KR101956867B1 - Lactobacillus rhamnosus CKDB009 having anti-obesity and cholesterol lowering effect and uses thereof - Google Patents

Abstract

Lactobacillus lambatus CKDB009 strain, a novel lactic acid bacterium, has an effect of reducing cholesterol and preventing obesity in the body. More specifically, the Lactobacillus lambatus CKDB009 strain has acid resistance and bile resistance, which are fundamental properties of probiotics, and does not produce intestinal harmful enzymes such as urease and gelatinase, resulting in hemolysis , It has excellent safety against intestinal environment. In particular, the Lactobacillus lymphosus CKDB009 strain has excellent cytotoxicity and cytotoxicity, and inhibits the differentiation of 3T3-L1, a lipid precursor cell, to inhibit peroximal proliferator-activated receptor-γ (PPARγ) and adipocyte binding protein (aP2). < RTI ID = 0.0 > (a2) < / RTI > In addition, the Lactobacillus lambosus CKDB009 strain reduces the body's cholesterol, inhibits lipid production in white adipocytes and reduces fat cell size, thereby reducing body mass index (BMI) It works effectively for numerical relaxation. Accordingly, the Lactobacillus lambosus CKDB009 strain of the present invention and the composition containing it can be industrially useful as a blood cholesterol reducing and anti-obesity functional food.

Description

Lactobacillus rhamnosus CKDB009 having anti-obesity and cholesterol lowering effect and uses thereof,

The following examples relate to lactic acid bacteria having cholesterol abatement and obesity relieving effect and their use, and more particularly to lactobacillus lambosus strain CKDB009 having a cholesterol reduction and obesity relieving effect, a probiotic preparation containing the same, Functional food compositions and pharmaceutical compositions.

Obesity is a metabolic disease and has been declared an object of the disease to be treated by the World Health Organization (WHO). Obesity is a condition in which excess fat tissue accumulates in the body, and the main reason for causing obesity is the increase in body weight due to ingestion of high calorie or high fat foods, lack of exercise, . However, in recent years, it has been reported that not only eating habit but also neuroendocrine system abnormality, drug, genetic cause and biochemical adverse reaction may cause obesity.

These lipogenic precursor cells that produce in vivo fat have been studied in vitro for 3T3-L1 and 3T3-F442A cells for many years and the process of differentiation of 3T3-L1 preadipocytes into adipocytes (PPARγ) and C / EBPα, which are regulated mainly on C / EBP-β and C / EBPδ, which are CCAAT / enhancer binding proteins. In particular, PPARγ and C / EBPα are key transcription factors that regulate lipogenesis. Among them, PPARγ is a kind of lipid sensing peroxisome proliferator-ctivated receptors. It is expressed by C / EBPα, And leads to accumulation of fat. These transcription factors and adipocyte regulatory factors promote the differentiation of adipocytes, and the expression levels of adipocyte-specific proteins such as adipocyte protein 2 (adipocyte protein 2) and adipocyte fatty acid binding protein 4 (adipocyte fatty acid binding protein 4) .

Meanwhile, as the risks of various complications arising from obesity and obesity are increasing, interest in obesity treatment is increasing not only in domestic but also in the world, but the treatment of obesity showing a definite therapeutic effect is insignificant. Moreover, the drugs used for the treatment of obesity have various side effects such as dry mouth, numbness, constipation, insomnia, nausea, dizziness and insomnia, and it is required to develop a therapeutic agent for obesity which has high affinity for human body .

Therefore, researches on probiotic lactic acid bacteria as a natural substance that can replace obesity therapy have been carried out, and various studies on the correlation between intestinal microflora and obesity have been published, and some probiotic lactic acid bacteria Have been published. The mechanism by which these probiotics affect the anti-obesity is to produce conjugated linoleic acid (CLA) (trans-10, cis-12 conjugated linoleic acid) or to improve the thermogenesis of brown fat, Or by acting as a fasting-induced adipose factor (FIAF), or by controlling the activity of the central pituitary gland.

In addition, it is known that such probiotic lactic acid bacteria have a function of lowering the absorption of external cholesterol taken in combination with bile acid and cholesterol, or having a cholesterol lowering effect in blood through self-produced bile acid degrading enzyme, . In particular, the mechanism of cholesterol-lowering by the Lactobacillus strain is that the released bile acid is deconjugated by the lactobacillus strain and is not reabsorbed to the liver, resulting in increased consumption of cholesterol, a raw material of bile acid biosynthesis, Is reduced.

As for the anti-obesity effect of the probiotic lactic acid bacteria and the effect of lowering the cholesterol of blood, Korean Patent Laid-Open No. 2010-0109661 discloses a novel Lactobacillus kvetetus strain having a blood cholesterol lowering effect and an obesity inhibitory effect, Limited and the effect has not reached a satisfactory level.

Therefore, it is required to research and develop new lactic acid bacteria having improved cholesterol abatement and obesity relieving effect.

SUMMARY 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 a novel lactic acid bacterium which is excellent in acid resistance and bile resistance, has safety, has cholesterol reducing ability, antioxidant ability and obesity- Lactobacillus rhamnosus CKDB009 strain was obtained and cell test for the above strain was confirmed to be free from cytotoxicity, reduction of intracellular fat content, inhibition of mRNA expression of Aipogenesis transcription factor, and animal experiment to determine body weight and body fat mass , Reduces liver and blood lipid content, inhibits the mRNA expression of obesity-related proteins, increases mRNA expression of lipid metabolism-related proteins, and has an improved anti-obesity effect that reduces histologically the size of fat cells The present inventors have completed the present invention.

Therefore, it is an object of the present invention to provide a novel lactic acid bacterium with reduced cholesterol and an anti-obesity effect.

In order to attain the above object, the present invention provides a lactic acid bacterium having cholesterol lowering and obesity prevention and improvement effect, which comprises 16s rRNA as a Lactobacillus rhamnosus CKDB009 strain containing the nucleotide sequence shown in SEQ ID NO: 1 : KCTC13249BP).

According to one aspect, the present invention provides Lactobacillus lambosus strain, which is a novel lactic acid bacterium isolated from neonatal feces.

According to another aspect of the present invention, there is provided a probiotic composition which is resistant to intestinal toxicity such as urease and gelatinase, exhibits acid resistance and biliary properties, which are fundamental properties of probiotics, Lactobacillus lansosus strain having cholesterol-lowering effect is provided.

According to another aspect of the present invention, the present invention provides a method for inhibiting the differentiation of 3T3-L1 cells into adipocytes by reducing the blood cholesterol significantly and inducing the peroxisome proliferator-activated receptor-γ (PPARγ ), an effect of inhibiting the expression of adipocyte binding protein (aP2), and an effect of reducing the size of adipocytes themselves.

According to another aspect, there is provided a method for reducing obesity in an animal model that induces obesity through high-fat, high-cholesterol diet, reducing body mass index (BMI), decreasing the weight of white adipose tissue and cholesterol content in white adipose tissue, The cholesterol content in the serum and the cholesterol content in the liver are decreased, the activity of the lipolytic enzyme in the white fat tissue is increased, the adipogenesis-related factors in the white fat tissue are decreased, and the histopathological analysis The present invention provides Lactobacillus lansos strains effective for reducing obesity and blood cholesterol levels by reducing the size of liver tissue and white adipose tissue.

The present invention also provides a probiotic preparation comprising at least one selected from the group consisting of the strain, the culture of the strain, the concentrate of the culture and the dried product as an active ingredient.

The present invention also provides a food and beverage comprising at least one selected from the group consisting of the strain, the culture of the strain, the concentrate of the culture and the dried product as an active ingredient.

The present invention also provides a health functional food composition for preventing or ameliorating an anti-inflammatory and metabolic disease comprising at least one member selected from the group consisting of the strain, the culture of the strain, the concentrate of the culture and the dried product do.

The present invention also provides a pharmaceutical composition for the prophylaxis or treatment of anti-inflammatory and metabolic diseases comprising, as an active ingredient, at least one selected from the group consisting of the strain, the culture of the strain, the concentrate of the culture and the dried product .

According to one aspect of the present invention, there is provided a composition prepared by culturing the strain to recover the microbial cells, lyophilizing and then pulverizing the powder to obtain a biocide. At this time, in the present invention, the culture broth is centrifuged, followed by rapid freezing in a -60 freezer, lyophilization under 0-45 temperature condition, followed by pulverization to obtain a powder, but not limited thereto, Various methods known in the art can be used in such a manner that the powder is obtained by lyophilization after drying.

