KR20180116173A - Novel Lactobacillus plantarum Ln4 strain and compositions for the prevention and treatment of obesity containing the same - Google Patents

Abstract

The present invention provides a Lactobacillus plantarum Ln4 strain which is probiotics separated from traditional fermented food and a purpose of the same and, specifically, to an antiobesic purpose of a Lactobacillus plantarum Ln4 strain. The Lactobacillus plantarum Ln4 strain divided from the traditional fermented food such as soybean paste, red chili paste, and soy sauce can be used as probiotics. The Lactobacillus plantarum Ln4 strain can suppress accumulation of fat in a fat cell and can indicate significant body fat reduction effect when being injected into an obese mouse model induced by high fat diet. Therefore, the Lactobacillus plantarum Ln4 strain can be used as an active ingredient of a composition for prevention, treatment, and improvement of obesity.

Description

Novel Lactobacillus plantarum Ln4 strain and compositions for the prevention and treatment of obesity containing the same.

The present invention is to provide the Lactobacillus plantarum Ln4 (Lactobacillus plantarum Ln4) strain, which is a probiotic isolated from traditional fermented foods, and uses thereof, and more particularly, to provide an anti-obesity use of the strain.

Obesity is a risk factor for major adult diseases, and is involved in the development of adult diseases such as high blood pressure, diabetes, arteriosclerosis, stroke, heart attack and various tumors (Wilson et al., 2005, Circulation 112: 3066-3072). , It is known that the risk of developing adult diseases due to obesity is 3 to 6 times higher than that of normal people. Therefore, obesity is not just an appearance problem, but a serious problem directly related to health. To prevent obesity, it is important to increase the amount of exercise while maintaining the current amount of meal. In the case of obesity, the cause can be identified and the body can maintain a normal metabolism while maintaining an appropriate weight through an appropriate diet. It is desirable to do so.

Meanwhile, in various Korean traditional fermented foods such as Kimchi, Doenjang, and Cheonggukjang, there are many lactic acid bacteria that have potential as probiotics, and many studies using lactic acid bacteria isolated from traditional fermented foods are also being conducted. In addition, various products using probiotics are being released. Probiotics are beneficial microorganisms that enter the body in a living state and provide immunity and various functions to help health. Most of the probiotics known to date are lactic acid bacteria, but also include Bacillus spp. or yeast. In order to be recognized as a probiotic strain, it must survive in gastric and bile acids and reach the small intestine. In addition, it must proliferate and settle in the intestine, exhibit immunity and various functions in the intestine, and prove its safety.

According to studies to date, it has been known that lactic acid bacteria isolated from fermented foods such as paste or kimchi can be used as probiotics, and can be applied as a pharmaceutical treatment through their various physiological functions. For example, in Korean Patent Registration No. 10-161653, a probiotic composition comprising a novel Lactococcus lactis KR-WW-2 strain isolated from flounder Sikhye is used to treat diseases caused by pathogens of farmed fish. It is disclosed that it can be used for treatment, and Korean Patent Laid-Open No. 10-2015-0106093 discloses that the Bacillus methylotrophicus C14 strain isolated from intestines can be used as a probiotic formulation and can be used as an antibacterial composition. I'm doing it. In addition to this, research for using various lactic acid bacteria as useful uses is ongoing.

Accordingly, the present inventors have tried to isolate lactic acid bacteria that can be used as probiotics from traditional fermented foods and to find their use. As a result, Lactobacillus plantarum Ln4 isolated from traditional fermented foods such as soybean paste, red pepper paste and soy sauce. The present invention by confirming that the strain can be used as a probiotic, and the strain can suppress the accumulation of fat in adipocytes and exhibit a significant body fat reduction effect when administered to an obese mouse model induced by a high fat diet. Was completed.

