellent strain of antidiabetic and anti-hyperlipidemic activity, it Lactobacillus brevis (Lactobacillus brevis ) JB PML-127 strain.

The Lactobacillus brevis (Lactobacillus brevis ) JB PML-127 strain was deposited at the National Institute of Agricultural Science and Technology, National Institute of Agricultural Science and Technology on May 22, 2014 (Accession No .: KACC91949P).

The present invention also provides a composition for an anti-diabetic or antihyperlipidemic comprising the strain or a culture thereof as an active ingredient.

In a composition according to an embodiment of the present invention the composition may preferably be in the form of a food, a food additive or a medicament, the food being selected from the group consisting of dairy products (milk, soy milk, processed milk), fermented milk (liquid yogurt, But are not limited to, be selected from the group consisting of drinks, meat, sausage, bread, chocolate, candy, snacks, confectionery, pizza, ramen, gum, ice cream, soup, beverage, alcoholic beverage and vitamin complex.

The food of the present invention may contain a functional food. In the functional food of the present invention, in addition to the above-mentioned active ingredients, various auxiliary ingredients may be added as necessary. In the case of the functional food of the present invention, vitamins such as vitamin A, vitamin B1, vitamin B2, vitamin B3, vitamin B6, vitamin B12, folic acid, vitamin C, vitamin D3 and vitamin E, Iron, magnesium, potassium, zinc, etc., or lactic acid bacteria.

In addition, among the functional foods of the present invention, health drinks may contain various flavors or natural carbohydrates as additional components such as ordinary beverages. Examples of the flavoring agent include natural sweetening agents such as tau Martin and stevia extract, and synthetic sweetening agents such as saccharin and aspartame. Examples of natural carbohydrates include monosaccharides such as glucose and fructose, disaccharides such as maltose and sucrose, polysaccharides such as dextrin and cyclodextrin, and sugar alcohols such as xylitol, sorbitol and erythritol.

When the strain obtained in the step of culturing the strain of the present invention or a culture solution thereof is used as a food additive, the strain or the culture thereof may be directly added, used in combination with other food or food ingredients, . The amount of the active ingredient to be mixed can be suitably determined according to its intended use (prevention, health or therapeutic treatment).

The present invention also provides a pharmaceutical product comprising the strain of the present invention or a culture solution thereof as an active ingredient. Since the above medicines have antidiabetic and antihyperlipidemic activities, diabetes and hyperlipidemia can be prevented or treated. The strain is preferably Lactobacillus brevis, more preferably Lactobacillus brevis JB PML-127.

 In addition, the present invention provides a method for producing a microbial preparation for antidiabetic and antihyperlipidemic comprising the step of culturing the strain. The method for culturing the strain of the present invention can be carried out according to a method commonly used in the art, and preferably, a culture medium supplemented with L-cysteine is used, but the method is not limited thereto.

The antidiabetic and antihyperlipidemic microbial preparations of the present invention can be produced using the Lactobacillus brevis JB PML-127 strain as an active ingredient. The anti-diabetic and antihyperlipidemic microbial preparations according to the present invention may be prepared as solutions, powders, suspensions, dispersions, emulsions, oily dispersions, pastes, dusts, dispersible materials or granules.

Hereinafter, the present invention will be described in detail with reference to examples. However, the following examples are illustrative of the present invention, and the present invention is not limited to the following examples.

Example  1: Explore traditional meju-derived lactic acid bacteria

1) Strain isolation

In order to separate lactic acid bacteria existing in traditional meju, commercially available meju was collected, homogenized Meju was uniformly pulverized, and sterilized physiological saline containing 0.85% sodium chloride was mixed, diluted by deciduous dilution method, and spread on BCP agar medium . A single colony which had turned yellow by the production of lactic acid in the BCP agar medium was separated and added to lactic acid bacteria MRS (Difco Laboratories, Detroit, MI, USA) agar medium (1.5% agar, w / v) containing 0.05% L-cysteine And the lactic acid bacteria were isolated.