The present invention provides a novel lactic acid bacterium having a cholesterol reducing effect and an obesity-suppressing effect in the body, and a probiotic preparation, a health functional food composition and a pharmaceutical composition containing the same.

The present invention also provides a lactic acid bacterium which does not produce intestinal harmful enzymes as a probiotics and is excellent in digestive juice resistance, antioxidative ability and cholesterol reducing ability, and probiotic preparations, health functional food compositions and pharmaceutical compositions containing them.

The present invention also provides a lactic acid bacterium having excellent effect of inhibiting the differentiation of lipid precursor cells and promoting cell differentiation, a probiotic preparation containing the same, a health functional food composition, and a pharmaceutical composition.

The present invention also relates to a lactic acid bacterium having excellent effects of reducing the accumulation of body weight and body fat, reducing the content of blood LDL cholesterol, decreasing the expression of obesity and lipid metabolism-related genes, and reducing the size of white lipids, and a probiotic preparation , A health functional food composition and a pharmaceutical composition.

In addition, the present invention provides a lactic acid bacterium which is industrially useful as a blood cholesterol and a functional food, a probiotic preparation containing the same, a health functional food composition and a pharmaceutical composition.

FIG. 1 is a graph showing the results of calculating the neutral fat content in 3T3-L1 cells according to the present invention when treated with the novel lactic acid bacteria.
FIG. 2 is a graph showing the results of confirming the sizes of lipids per control and treatment by staining 3T3-L1 cells with the novel lactic acid bacteria of the present invention. FIG.
FIG. 3 is a graph showing the results obtained by dissolving cells to confirm the fat accumulation in 3T3-L1 cells and confirming their absorbance by treating the novel lactic acid bacteria of the present invention.
FIG. 4 is a graph showing the expression levels of PPARγ, C / EBPα, and aP2 among mRNAs involved in lipid differentiation in 3T3-L1 cells in the treatment with the novel lactic acid bacteria of the present invention.
FIG. 5 is a graph showing the effect of the lactic acid bacterium powder of the present invention on body weight, percent body weight gain (%), and BMI index.
6 is a graph showing the effect of the lactic acid bacterium powder ingestion of the present invention on the weight of white adipose tissue of an animal.
FIG. 7 is a graph showing the effect of the lactic acid bacteria powder ingestion of the present invention on total triglyceride, total cholesterol, HDL-cholesterol and LDL / VLDL-cholesterol content in serum.
8 is a graph showing the effect of the lactic acid bacterium powder ingestion of the present invention on triglyceride and total cholesterol content in liver tissue.
9 is a graph showing the effect of the lactic acid bacterium powder ingestion of the present invention on the activity of lipolytic enzyme in white adipose tissue.
10 is a graph showing the effect of the lactic acid bacterium powder ingestion of the present invention on the expression of PPARγ, C / EBPα, which is a fat-differenting cell transcription factor in white adipose tissue.
FIG. 11 is a graph showing the effect of the lactic acid bacterium powder ingestion of the present invention on the expression of UCP-1 and UCP-2, heat-related genes in white adipose tissue.
12 is a graph showing the effect of the lactic acid bacterium powder ingestion of the present invention on the expression of CYP7A1, a factor involved in homeostasis of cholesterol and bile acid in liver tissue.
13 is a graph showing the results of histological examination of the influence of the lactic acid bacteria powder ingestion of the present invention on lipid perforation in liver tissue.
FIG. 14 is a diagram showing the results of histological confirmation of the effect of the lactic acid bacterium powder ingestion of the present invention on lipid perforation in white adipose tissue.

Hereinafter, embodiments according to the present invention will be described in detail with reference to the accompanying drawings. However, the present invention is not limited to or limited by the embodiments. In addition, the same reference numerals shown in the drawings denote the same members.

Also, terminologies used herein are terms used to properly represent preferred embodiments of the present invention, which may vary depending on the viewer, the intention of the operator, or the custom in the field to which the present invention belongs. Therefore, the definitions of these terms should be based on the contents throughout this specification.

In order to achieve the object of the present invention, the present invention provides a Lactobacillus rhamnosus CKDB009 strain having cholesterol reduction and obesity inhibitory activity in the body.

The present inventors have succeeded in isolating lactic acid bacteria from a sample collected from the neonatal feces and isolating the lactic acid bacteria, and found that the lactic acid bacteria isolated had the acid resistance, bile resistance, safety, cholesterol lowering ability, antioxidant ability, cytotoxicity, Analysis of mRNA expression revealed that the isolated lactic acid bacterium exhibits excellent acid resistance and biliary properties, has safety, has excellent cholesterol reducing ability and antioxidant ability, has no cytotoxicity, reduces the intracellular fat content, , The isolated strain containing the nucleotide sequence shown in SEQ ID NO: 1 as 16s rRNA was designated as Lactobacillus lambosus CKDB009 and deposited in the genetic bank of the Korea Biotechnology Research Institute (accession number: KCTC13249BP).

The strain according to an embodiment of the present invention is a strain selected from the group consisting of D-arabinose, ribose, D-galactose, D-glucose, D-fructose, D- mannose, L- sorbose, rhamnose, dulcitol, mannitol, Glucoside, amigalline, arbutin, salicin, cellobiose, maltose, lactose, saccharose, trehalose, meleletose, beta -gentiobiose, D-tagatose But are not limited to, one or more sugars selected from the group of glucose, glucose, L-fucose, and gluconate.

The strain according to an embodiment of the present invention has acid resistance and biliary cholesterol and does not produce at least one intestinal harmful enzyme selected from the group consisting of urease and gelatinase, But is not limited to, it is characterized by excellent safety against intestinal environment.

The strain according to an embodiment of the present invention is characterized by having a cholesterol-reducing ability and an antioxidant ability, but is not limited thereto.

The strain according to one embodiment of the present invention has no cytotoxicity and is selected from the group consisting of peroximal proliferator-activated receptor-γ (PPARγ) and adipocyte binding protein (aP2) by inhibiting differentiation of 3T3-L1, But is not limited to, having the effect of reducing the expression of one or more obesity-related biochemical markers.

The strain according to one embodiment of the present invention is characterized in that it has an effect of lowering blood cholesterol in the body, inhibiting lipid production in white adipose tissue and decreasing adipocyte size, thereby reducing body weight.

The present invention also provides a probiotic preparation comprising as an active ingredient at least one member selected from the group consisting of the Lactobacillus lambosus CKDB009 strain, the culture of the strain, the concentrate of the culture, and the dried product.

The Lactobacillus lambosus CKDB009 strain of the present invention has excellent acid resistance and biliary cholesterol and does not produce intestinal toxic enzymes and does not cause hemolysis, thereby having excellent safety against intestinal environment and thus being useful as probiotics Can be used.

The probiotic agent can be prepared and administered in various formulations and methods according to methods known in the art. For example, the Lactobacillus lambosus CKDB009 strain of the present invention, the culture thereof, the concentrate of the culture broth or the dried product thereof may be mixed with a carrier commonly used in the pharmaceutical field to prepare powders, liquids and solutions, for example, in the form of tablets, capsules, syrups, suspensions or granules, and the like. Examples of the carrier include, but are not limited to, binders, lubricants, disintegrants, excipients, solubilizers, dispersants, stabilizers, suspending agents, coloring matters and fragrances. The administration dose can be appropriately selected depending on the degree of absorption of the active ingredient in the body, the inactivation rate, the excretion rate, the age, sex, strain, condition and severity of the disease.

The present invention also relates to a pharmaceutical composition for preventing or ameliorating an anti-inflammatory and metabolic disease comprising at least one selected from the group consisting of the above-mentioned Lactobacillus laminosus CKDB009 strain, a culture of the strain, a concentrate of the culture, To provide a food composition.