Accordingly, the present inventors can use the Lactobacillus plantarum Ln4 (Lactobacillus plantarum Ln4) strain isolated from traditional fermented foods as a probiotic, and when administered to an obese mouse model induced by a high fat diet, a significant anti-obesity effect is achieved. The present invention was completed by confirming that it can be represented.

Accordingly, an object of the present invention is to provide a novel Lactobacillus plantarum Ln4 (Lactobacillus plantarum Ln4) strain deposited with accession number KCCM 11897P.

In addition, another object of the present invention is to provide a probiotic formulation comprising the strain.

In addition, another object of the present invention is to provide a pharmaceutical use of the strain.

In order to achieve the above object, the present invention provides a Lactobacillus plantarum Ln4 (Lactobacillus plantarum Ln4) strain deposited with accession number KCCM 11897P.

In addition, the present invention provides a probiotic preparation containing as an active ingredient the Lactobacillus plantarum Ln4 strain deposited with accession number KCCM 11897P, a culture solution thereof, or a mixture thereof.

In addition, the present invention provides a pharmaceutical composition for the prevention and treatment of obesity, containing the Lactobacillus plantarum Ln4 strain deposited as an accession number KCCM 11897P, a culture medium thereof, or a mixture thereof as an active ingredient.

In addition, the present invention provides a health functional food for preventing and improving obesity, containing the Ln4 strain of Lactobacillus plantarum Ln4 deposited under the accession number KCCM 11897P, a culture medium thereof, or a mixture thereof as an active ingredient.

In a preferred embodiment of the present invention, the strain may have a 16S rRNA nucleotide sequence represented by SEQ ID NO: 1.

In another preferred embodiment of the present invention, the strain is characterized by having acid resistance, bile resistance, intestinal adhesion and antibiotic resistance.

In another preferred embodiment of the present invention, the Lactobacillus plantarum Ln4 strain may exhibit an effect of inhibiting fat accumulation and reducing body fat.

Lactobacillus plantarum Ln4 (Lactobacillus plantarum Ln4) strain isolated from traditional fermented foods such as doenjang, red pepper paste and soy sauce can be used as a probiotic, which can inhibit the accumulation of fat in adipocytes and induces a high fat diet. When administered to an obese mouse model, it can exhibit a significant body fat reduction effect, so the Lactobacillus plantarum Ln4 strain of the present invention can be used as an active ingredient of a composition for preventing, treating and improving obesity.

1 is a schematic diagram of a Lactobacillus plantarum Ln4 strain.
2 shows the effect of inhibiting fat accumulation in adipocytes according to Lactobacillus plantarum Ln4 treatment.
Figure 3 shows the effect of weight control according to the administration of Lactobacillus plantarum Ln4 in an animal model of obesity and metabolic disease induced by a high fat diet.
Figure 4 shows the effect of reducing body fat according to the administration of Lactobacillus plantarum Ln4 in an animal model of obesity and metabolic disease induced by a high fat diet.

Hereinafter, the present invention will be described in detail.

The present invention provides a Lactobacillus plantarum Ln4 (Lactobacillus plantarum Ln4) strain deposited with accession number KCCM 11897P.

In addition, the present invention provides a probiotic preparation containing the Lactobacillus plantarum Ln4 strain, a culture solution thereof, or a mixture thereof as an active ingredient.

It is preferable that the "Lactobacillus plantarum Ln4 strain" of the present invention has a 16S rRNA nucleotide sequence represented by SEQ ID NO: 1.

The "Lactobacillus plantarum Ln4 strain" of the present invention preferably has acid resistance, bile resistance, intestinal adhesion and antibiotic resistance, but is not limited thereto.