2) Identification using 16S rRNA sequencing

Identification was carried out using 16S rRNA sequencing to isolate the food - derived lactic acid bacteria by molecular biology. DNA was isolated from the cultured cells using Wizard Genomic DNA Prep. (5'-AGA GTT TGA TCC TGG CTC AG-3 ': SEQ ID NO: 2) and reverse primer (SEQ ID NO: 2) were used to amplify 16S rDNA using a genomic DNA kit (GeneAll Biotechnology, 1492R) (5'-TAC GGT TAC CTT GTT ACG ACT T-3 ': SEQ ID NO: 3). The PCR reaction products were confirmed by electrophoresis on 2% agarose gel and purified using GeneAllR Gel SV kit (GeneAll Biotechnology, Korea) for better gene sequencing quality and used for sequencing. For DNA sequencing, the nucleotide sequence of the amplified PCR products was analyzed using a bacterial 16S rRNA 27F, 1492R primer using an automated sequencer (ABI 3130). The analysis of homology of the DNA sequence of SEQ ID NO: 1 obtained from the isolated lactic acid bacteria was carried out using the NCBI BLAST N program (http: //www.ncbi.nlm.nihgov/blast/). Lactobacillus brevis ( Lactobacillus brevis ), which was named Lactobacillus brevis JB PML-127.

Example  2: Safety evaluation

Cytotoxicity was confirmed by the cell viability of the sample in normal kidney epithelial cell line (293T cells). Plant-derived Lactobacillus brevis brevis) JB PML-127 to check the strength of the normal cell line, a human kidney epithelial cell line to (Human kidney epithelial cell line) in 293T cells 3- (4,5-dimethylthiazol-2- yl) -5- (3-carboxy in (MTS, promega, USA) assay for the survival rate of the cells. The culture supernatant of JB PML-127 was treated with 0.1%, 0.3% and 0.5% concentration of 293T cells to confirm toxicity. As a result, it was confirmed that the culture supernatant did not exhibit cytotoxic effect up to the concentration of 0.5% (Fig. 1).

Example  3: Anti-diabetic  Functional evaluation

1)? -Glucosidase (AGI) inhibitory activity

In this experiment, the food-derived lactic acid bacteria Lactobacillus brevis In order to confirm the AGI activity of JB PML-127, activity evaluation was carried out using the culture supernatant of the strain. The culture supernatant was used after 0.22 ㎛ filter. (PH 7.0) and 2.5 mM ρ-nitrophenyl a-glucopyranoside dissolved in buffer were added, and the mixture was reacted at 37 ° C. for 20 minutes. Then, the mixture was reacted at 405 nm The activity was measured by measuring the absorbance. As a result, it was confirmed that the culture broth of Lactobacillus brevis JB PML-127 showed much higher AGI activity than acarbose as a positive control (Fig. 2).

2) 2-NBDG uptake into muscle cells

In type 2 diabetes, insulin resistance occurs in peripheral tissues including muscle and adipose tissue due to insulin absorption and secretory disorder, and the disturbance of blood glucose absorption in these peripheral tissues leads to an increase in blood glucose level. When the insulin binds to the insulin receptor, the cell induces a signal transduction cascade that causes glucose to enter the cell via GLUT4 (Glucose transporter-4). Therefore, in order to evaluate blood glucose uptake in C2C12 muscle cells, 2-NBDG was treated to measure the amount of glucose introduced into muscle cells by fluorescence.

In order to confirm the intracellular glucose absorption function of Lactobacillus brevis JB PML-127 strain, the degree of glucose absorption was evaluated using the culture supernatant of the strain. The C2C12 cell line, which is a muscle cell, was cultured in a 96 well plate at 37 ° C in a 5% CO ₂ incubator for 24 hours, and then the medium was changed to a no glucose medium and starved. The culture supernatant and 2-N- (7-nitrobenz-2-oxa-1,3-diazol-4-yl) amino-2-deoxy-d-glucose (2-NBDG) were added thereto. After the culture medium was removed, the cells were washed with PBS, and 100 쨉 l of PBS was dispensed and measured using a fluorescence micro-reader (EX / EM = 488/535 nm). As a result, it was confirmed that Lactobacillus brevis JB PML-127 strain had a higher intracellular glucose level than control (Fig. 3).

Example  4: Evaluation of antihyperlipidemic function

1) Measurement of BSH (Bile salt hydrolase) activity

In recent years, the measurement of BSH (bile salt hydrolase) activity has been studied as a mechanism of lowering the cholesterol level of lactic acid bacteria in blood. Therefore, the method of measuring complex bile acid degradation activity was used as an evaluation method for measuring the cholesterol function of lactic acid bacteria isolated from native resources.