The health functional food composition of the present invention may be prepared in any one form selected from the group consisting of tablets, granules, powders, capsules, liquid solutions and rings. The health functional food composition according to the present invention may be formulated into powders, liquids, tablets, soft capsules, granules, tea bags, instant tea or drink form containing the Lactobacillus lambosus CKDB009 strain as an active ingredient have. The content of the Lactobacillus laminosus CKDB009 strain as an active ingredient can be appropriately determined depending on the intended use (for prevention or improvement). Generally, the amount of the Lactobacillus laminosus CKDB009 strain contained in the food composition may be 0.1 to 90% by weight of the total food weight. However, in the case of long-term consumption intended for health and hygiene purposes or for health control purposes, the amount may be less than the above range. In addition, the food composition according to the present invention may contain, in addition to the lactobacillus rhamnosus CKDB009 strain, other ingredients capable of synergistic effect to the main effect within a range not impairing the intended main effect of the present invention Do.

The food composition formulated in this form can be added directly to the food or used together with other food or food ingredients, and can be suitably used according to conventional methods. Examples of food products include dairy products including drinks, meat, sausage, bread, biscuits, rice cakes, chocolate, candies, snacks, confectionery, pizza, ramen, other noodles, gums, ice cream, soups, , Dairy products and dairy products, and all health functional foods in the ordinary sense are included.

When the food composition of the present invention is a beverage, it contains Lactobacillus lambosus CKDB009 strain as an essential ingredient at the indicated ratio, and there are no particular restrictions on other ingredients used for the purpose of producing other beverages, A flavoring agent or a natural carbohydrate as an additional ingredient. Examples of such natural carbohydrates include monosaccharides such as glucose, fructose, and the like; Disaccharides such as maltose, sucrose and the like; And polysaccharides, for example, conventional sugars such as dextrin, cyclodextrin and the like, and sugar alcohols such as xylitol, sorbitol and erythritol. Natural flavors and synthetic flavors and the like can be used as the flavors other than those described above. The ratio of the natural carbohydrate is generally about 1 to 20 g, preferably about 5 to 12 g per 100 ml of the composition of the present invention.

In addition, the food composition of the present invention can be used as a food composition containing various nutrients, vitamins, minerals (electrolytes), flavorings such as synthetic flavors and natural flavors, colorants and enhancers (cheese, chocolate etc.), pectic acid and its salts, Salts, organic acids, protective colloid thickeners, pH adjusting agents, stabilizers, preservatives, glycerin, alcohols, carbonating agents used in carbonated beverages and the like. In addition, the food composition of the present invention may contain natural fruit juice and pulp for the production of fruit juice drinks and vegetable drinks. These components may be used independently or in combination. The proportion of such additives is not so important, but is generally selected in the range of 0.1 to about 20 parts by weight per 100 parts by weight of the Lactobacillus laminosus CKDB009 strain of the present invention.

The present invention also relates to a pharmaceutical composition for the prevention or treatment of anti-inflammatory and metabolic diseases comprising as an active ingredient at least one member selected from the group consisting of the above-mentioned Lactobacillus laminosus CKDB009 strain, the culture of said strain, the concentrate of said culture and the dried product Lt; / RTI >

The amount of the pharmaceutical composition for preventing or treating anti-inflammatory and metabolic diseases used in the present invention should be a pharmaceutically effective amount. As used herein, the term "pharmaceutically effective amount" means an amount sufficient to treat a disease at a reasonable benefit / risk ratio applicable to medical treatment and the effective dose level will vary depending on the species and severity, The type of virus being infected, the activity of the drug, the sensitivity to the drug, the time of administration, the route of administration and rate of release, the duration of the treatment, factors including co-administered drugs and other well known factors in the medical field. Effective amounts may vary depending on the route of treatment, the use of excipients, and the likelihood of use with other agents, as will be appreciated by those skilled in the art.

The pharmaceutical compositions for the prevention or treatment of anti-inflammatory and metabolic diseases of the present invention may be formulated into pharmaceutical formulations using methods well known in the art so as to provide rapid, sustained or delayed release of the active ingredient after administration to the mammal . In the preparation of the formulations, it is preferred that the active ingredient is mixed with or diluted with the carrier, or enclosed in a carrier in the form of a container.

Accordingly, the pharmaceutical composition for the prevention or treatment of anti-inflammatory and metabolic diseases of the present invention may be formulated into oral preparations such as powders, granules, tablets, capsules, suspensions, emulsions, syrups and aerosols, external preparations and patches And may further comprise suitable carriers, excipients or diluents conventionally used in the preparation of the compositions.

Examples of carriers, excipients and diluents that can be included in the pharmaceutical composition of the present invention include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium Cellulose, methylcellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil. In the case of formulation, a diluent or excipient such as a filler, an extender, a binder, a wetting agent, a disintegrant, or a surfactant is usually used.

Hereinafter, the present invention will be described in more detail with reference to Examples. It is to be understood by those skilled in the art that these embodiments are merely illustrative of the present invention and that the scope of the present invention is not construed as being limited by these embodiments.

Ⅰ. Isolation and Identification of Lactic Acid Bacteria

Example 1. Isolation of lactic acid bacteria

The Lactobacillus lambosus CKDB009 strain used in the present invention was obtained by collecting samples from the neonatal feces. To isolate the lactic acid bacteria in the neonatal feces, the cells were suspended in sterilized anaerobic water, decidualized, plated on MRS (ManRogosaSharpe) solid medium, cultured at 37 ° C for 48 hours, and then purified. Biochemical and molecular biochemical identification of isolates was performed. As a result, 135 strains identified as Lactobacillus, Lactococcus, Luconobacter and Pediococcus were obtained, and acid tolerance and biliary exudation test were carried out to confirm resistance to digestive juices as a probiotic strain.

Example 2. Identification and characterization of Lactobacillus lambosus CKDB009 (Lactobacillus rhamnosus CKDB009)

<2-1> Identification of microorganism (glycosylation analysis and identification of 16s rRNA)

1) Usability analysis

The lactic acid bacteria selected as described above were tested for their sugar availability using an API CHL50 kit. As a result of the analysis, as shown in Table 1, D-arabinose, ribose, D-galactose, D-glucose, D-fructose, D- mannose, L- sorbose, rhamnose, dulcitol, mannitol, sorbitol, but are not limited to, α-methyl-D-glucoside, N- acetylglucosamine, amigalline, arbutin, salicin, cellobiose, maltose, lactose, saccharose, trehalose, meleletose, L-fucose, and gluconate.

Glycosylation of Lactobacillus lambosus CKDB009 temperament reaction temperament reaction Blank - Esculin - Glycerol - Salincin + Erythritol - Cellobiose + D-arabinose w maltose + L-arabinose - Lactose + Ribose + Melibiose - D-xylose - Saccharose w L-xylose - Trehalose + Andonitor - Inulin - beta -methyl-xyloside - Meletitose + D-galactose + D-raffinose - D-glucose + Amidon - D-fructose + Glycogen - D-mannose + Xylitol - L-sorbose + β-Gentiobiose + Rhamnoose w D-Turanos - Dallitol + D-Rick Source - Inositol - D-Tagatose + Mannitol + D-fucose - Sorbitol + L-fucose + alpha -methyl-D-mannose - D-arabitol - alpha -methyl-D-glucoside + L-arabitol - N-acetylglucosamine + Gluconate w Amigalline + 2-keto-gluconate - Arbutin + 5-keto-gluconate -

2) Identification of 16s rRNA

The colonies grown on the MRS medium were harvested and double stranded DNA sequencing (Solgent, Korea) was performed. The obtained sequence was searched by BLAST and strains were identified. As a result, 100% homology with Lactobacillus rhamnosus was confirmed. As a result, it was confirmed that the novel microorganism of the present invention belonged to Lactobacillus rhamnosus species. The results of rRNA sequencing are shown below.