Specifically, the acid resistance indicates that it can exhibit a survival rate of 99% or more for 3 to 5 hours even in a medium having a pH of 2 to 3, and the bile resistance may exhibit a survival rate of 80% or more for 24 hours in an environment similar to human bile. It is desirable to have. In addition, it shows the ability to bind the intestinal adhesion ability to a large intestine cell at a significant level. In order to use the Lactobacillus plantarum Ln4 strain of the present invention as a probiotic preparation, the effect may be reduced or enhanced through maintaining the viability of the strain even when administered orally in the body, so that it can survive in the environment in the digestive tract. Significant effects can be exhibited in those having acid resistance, bile resistance, and intestinal adhesion ability. In addition, the antibiotic resistance is preferably to have resistance to zetamicin, ciprofloxacin, kanamycin, and streptomycin, but is not limited thereto.

In a specific embodiment of the present invention, the present inventors isolated lactic acid bacteria that can be used as probiotics from traditional fermented foods, and isolated a new strain containing 16S rRNA composed of the nucleotide sequence shown in SEQ ID NO: 1, and homology thereof Through analysis, it was confirmed that it was a novel strain of Lactobacillus plantarum Ln4 (FIG. 1), and it was deposited with the Korea Microbial Conservation Center (KCCM) on September 22, 2016 (Accession No. KCCM 11897P).

In addition, as a result of confirming the characteristics of Lactobacillus plantarum Ln4, it was confirmed that Lactobacillus plantarum Ln4 exhibits acid resistance, bile resistance, intestinal adhesion and resistance to some antibiotics. It was confirmed that it does not produce and does not show toxicity to normal cells (Tables 1 to 5), and through these characteristics, the Lactobacillus plantarum Ln4 strain of the present invention can be used in a probiotic preparation in the form of a live or apoptotic body. It was confirmed to be possible.

Therefore, the Lactobacillus plantarum Ln4 strain of the present invention is a novel strain that is safe for normal cells and has characteristics of acid resistance, bile resistance, and intestinal adhesion, so that the strain, its culture solution, or a mixture thereof is used as a probiotic formulation. It can be usefully used as an active ingredient of.

In addition, the present invention provides a pharmaceutical composition for the prevention and treatment of obesity, containing the Lactobacillus plantarum Ln4 strain deposited as an accession number KCCM 11897P, a culture medium thereof, or a mixture thereof as an active ingredient.

It is preferable that the "Lactobacillus plantarum Ln4 strain" of the present invention has a 16S rRNA nucleotide sequence represented by SEQ ID NO: 1.

The "Lactobacillus plantarum Ln4 strain" of the present invention preferably has acid resistance, bile resistance, intestinal adhesion and antibiotic resistance, but is not limited thereto.

The "Lactobacillus plantarum Ln4 strain" of the present invention may exhibit a therapeutic effect on obesity through the effect of inhibiting fat accumulation and reducing body fat.

In another specific embodiment of the present invention, in order to confirm the anti-obesity effect of the Lactobacillus plantarum Ln4 strain, the present inventors constructed a mouse model in which metabolic syndrome was induced through a high fat diet. When the Lactobacillus plantarum Ln4 strain was administered to the mouse model, the accumulation of fat in adipocytes was suppressed, and when administered to an obese mouse model induced by a high fat diet, it was confirmed that a significant body fat reduction effect could be exhibited. Was (Fig. 2 to Fig. 4).

Therefore, when the Lactobacillus plantarum Ln4 strain of the present invention is administered as a viable cell, it is possible to suppress the accumulation of fat in adipocytes, and exhibit a significant anti-obesity effect by exhibiting a body fat reduction effect. Bacillus plantarum Ln4 strain can be used as an active ingredient in a pharmaceutical composition for preventing and treating obesity.

The microbial preparation of the present invention may be prepared according to a conventional method for preparing a probiotic composition, and generally, may be in the form of a culture suspension or a dry powder. In addition, one or two or more pharmaceutically acceptable conventional carriers or one or two or more additives are selected in an effective amount of the Lactobacillus plantarum Ln4 strain, or a culture medium thereof, which is the main component, to obtain a composition of a conventional formulation. Can be manufactured.