The BSH activity measurement was performed using a positive control (PC) and the standard strain Lactobacillus acidophilus KCTC 3164, negative control (NC), Escherichia coli coli ) ACTC 8739, and Lactobacillus brevis, a lactic acid bacterium isolated from native sources brevis JB PML-127 was used to measure BSH activity. Of the lactic acid bacteria in disc diffusion method (Lactobacilli) MRS (Control), lactic acid bacteria (Lactobacilli) of the MRS contain (Taurodeoxycholic acid) TDCA MRSBA-T , lactic acid bacteria (Lactobacilli) MRS contain (Glycodeoxycholic acid) GDCA the MRSBA-G plate medium The cells were incubated at 37 ° C for 72 hours. The diameters of the acicular or opaque white granular weevils formed around the discs were measured and compared. The results are shown in FIG. As a result, the negative control group (NC) showed no acellular or opaque white granular bacilli. In the positive control group (PC), both of the two types of complex bile acids, TDCA and GDCA, Were observed. Lactobacillus brevis JB PML-127 showed no decomposition activity in TDCA (Taurodeoxycholic acid) and only degradation activity in GDCA (Glycodeoxycholic acid). In particular, it was confirmed that the acupuncture formed was 2.6 times larger than that in the positive control, and it was confirmed that opaque white granular bacilli were formed around the acupuncture conversion.

2) Measurement of cholesterol removal effect

Studies have been carried out to demonstrate the beneficial effects of Lactobacilli and Bifidobacteria on lipid metabolism after the fermented milk containing lactic acid bacteria has been shown to have a cholesterol-lowering effect in the human body over the last several decades. Studies have shown that some lactic acid bacteria have a cholesterol-lowering effect in the host.

Little is known about the mechanism of the direct effect of fermented milk on reducing cholesterol, but it has been reported that cholesterol is reduced in vitro by absorption or assimilation of cholesterol by bacterial cells. They found that cholesterol uptake by Lactobacillus acidophilus occurs when cultured in the presence of bile, an environment similar to the body. To measure the cholesterol removal effect, the standard strain Lactobacillus ( Lactobacillus acidophilus ) KCTC 3164 and Lactobacillus brevis ( Lactobacillus brevis, brevis JB PML-127 was used. Cholesterol was extracted from the culture supernatant after incubation for 24 hours in a 1% Lactobacilli MRS liquid medium supplemented with water-soluble cholesterol and oxgall. The water soluble cholesterol and the sample, which are standard substances, were measured at OD ₅₅₀ using a spectrophotometer, and the results are shown in FIG. As a result, the positive control group (PC) showed about 25% cholesterol reduction effect. Compared with the positive control group, Lactobacillus brevis JB PML-127 showed a 0.7-fold reduction in cholesterol.

Agricultural Biotechnology Research Institute KACC91949P 20140522

<110> JEONJU BIOMATERIALS INSTITUTE <120> Lactobacillus brevis JB PML-127 strain having anti-diabetic and          anti-hyperlipidemia activity and uses thereof <130> PN14138 <160> 3 <170> Kopatentin 2.0 <210> 1 <211> 229 <212> DNA <213> Lactobacillus brevis <400> 1 aaccggagaa gtgcatcgga aactgggaga cttgagtgca gaagaggaca gtggaactcc 60 atgtgtagcg gtggaatgcg tagatatatg gaagaacacc agtggcgaag gcggctgtct 120 agtctgtaac tgacgctgag gctcgaaagc atgggtagcg aacaggatta gataccctgg 180 tagtccatgc cgtaaacgat gagtgctaag tgttggaggg tttccgccc 229 <210> 2 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 2 agagtttgat cctggctcag 20 <210> 3 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 3 tacggttacc ttgttacgac tt 22

Claims (4)

Lactobacillus brevis JB PML-127 strain (Accession No. KACC91949P) having antidiabetic and antihyperlipidemic activity. A composition for an anti-diabetic or antihyperlipidemic comprising the strain of claim 1 or a culture thereof as an active ingredient. The composition according to claim 2, wherein the composition is in the form of a food, a food additive or a medicine. A method for producing a microbial preparation for antidiabetic and antihyperlipidemic comprising culturing the strain of claim 1.

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