TGGTTCAGGATGAACGCGGGCGGCGTCCTTAATACATGCAAGTCGAAGGAGTTCTGATTA

TTGAAAGGTGCTTGCATCTTGATTTAATTTTGAACGAGTGGCGGACGGGTGAGTAACACG

TGGGTAACCTGCCCTTAAGTGGGGGATAACATTTGGAAACAGATGCTAATACCGCATAAA

TCCAAGAACCGCATGGTTCTTGGCTGAAAGATGGCGTAAGCTATCGCTTTTGGATGGACC

CGCGGCGTATTAGCTAGTTGGTGAGGTAACGGCTCACCAAGGCAATGATACGTAGCCGAA

CTGAGAGGTTGATCGGCCACATTGGGACTGAGACACGGCCCAAACTCCTACGGGAGGCAG

CAGTAGGGAATCTTCCACAATGGACGCAAGTCTGATGGAGCAACGCCGCGTGAGTGAAGA

AGGCTTTCGGGTCGTAAAACTCTGTTGTTGGAGAAGAATGGTCGGCAGAGTAACTGTTGT

CGGCGTGACGGTATCCAACCAGAAAGCCACGGCTAACTACGTGCCAGCAGCCGCGGTAAT

ACGTAGGTGGCAAGCGTTATCCGGATTTATTGGGCGTAAAGCGAGCGCAGGCGGTTTTTT

AAGTCTGATGTGAAAGCCCTCGGCTTAACCGAGGAAGTGCATCGGAAACTGGGAAACTTG

AGTGCAGAAGAGGACAGTGGAACTCCATGTGTAGCGGTGAAATGCGTAGATATATGGAAG

AACACCAGTGGCGAAGGCGGCTGTCTGGTCTGTAACTGACGCTGAGGCTCGAAAGCATGG

Gt;

GGAGGGTTTCCGCCCTTCAGTGCCGCAGCTAACGCATTAAGCATTCCGCCTGGGGAGTAC

GACCGCAAGGTTGAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTG

GTTTAATTCGAAGCAACGCGAAGAACCTTACCAGGTCTTGACATCTTTTGATCACCTGAG

AGATCAGGTTTCCCCTTCGGGGGCAAAATGACAGGTGGTGCATGGTTGTCGTCAGCTCGT

GTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTATGACTAGTTGCCAGCA

TTTAGTTGGGCACTCTAGTAAGACTGCCGGTGACAAACCGGAGGAAGGTGGGGATGACGT

CAAATCATCATGCCCCTTATGACCTGGGCTACACACGTGCTACAATGGATGGTACAACGA

Gt;

GCAACTCGCCTACACGAAGTCGGAATCGCTAGTAATCGCGGATCAGCACGCCGCGGTGAA

TACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCAT

<2-2> Microbial characteristics

The characteristics of the novel Lactobacillus lambosus CKDB009 according to the present invention are as follows.

1) Types of bacteria

When cultured in MRS agar plate medium at 37 ° C for 48 hours, the morphology of the bacteria

① Cell shape: Bacillus

② Mobility: None

③ Spore forming ability: None

④ Gram staining: positive

2) Form of colony

MRS agar plate culture medium at 37 ℃ for 48 hours

① Shape: Circular

② Bump: convex

③ Surface: smooth (smooth)

④ Color: milky white

3) Physiological properties

① Growth temperature

Growth possible Growth temperature: 15 ~ 40 ℃

Optimum growth temperature: 36 ~ 38 ℃

② Growth pH

Growth possible pH: 4.6 ~ 7.5

Optimum pH: 6.0 ~ 7.0

③ Effect on oxygen: Tumor anaerobic

4) Catalase: negative

5) Whether to generate gas: negative

6) Indole production: negative

7) Lactic acid production: positive

8) Biomolecular production: negative

The new strain isolated from the feces of the newborn on the basis of the microorganism identification result and the characteristics of the microorganism was designated as Lactobacillus rhamnosus CKDB009 and was deposited at Korean Biotechnology Research Institute (Accession No .: KCTC13249BP) on Apr. 12, 2017 &Lt; / RTI &gt;

Ⅱ. Characteristics of novel lactic acid bacteria

Example 3 Digestive Fluid Resistance

<3-1> Acid resistance

The acid resistance of the lactic acid bacteria including the Lactobacillus lambosus CKDB009 strain was confirmed. After sterilization of the MRS liquid medium adjusted to pH 2.5 with hydrochloric acid, pepsin was added at a concentration of 1,000 units / mL to prepare an acidic medium. Each strain was inoculated 1% in the medium. To measure the initial number of bacteria, the mixture was decanted into anaerobic dilution (pH 6.2), and 1 ml of the solution was dispensed. After pouring with MRS solid medium, the cells were cultured at 37 ° C for 48 hours. The MRS liquid medium at pH 2.5 inoculated with the strain was incubated for 3 hours in a 37 ° C water bath and cultured in MRS solid medium in the same manner as above. The colonies that appeared after incubation were counted to determine resistance to pH. The acid resistance was evaluated as the strain having the acid resistance when the viable count from the acid solution after 3 hours was converted into the log value to 0.5 log or less or when the growth rate was higher than the original number.

As a result, it was confirmed that 60 strains of 135 kinds of lactic acid bacteria have high acid resistance at pH 2.5 and Lactobacillus lambatus CKDB009 has high acid resistance (Table 2).

&Lt; 3-2 >

Bacterial bile resistance was confirmed for 60 lactic acid bacteria having acid resistance including Lactobacillus lambatus CKDB009 strain. To confirm the biliary properties, 0.3% OXGAL (Sigma, USA) was added to the MRS liquid medium using the method of Gilliland and Walker (1990) to inoculate 1% of the second-passaged lactic acid bacteria. In the same manner as that of acid-fast bacilli, the number of live cells at 0 hours and 24 hours is measured. The evaluation method was also evaluated to have bacteriostasis when the number of survivors from the insemination solution after 24 hours was converted to a log value and the number of bacteria was decreased to 0.5 log or less compared with the original number of bacteria. As a result, it was confirmed that the 60 bacterias with high acid resistance were found to have a high bite resistance, and the bile bacterium of Lactobacillus lambatus CKDB009 was also high (Table 2).

List of excellent acid-resistant and biliary-resistant bacteria Strain number Species Strain number Species 7 Le. mesenteroides 153 L. rhamnosus 12 P. pentosaceus 158 L. gasseri 18 L. rhamnosus 161 L. gasseri 19 L. reuteri 162 L. reuteri 21 L. rhamnosus 163 L. rhamnosus 28 L. rhamnosus 164 L. gasseri 29 L. reuteri 169 L. reuteri 31 L. reuteri 174 L. rhamnosus 32 L. rhamnosus 178 L. rhamnosus 34 L. reuteri 183 L. rhamnosus 35 L. rhamnosus 193 Le. mesenteroides 36 L. reuteri 197 Le. mesenteroides 39 L. plantarum 203 L. plantarum 40 L. plantarum 215 L. plantarum 51 L. reuteri 218 L. rhamnosus 57 L. rhamnosus 228 L. plantarum 58 P. pentosaceus 241 L. plantarum 62 Lc. lactis 244 L. paracasei 64 L.sakei 260 L. fermentum 70 L. casei 263 L. rhamnosus 71 Le. paramesenteroides 267 L. gasseri 81 L. rhamnosus 275 P. pentosaceus 82 L. rhamnosus 279 P. pentosaceus 83 L. reuteri 289 L. plantarum 86 L. rhamnosus (CKDB009) 296 P. pentosaceus 91 L. rhamnosus 337 L. casei 93 L. plantarum 344 L. plantarum 105 L. sakei 345 Lc. lactis 106 L. gasseri 346 Lc. lactis 123 L. rhamnosus 347 Lc. lactis

Example 4. Safety check

In order to confirm the safety of the lactic acid bacteria in which digestive juice resistance was confirmed, it was confirmed whether or not hemolysis, gelatinase and urease were produced. First, the hemolysis test to confirm that it is a non-pathogenic bacterium was performed by inoculating the 60 strains isolated in the blood agar medium and incubating the cells at 37 ° C for 24 hours. Gelatinase production was determined by preparing a gelatin nutrient medium containing 0.3 g of meat, 0.5 g of peptone, 12 g of gelatin and 100 ml of MRS medium, inoculating 60 strains, culturing at 37 ° C for 48 hours, The mixture was allowed to stand in the refrigerator at 4 ° C for about 4 hours to confirm the solidification of the medium. When the medium did not solidify, the liquefaction reaction was judged to be positive. In addition, agar medium containing urea and phenol red as an indicator of urease production was prepared, and it was confirmed whether or not the agar medium was produced through the color change of the indicator component for ammonia production after inoculation of the strain. As a result, it was confirmed that 60 types of lactic acid bacteria, including Lactobacillus lambosus CKDB009, which is a patent deposited lactic acid bacterium of the present invention, did not produce gelatinase and urease.

Example 5. In vitro test

<5-1> Confirmation of cholesterol lowering ability

Forty-eight lactic acid bacteria, except for some of the 60 lactic acid bacteria that have been confirmed to be safe, including Lactobacillus lambosus CKDB009 and the commercial strain Lactobacillus rhamnosus GG (LGG), were tested for cholesterol reduction and functionalities as an anti-obesity probiotic strain Cholesterol lowering ability test was conducted to confirm the test. MRS-THIO broth was prepared by adding 0.2% sodium thioglycolate and 0.3% oxgall to MRS, sterilized and adding 1 ml of cholesterol micelle (cholesterol 0.091 mg / ml in 0.4 mM sucrose solution) to 9 ml of MRS-THIO broth. The selected strains were inoculated at 1% and cultured at 37 ° C for 24 hours. The amount of cholesterol in the culture supernatant was quantified to confirm cholesterol reducing ability. At this time, MRS-THIO medium not inoculated with bacteria was used as a control. To identify cholesterol-lowering ability, selected strains were cultured in MRS-THIO medium for 24 hours and centrifuged at 11,000 rpm for 15 minutes. Then, 3 mL of 95-97% ethanol and 2 mL of 50% KOH were added to 2 mL of the supernatant. 60 &lt; 0 &gt; C) for 10 minutes. After cooling at room temperature, 5 mL of hexane was added and allowed to stand for 15 minutes. 4 mL of hexane layer was separated and 4 mL of o-phtalaldehyde (0.55 mg / acetic acid 1 mL) was added to the tube dried with nitrogen gas. After 10 minutes of reaction, 2 mL of sulfuric acid was added and reacted for 10 minutes. Light intensity was measured. From the measured absorbance, the amount of cholesterol was determined by using a cholesterol standard curve. The degree of reduction of cholesterol was calculated as a% reduction rate.

<Formula 1>

As a result, cholesterol lowering ability of Lactobacillus rhamnosus GG (LGG) strain, which is a representative commercial comparative strain, was confirmed to be 6.442%, and cholesterol lowering ability of the isolated Lactobacillus lambosus CKDB009 among the 48 isolated lactic acid bacteria was 45.890% (Table 3).

Cholesterol lowering activity measurement result Strain number Cholesterol lowering
activity (%) Strain number Cholesterol lowering
activity (%) LGG 6.442 CKDB009 45.890 70 10.682 93 41.945 279 10.097 203 41.194 29 9.983 64 36.328 296 8.464 36 35.843 71 7.748 163 34.870 241 6.968 346 30.017 62 6.327 161 29.663 275 5.946 123 27.582 51 5.737 39 27.333 12 5.083 158 27.138 58 4.895 105 24.183 40 4.591 83 22.224 197 4.526 260 19.586 35 4.108 263 17.432 344 3.862 164 15.058 228 3.389 267 14.517 32 3.122 244 12.691 183 1.949 193 12.198 82 1.519 18 12,000 81 1.468 106 11.754 347 1.437 289 11.145 345 1.228 57 11.093 7 0.458 19 10.792 337 0.356

<5-2> Confirmation of antioxidant activity

Antioxidant activity was confirmed against 48 kinds of lactic acid bacteria except for the same strains of several newly isolated lactic acid bacteria including some of the newly isolated lactobacillus strains including the above-mentioned Lactobacillus lambatus CKDB009 and the commercial strain Lactobacillus lambosus GG (LGG) Respectively. First, DPPH (2,2 Diphenyl 1 picrylhydrazyl) was dissolved in methanol to a concentration of 0.2 mM and diluted with distilled water to obtain an absorbance value of 1.0-1.2 at 517 nm using a spectrophotometer to prepare a DPPH solution. The lactic acid bacteria were inoculated on the MRS medium and cultured at 37 ° C for 18 hours. The culture broth was centrifuged to recover the pellet. The collected cells were suspended in PBS and the absorbance was adjusted to 1.0-1.2 to prepare a sample. 800 μl of the sample and 1 ml of the DPPH solution were mixed and reacted at 37 ° C. for 30 minutes under dark conditions. Then, the absorbance was measured at 517 nm using a spectrophotometer. As a positive control for comparison of antioxidant ability, 0.2 mM ascorbic acid aqueous solution (3.5229 g / 100 mL) which is known to have high antioxidant activity was used. As a control, scavenging activity (%) was confirmed by reacting the same amount of supernatant with PBS in DPPH solution.

<Formula 2>

As a result, the antioxidant activity of Lactobacillus lambusus GG strain, which is a commercial comparative strain, was confirmed to be 11.25%, and the antioxidative activity of Lactobacillus rhamnosus CKDB009 of 48 isolates was 15.12%, which is superior to commercial strain LGG , And antioxidant capacity among the total isolated lactic acid bacteria belonged to the upper group (Table 4).

Antioxidant activity measurement results Strain number Scavenging activity (%) Strain number Scavenging activity (%) LGG 11.25 Ascorbic acid 90 7 34.65 244 9.45 279 31.05 70 9 105 29.34 93 8.64 51 16.74 82 7.38 CKDB009 15.12 71 7.11 35 14.13 62 6.93 83 14.04 106 6.12 36 12.87 289 6.12 158 12.69 164 5.94 347 12.51 203 5.67 29 12.51 346 5.58 267 12.15 161 5.04 260 12.15 345 4.77 40 12.06 241 3.6 39 11.88 228 3.42 344 11.07 197 3.06 18 10.98 19 2.97 58 10.71 163 2.52 32 10.62 275 2.25 337 10.44 193 1.98 12 10.17 263 1.98 64 9.99 183 1.98 81 9.9 123 0.99 57 9.54 296 0.9

The oxygen that enters the body through the breathing process and food intake is used in various metabolic processes. The active oxygen produced in this process is combined with lipids in the body to produce lipid peroxidation. It circulates in the blood, and produces allergic sclerosis, cerebral hemorrhage, myocardial infarction, obesity &Lt; / RTI &gt; Therefore, antioxidant activity to remove active oxygen is important for the treatment and prevention of obesity and atherosclerosis patients. Therefore, Lactobacillus lambatus CKDB009, which has high antioxidant ability and cholesterol reducing ability, was finally selected as an anti-obesity probiotic.

Example 6. In vitro cell application experiment

As the probiotic lactic acid bacteria, Lactobacillus rhamnosus CKDB009 and the patented strain Pediococcus pentosaceus KID7, which has already been confirmed to have a cholesterol-lowering effect, and Lactobacillus rhamnosus ATCC9595, a deposit strain, Lowering and anti-obesity efficacy. Since the growth of cells can be inhibited by bacteria when living viable cells are treated on cells, all the lactic acid bacteria in the treatment area were tested in the form of dead cells. In the present invention, mouse-derived 3T3-L1 cells (pre-adipocyte cells, fat precursor cells) were used and used as a control for all experiments. Because obesity is caused by adipogenesis and increased adipocyte cells due to increased accumulation of intracellular triglycerides, the anti-obesity effect was verified by measuring the adipogenesis gene and lipid accumulation of 3T3-L1 cells.

<6-1> Cytotoxicity check

The cytotoxicity of 3T3-L1 cells to MTT (3- (3-tert-butyloxycarbonyl) -3H-pyrazole) was measured using heat-treated cells (10 ⁵ CFU / mL) of the novel lactobacillus Lactobacillus lambosus CKDB009, Pediococcus pentosaceus KID7 and Lactobacillus lambosus ATCC9595. [4,5-Dimethylthiazol-2-yl] -2,5-diphenyl-tetrazolium bromide method. The MTT method is a colorimetric assay that measures the viability of cells using tetrazolium, a yellow, water soluble substrate, which is reduced to a purple non-aqueous formazan by mitochondrial dehydrogenase, mitochondrial succinate dehydrogenase. MTT (3- [4,5-Dimethylthiazol-2-yl] -2,5-diphenyl-tetrazolium bromide) was diluted with sterilized distilled water to a concentration of 12 mM and filtered through a 0.2 μm filter to prepare MTT stock solution MTT assay was performed while blocking light. First, when the cells were grown in a 100 mm ² cell culture dish, the culture medium was removed, and the cells were removed and centrifuged at 1000 rpm for 3 minutes to seed cells in a 96-well plate at a concentration of 1.0 × 10 ⁴ cells / well 24 hours were incubated _{(37 ℃, 5% C0 2} ). At this time, 10% heat-inactivated bovine serum (BS) and 1% antibiotic (Penicillin, Streptomycin) were added to the maintenance medium, DMEM (Dulbeco's Modified Eagle's Medium, Respectively. After 24 hours, the cell adhesion was confirmed, and the well medium was replaced with 11 heat killed cells (10 ⁵ CFU / mL in medium) and a maintenance medium mixture and cultured for 24 hours and 48 hours , 5% C0 _2). After incubation for 24 hours and 48 hours, the medium was removed and replaced with a mixture of 10 μl MTT stock solution and 100 μl phenol red free DMEM. After incubation for 4 hours in the dark (37 ° C, 5% C0 ₂ ) Respectively. Then, 50 μl of DMSO solution was added to each well, incubated for 30 min in a dark state, and then absorbance was measured at 570 nm using a spectrophotometer.

As a result, there was no difference in the absorbance of all three species when compared with the control. As the decrease in absorbance means a decrease in cell viability, it was confirmed that the three heat-killed cells were not cytotoxic to 3T3-L1 cells.

<6-2> Identification of effects on adipocyte differentiation

Inhibition effect on the differentiation of 3T3-L1 cells into adipocytes by heat-treated cells (10 ⁸ CFU / mL) of the novel lactobacillus Lactobacillus lambosus CKDB009, Pediococcus pentosaceus KID7 and Lactobacillus lambosus ATCC 9595 Respectively. After completion of Day 8 differentiation, the content of triglyceride (TG) was measured and stained with Oil red O stain.

The intracellular TG content was calculated by calculating the percentage of TG in the control from 100 (FIG. 1). The ATCC9595 and CKDB009 treatments showed a marked decrease in intracellular TG content compared to the control, and the tendency was also decreased in KID7 treatment.

In addition, 3T3-L1 cells were stained with Day 8 differentiation and observed with a microscope. As a result, it was confirmed that red cell staining of a large amount of fat droplets was well induced in adipocyte differentiation. In the treatment with CKDB009, (Fig. 2). &Lt; tb &gt; &lt; TABLE &gt;

After microscopic observation, the stained cells were dissolved in 100% isopropanol to quantitate fat accumulation, and the OD value was measured and shown in FIG. CKDB009 significantly reduced fat accumulation compared to the control.

<6-3> Determination of mRNA expression level of adipocyte differentiation-related protein

When the heat-treated cells (10 ⁸ CFU / mL) of the novel lactic acid bacteria Lactobacillus lambosus CKDB009, Pediococcus pentosaceus KID7 and Lactobacillus lambosus ATCC 9595 were treated with 3T3-L1 cells, the protein associated with adipogenesis, PPARγ , C / EBPα, and aP2 genes were compared in each treatment.

Adipogenesis transcription factors play an important role in the differentiation of adipose precursor cells. In particular, CCAAT / enhancer binding protein-alpha (C / EBPα), peroximal proliferator-activated receptor-γ (PPARγ) and adipocyte binding protein (aP2) Because it is a transcription factor, it can be used as a biochemical indicator in promoting adipocyte differentiation and inducing or inhibiting obesity.

In this study, the expression of PPARγ was significantly decreased in CKDB009 treated group, and the expression of C / EBPα was significantly decreased in ATCC9595 treated group and aP2 expression was significantly decreased in CKDB009 treated group. Overall, the CKDB009 treatment showed a tendency to decrease the fat precursor cell transcription factor compared to the control (Fig. 4).

Ⅲ. Animal experiment using obesity induction model

Example 7. Preparation of lactic acid bacteria powder

Animal experiments were conducted on the Lactobacillus lambatus CKDB009 as a probiotic lactic acid bacterium and the patented strain Padiococcus pentosaceus KID7, which has been proven in cholesterol-lowering ability in an animal model of hyperlipidemia. The two strains were inoculated into a liquid medium suitable for each strain in a conventional manner and cultured at 37 DEG C for 18 hours. Activated bacteria were recovered by centrifugation and then washed. The collected cells were rapidly frozen at -60 ° C, lyophilized at 0-45 ° C, pulverized and stored at 4 ° C. For the oral administration of the two lactic acid bacteria in a fresh state, they were suspended in PBS to a concentration of 10 ¹⁰ CFU / 100 μl before administration.

Example 8. Experimental animal and experimental design

<8-1> Materials and reagents

Animal experiments were conducted to confirm the cholesterol lowering and anti-obesity effects of the novel lactic acid bacteria. The powdered Lactobacillus lambosus CKDB009 and podiococcus pentosaceus KID7 strains were used, and high cholesterol and anti-obesity induction were regulated through diet.

<8-2> Experimental animals and treatment

The experimental groups were divided into four groups: Normal Diet (ND group), Negative control group (High fat Diet, HD group), Pediococcus pentosaceus KID7 monoclonal antibody and Lactobacillus lambosus CKDB009 monoclonal antibody . The conditions and abbreviations of each experimental group are shown in Table 5 below. Sixteen male C57BL / 6 mice were purchased from Sam Taco Bio Korea and the darkness was controlled at 12 hours intervals. The temperature was 23 ± 2 ℃ and the humidity was 50 ~ 60 %. Feed and water were allowed to eat freely. The initial mean weights of the experimental animals were not different between the four groups, and were adjusted to the normal diet for one week and then subjected to experimental treatment.

Anti-obesity effect validation model Experimental group Diet Treatment Group 1 (ND) Normal diet - Group 2 (HD) High fat, high CHO diet - Group 3 (KID7) High fat, high CHO diet P. pentosaceus KID7 1 x 10 &lt; 10 ^&gt; CFU / day Group 4 (CKDB009) High fat, high CHO diet L. rhamnosus CKDB009 1 x 10 ¹⁰ CFU / day

For experimental diets, D12450B (10 kcal% fat) was used for the normal diet and D08062402 (45 cal% fat, 1.25% cholesterol) for the high fat and high cholesterol diet was purchased from Sam Taco Bio Korea. The dietary composition used is shown in the table below (Table 6). Experimental period was 12 weeks. Lactobacillus was sacrificed after oral administration by suspending lactobacillus powder in PBS at the same time once a day during the whole experimental period.

Animal Experimental Dietary Composition High-fat-high-cholesterol diet (HD, D08062402) Normal diet
(ND, D12450B) gm (%) kcal (%) gm (%) kcal (%) Nutrient content in feed Protein 23.4 20 19.2 20 Carbohydrate 40.9 35 67.3 70 Fat 23.3 45 4.3 10 Ingredient gm kcal gm kcal Feed composition Casein 200 800 200 800 L-Cysteine 3 12 3 12 Corn starch 72.8 291 506.2 2024.8 Maltodextrin 100 400 125 500 Sucrose 172.8 691 68.8 275.2 Cellulose 50 0 50 0 Soybean oil 25 225 25 225 Lard 177.5 1598 20 180 Mineral mix 10 0 10 0 Dicalclium phosphate 13 0 13 0 Calcium carbonate 5.5 0 5.5 0 Potassium citrate 16.5 0 16.5 0 Vitamin mix 10 40 10 40 Choline bitartrate 2 0 2 0 Cholesterol 10.7 0 0 0 Total 868.85 4057 1055.05 4057

Example 9. Effect on body weight

The experimental animals measured body weight at regular intervals once a week during the treatment period. The weight gain (%) was calculated by the following equation (3) as the weight value for 0th and 12th weeks.

<Formula 3>

BMI of the experimental animals was calculated by measuring the body weight and body length of the rats before sacrifice from the nose to the tail starting point,

<Formula 4>

As a result, the body weight of the high fat and high cholesterol diet group was increased about 2.5 times as compared with that of the normal diet group, so that it was confirmed that the induction of the obese mouse model was successful. In comparison with the high fat, high cholesterol diet group, the body weight of the group treated with CKDB009 or KID7 was significantly lower than that of the high fat and high cholesterol diet group. BMI (kg / m ² ) was significantly higher in the high fat and high cholesterol diet group than in the normal diet group, and in the CKDB009 and KID7 treated groups, the high fat and high cholesterol diet was decreased compared to the control. Especially, in the group treated with CKDB009, the normal diet was greatly reduced to the same level as the group (FIG. 5).

Example 10. Effect on adipose tissue

<10-1> Weight of white adipose tissue

The white adipose tissue (eWAT), the visceral white adipose tissue (eWAT), and the inguinal white adipose tissue (iWAT), the subcutaneous white adipose tissue were separated from the sacrificed white adipose tissue. The total white adipose tissue weights were the combined weight of eWAT and iWAT. The results showed that eWAT, iWAT and total WAT were increased in the high fat and high cholesterol diet group compared to the normal diet group. However, in the group treated with CKDB009 and KID7, the weight of iWAT was significantly lowered in the high fat and high cholesterol diet compared with the control, and as a result, the total WAT was also decreased (FIG. 6).

Example 11. Lipid content in serum and liver tissue

Blood from all sacrificed laboratory animals was collected by cardiac puncture. Blood was centrifuged at 3,000 rpm for 15 minutes in serum separate tube, and serum (supernatant) was separated from the tube and lipid content was measured.

<11-1> Lipid content in serum

The serum triglyceride (TG), total cholesterol (TC), high density lipoprotein-cholesterol (HDL-C) and LDL / VLDL-cholesterol cholesterol, LDL / VLDL-C) were measured using commercially available kits. HDL-C and LDL / VLDL-C are HDL and LDL / VLDL Quantification Colorimetric / Fluorometric Kits (Bio vision), TC is Total Cholesterol and Cholesteryl Esterase Quantification Colorimetric / Fluorometric Assay Kit (Bio vision), and TG is Triglyceride Quantification Colorimetric / Fluorometric Kit Fluorometric Kit (Bio vision). The serum triglyceride, total cholesterol, HDL-cholesterol, and LDL / VLDL-cholesterol content were analyzed to determine the lipid metabolism in the blood, and it is shown in FIG. Concentrations of triglyceride were not different in all groups including normal dietary group and high fat and high cholesterol diet group, but total cholesterol concentration was 177.5 ± 5.6mg / dL in normal dietary group, 316.3 ± 5.6mg / dL in high fat and high cholesterol dietary group, 12.1 mg / dL, total cholesterol was significantly increased. On the other hand, the CKDB009 treatment group was 195.7 ± 11.2 mg / dL and the KID7 treatment group was 224.2 ± 13.2 mg / dL, which was significantly lower than the high fat and high cholesterol diet group.

There was no significant difference in the HDL-cholesterol concentration between the normal diet group and the high fat / high cholesterol diet group, but the LDL / VLDL-cholesterol level was 26.8 ± 1.9 mg / dL in the normal dietary group, The serum LDL / VLDL-cholesterol level was significantly increased in the high cholesterol diet group (51.9 ± 3.6 mg / dL). On the other hand, the CKDB009 treatment group was 30.0 ± 2.4 mg / dL and the KID7 treatment group was 34.3 ± 3.6 mg / dL, which was significantly lower than the high fat and high cholesterol diet group.

<11-2> Liver lipid content

The concentration of triglyceride (TG) in liver tissue was enzymatically hydrolyzed with glycerol and free fatty acid, and the released glycerol was indirectly measured by colorimetric method. To measure TG in liver tissue, 1 mL of 5% Tween 20 was added to 50 mg liver tissue, homogenized using a microhomogenizer, heated in a water bath at 80-100 ° C for 2-5 minutes, And all of the TG in the liver tissue was solubilized. Then, the supernatant was recovered by centrifugation at 15,000 rpm for 2 minutes, and the TG content of the supernatant was measured using a Triglyceride Quantification Colorimetric / Fluorometric Kit (Bio vision). The TG content was expressed as TG (μg) contained in 1 mg of liver tissue. Free cholesterol and cholesteryl esters were measured by colorimetric method to analyze total cholesterol (TC) of liver tissue. To this end, 1 mL of cholesterol extraction buffer (Chloroform: Isopropanol: Tween 20 = 7: 11: 0.1) was added to 50 mg liver tissue, homogenized using a microhomogenizer, centrifuged at 15,000 rpm for 10 min and all supernatants Organic phase) was transferred to a new tube. To remove chloroform, air drying was performed at 50 ° C. To remove remaining organic solvent, 1 ml of cholesterol assay buffer was added, followed by sonication, and homogenized until the buffer was poured. Total Cholesterol and Cholesteryl Esterase Quantification The TC content of the homogenate was measured using a Colorimetric / Fluorometric Assay Kit (Bio vision). The TC content was expressed as TC (μg) contained in 1 mg of liver tissue.

High-fat, high-cholesterol diets increase blood cholesterol and accumulate fat in the liver to form fatty liver. In order to evaluate the efficacy of the strain against dietary fat induced by diet, the triglyceride and total cholesterol content in liver tissues were measured and shown in FIG. 8 together with the control. The concentration of triglyceride was not significantly different in all groups including the normal dietary group and the high fat and high cholesterol dietary group, but the total cholesterol concentration was 2.6 ± 0.1 μg / mg in the normal dietary group and the high fat and high cholesterol dietary group 5.5 ± 0.2 μg / mg, indicating a significant increase in total cholesterol. On the other hand, the CKDB009 treatment group was 4.3 ± 0.2 μg / mg, and the KID7 treatment group was 4.1 ± 0.3 μg / mg, which was significantly lower than the high fat and high cholesterol diet group.

Example 12. Fattyzing enzyme activity

<12-1> Fatty enzyme activity in white adipose tissue

The content of HSL (hormone sensitive lipase) in white adipose tissue was measured. HSL is an enzyme secreted by adipose tissue and hydrolyzes the triglyceride stored in adipose tissue to fatty acid. Therefore, when the expression of HSL factor is increased, it is possible to decompose fat in adipose tissue to alleviate obesity. Therefore, the degree of increase of expression of HSL factor in lactic acid bacteria treatment was compared with that of the control group. First, in order to measure HSL activity in white adipose tissue, 1 mL of PBS is added to 100 mg tissue and homogenized using a microhomogenizer. It is dissolved at room temperature after overnight incubation at -20 ℃ to disrupt the tissue membrane. After overnight incubation at -20 ° C, slowly dissolve at room temperature and centrifuge for 5 minutes at 5,000g at 2-8 ° C. HSL activity of the supernatant was measured using Mouse Hormone-Sensitive Lipase (LIPE) ELISA Kit (CUSABIO). HSL activity was expressed as HSL Unit (U) per mg of white adipose tissue. As a result, HSL (hormone sensitive lipase) activity was increased in the CKDB009 treatment group as compared to the high fat / high cholesterol diet group (FIG. 9).

Example 13. Expression of mRNA of fat-related protein

Liver, brown fat, and white adipose tissue were rapidly frozen in liquid nitrogen after extraction and stored at -80 ° C until used for analysis. Each tissue was homogenized in 5% Tween 20 solution using a microhomogenizer and then total RNA was extracted using GeneJET RNA purification kit (Thermo scientific). High capacity cDNA reverse transcription kit (Applied biosystems) is used to synthesize cDNA. Real-time PCR was performed using the KAPA SYBR Fast qPCR kit (Kapabiosystem) using the CFX96 Touch ™ Real-Time PCR Detection System. GAPDH was used as a housekeeping gene to quantify the expression level of the target gene. The primer sequence of the target gene is shown in Table 7.

Real time PCR target gene primer sequence Primer Sequence Temperature PPARγ Forward 5'-GGAAGACCACTCGCATTCCTT-3 ' 61.0 DEG C Reverse 5'-GTAATCAGCAACCATTGGGTCA-3 ' C / EBPα Forward 5'-GCGGGAACGCAACAACATC-3 ' 61.0 DEG C Reverse 5'-GTCACTGGTCAACTCCAGCAC-3 ' UCP-1 Forward 5'-CAAAAACAGAAGGATTGCCGAAA-3 ' 64.3 DEG C Reverse 5'-TCTTGGACTGAGTCGTAGAGG-3 ' UCP-2 Forward 5'-ATGGTTGGTTTCAAGGCCACA-3 ' 59.5 DEG C Reverse 5'-TTGGCGGTATCCAGAGGGAA-3 ' CYP7A1 Forward 5'-GAACCTCCTTTGGACAACGGG-3 ' 59.6 DEG C Reverse 5'-GGAGTTTGTGATGAAGTGGACAT-3 ' GAPDH Forward 5'-GTATGACTCCACTCACGGCAAA-3 ' 58.5 DEG C Reverse 5'-GGTCTCGCTCCTGGAAGATG-3 '

<13-1> Expression of mRNA of adipogenesis and thermogenesis-related proteins in white adipose tissue

Protein mRNA expression associated with adipogenesis and thermogenesis was confirmed in white adipose tissue. The expression levels of PPARγ, C / EBPα, and UCP-1 and UCP-2, genes related to adipogenesis, were compared and evaluated. Peroxisome proliferator-activated receptor-γ (PPARγ) and CCAAT / enhancer binding protein-alpha (C / EBPα) are adipogenesis-inducing transcription factors that induce adipocyte differentiation and induce obesity. Thus, a decrease in the expression of PPARγ and C / EBPα means that obesity is alleviated. As shown in FIG. 10, the expression of PPAR gamma was significantly increased in the high fat / high cholesterol diet group compared to the normal diet group. On the other hand, the CKDB009 and KID7 treatment groups were significantly reduced to the same level as the normal group. In the case of C / EBPα, the expression level in the high fat and high cholesterol diet group was more than three times higher than that in the normal group. On the other hand, the CKDB009 treatment group was significantly reduced to the same level as the normal group. However, the group treated with KID7 showed no significant difference from the group with high fat and high cholesterol diet.

UCPs (Uncoupling proteins) belong to the proton transporter family located in the mitochondrial inner membrane. Among UCP family, UCP-1 participates in thermogenesis by uncoupling respiration from ATP synthesis through short-circulating proton flow in mitochondria. UCP-2 is involved in the regulation of energy metabolism and reactive oxygen species (ROS). In this study, the expression of UCP-1 was significantly reduced in the high-fat and high-cholesterol diet groups compared to the control group. There was no significant difference in the other treatment groups between the high fat and high cholesterol diet groups. In the case of UCP-2, the normals were significantly increased in the high-fat and high-cholesterol diet group compared to the group. On the other hand, the CKDB009 treatment group was significantly reduced to the same level as the normal group. However, the group treated with KID7 showed no significant difference from the group with high fat / high cholesterol diet (Fig. 11).

<13-2> Expression of CYP7A1 mRNA in lipid metabolism-related proteins in liver tissue

The level of mRNA expression of the CYP7A1 gene, a protein related to lipid metabolism in liver tissue, was compared between the groups. CYPA1 (Cholesterol 7 alpha-hydroxylase) is a factor secreted by the liver and is involved in the homeostasis of cholesterol and bile acid as a rate-limiting enzyme in the process of converting cholesterol into bile acid. In other words, CYP7A1 acts as a cholesterol bile acid to increase excretion, thereby reducing the amount of cholesterol accumulated in the liver. The effect of the strains on the expression of CYP7A1 in obese rat models was not found to show any significant difference between the control group and the high fat / high cholesterol diet group. However, CKDB009 strain showed a tendency to increase compared with the high fat / high cholesterol diet group, and the expression of CYP7A1 was significantly increased in the KID7 treatment group (FIG. 12).

Example 14. Histopathological analysis of tissue

Liver tissue and white adipose tissue were extracted and stored in 10% formaldehyde for some oil red O, H & E staining.

<14-1> Histopathological analysis of liver tissue

In order to analyze histopathologically the effect of the strains selected in this experiment on fatty liver induced by high fat and high cholesterol diet, oil red O was stained and pathological analysis was conducted.

For this purpose, the liver tissue was removed from the mouse and transferred to a 20% sucrose solution, followed by overnight incubation at 4 ° C. Cryosections were made on slide glass, transferred to 10% formaldehyde in PBS, and allowed to stand at 23 ° C for 1 hour. After washing twice with water, it was transferred to a 0.1% Oil red O solution (in 75% isopropanol), stained at 23 ° C for 2 hours, washed with water to remove unlabeled dye, Respectively. Oil red O staining of liver tissues showed no reddish fat droplets in the liver of the normal diet group but a large amount of reddish fat droplets were observed in the high fat and high cholesterol diet groups. CKDB009 and KID7 treatment groups showed a marked decrease in red stained fat droplets, indicating that fat accumulation was inhibited (FIG. 13).

<14-2> Histopathological analysis of white adipose tissue

H & E staining was performed to analyze the histopathological effects of the strains selected in this experiment on the fat cell size increased by the high fat and high cholesterol diet.

White fat was extracted from the mouse, fixed in 10% formaldehyde, dehydrated in ethanol, and washed in xylene to prepare a paraffin block. 5 μm thickness, placed on a slide glass, allowed to stand in Harris' hematoxylin for 2 minutes and 30 seconds, washed with water, immersed in acid alcohol, ammonia water 2-3 times in succession, placed in water and allowed to stand for 10 minutes. After staining with Eosin-phloxine for 2 minutes at room temperature, the stained slides were transferred to 95% alcohol and xylene in order to remove unlabeled dyes. The slide was dried and observed under a light microscope at 100x magnification. H & E staining of white adipose tissue showed an increase in adipocyte size in the high fat / high cholesterol diet group compared to the normal diet group. On the other hand, in the CKDB009 and KID7 treatment groups, it was confirmed that the fat cells were reduced in size compared to the high fat / high cholesterol diet group (Fig. 14).

IV. Production of lactic acid bacteria

Example 15: Preparation of a healthy functional food raw material containing Lactobacillus lambosus CKDB009 as an active ingredient

In the above animal test, a health functional ingredient starch containing Lactobacillus lambosus CKDB009 Lactobacillus which inhibits obesity induced by high fat high cholesterol diet as an active ingredient was prepared. Lactobacillus lambsosus CKDB009 strain was cultured by setting the culture medium and culture conditions optimized. After collecting the cells, maltodextrin 5-50 (volume / weight)% or trehalose 5-50 (volume / weight)% or cellulose 5-50 (volume / weight)% was added to the concentrate to lyophilize and pulverize the lactic acid bacteria Respectively.

While the present invention has been particularly shown and described with reference to exemplary embodiments thereof, it is to be understood that the invention is not limited to the disclosed exemplary embodiments. For example, it is to be understood that the techniques described may be performed in a different order than the described methods, and / or that components of the described systems, structures, devices, circuits, Lt; / RTI &gt; or equivalents, even if it is replaced or replaced.

Therefore, other implementations, other embodiments, and equivalents to the claims are also within the scope of the following claims.

Claims (10)

Lactobacillus rhamnosus CKDB009 strain (accession number: KCTC13249BP), which reduces cholesterol and alleviates obesity symptoms, and reduces LDL-cholesterol levels and reduces cholesterol in blood, is a strain of Lactobacillus lambosus CKDB009.
The method according to claim 1,
The strain has acid resistance and bile resistance and does not produce one or more intestinal harmful enzymes selected from the group consisting of urease and gelatinase and does not cause hemolysis, Lactobacillus lambosus CKDB009 strain characterized by excellent safety.
The method according to claim 1,
Wherein said strain has cholesterol reducing ability and antioxidant ability.
The method according to claim 1,
The strain is cytotoxic, and inhibits the differentiation of 3T3-L1, a lipogenic precursor cell, to produce at least one obesity-related biochemical marker selected from the group consisting of peroximal proliferator-activated receptor-γ (PPARγ) and adipocyte binding protein Lactobacillus Lymphosus CKDB009 strain having an effect of reducing the expression of the factor.
The method according to claim 1,
Wherein said strain inhibits lipid production in white adipocytes and has an effect of reducing body fat by reducing the fat cell size, thereby producing a lactobacillus lambosus strain CKDB009.
A probiotic formulation comprising at least one selected from the group consisting of a strain of any one of claims 1 to 5, a culture of the strain, a concentrate of the culture, and a dried product as an active ingredient.
A pharmaceutical composition comprising at least one member selected from the group consisting of a strain of any one of claims 1 to 5, a culture of the strain, a concentrate of the culture, and a dried product as an active ingredient and having a cholesterol reducing and obesity- Food composition.
8. The method of claim 7,
Wherein the health functional food composition is prepared by lyophilizing the strain to form a live bacterium.
A pharmaceutical composition comprising at least one member selected from the group consisting of a strain of any one of claims 1 to 5, a culture of the strain, a concentrate of the culture, and a dried product as an active ingredient and having an effect of alleviating cholesterol and obesity Composition.
10. The method of claim 9,
Wherein the pharmaceutical composition is prepared by lyophilizing the strain to form a live bacterium.

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