The carrier can be used by selecting one or two or more of diluents, lubricants, binders, disintegrants, sweeteners, stabilizers, and preservatives, and one or two or more of flavors, vitamins, and antioxidants can be selected as additives. Can be used.

In the present invention, all pharmaceutically acceptable carriers and additives can be used, and specifically, as a diluent, lactose monohydrate, trehalose, cornstarch, soybean oil, Microcrystalline cellulose or mannitol (D-mannitorl) is preferred, magnesium stearate or talc is preferred as a lubricant, and polyvinyipyrrolidone (PVP: polyvinyipyrolidone) or hydroxypropyl as a binder. It is preferable to select from cellulose (HPC: hydroxypropylcellulose). In addition, the disintegrant is preferably selected from carboxymethylcellulose calcium (Ca-CMC), sodium starchglycolate, pollacrylin potassium, or cross-linked polyvinylpyrrolidone. And, the sweetener is selected from white sugar, fructose, sorbitol, or aspartame, and the stabilizer is carboxymethylcellulose sodium (Na-CMC), beta-cyclodextrin, and white wax. (white bee's wax) or xanthan gum, and the preservative is methyl p-hydroxy benzoate (methlparaben), propyl p-hydroxybenzoate (propylparaben), or potassium sorbate ( potassium sorbate).

In addition, the present invention provides a health functional food for preventing and improving obesity, containing the Ln4 strain of Lactobacillus plantarum Ln4 deposited under the accession number KCCM 11897P, a culture medium thereof, or a mixture thereof as an active ingredient.

It is preferable that the "Lactobacillus plantarum Ln4 strain" of the present invention has a 16S rRNA nucleotide sequence represented by SEQ ID NO: 1.

The "Lactobacillus plantarum Ln4 strain" of the present invention preferably has acid resistance, bile resistance, intestinal adhesion and antibiotic resistance, but is not limited thereto.

The "Lactobacillus plantarum Ln4 strain" of the present invention may exhibit a therapeutic effect on obesity through the effect of inhibiting fat accumulation and reducing body fat.

When the Lactobacillus plantarum Ln4 strain of the present invention is administered as a live cell, it can suppress the accumulation of fat in adipocytes, and when administered to an obese mouse model induced by a high fat diet, it can exhibit a significant body fat reduction effect. Therefore, the Lactobacillus plantarum Ln4 strain of the present invention can be used as an active ingredient in a health functional food for preventing and improving obesity.

The food composition according to the present invention can be prepared in various forms according to conventional methods known in the art. General foods include, but are not limited to, beverages (including alcoholic beverages), fruits and processed foods thereof (e.g., canned fruit, canned food, jam, marmalade, etc.), fish, meat and processed foods thereof (e.g. ham, sausage) Corn beef), breads and noodles (e.g. udon, soba, ramen, spaghetti, macaroni, etc.), fruit juice, various drinks, cookies, syrup, dairy products (e.g. butter, cheese, etc.), edible vegetable oil, margarine Vegetable proteins, retort foods, frozen foods, various seasonings (eg, miso, soy sauce, sauce, etc.) can be prepared by adding the Ln4 strain of Lactobacillus plantarum of the present invention. In addition, nutritional supplements are not limited thereto, but may be prepared by adding the Ln4 strain of Lactobacillus plantarum of the present invention to capsules, tablets, pills, and the like. In addition, the health functional food is not limited thereto, but for example, the Lactobacillus plantarum Ln4 strain of the present invention itself is prepared in the form of tea, juice, and drinks, and liquefied, granulated, It can be encapsulated and powdered to be consumed. In addition, in order to use the Ln4 strain of Lactobacillus plantarum of the present invention in the form of a food additive, it may be prepared in the form of a powder or a concentrate. In addition, it can be prepared in the form of a composition by mixing the Lactobacillus plantarum Ln4 strain of the present invention with a known active ingredient known to have an effect of preventing and improving obesity.

In the case of using the Ln4 strain of Lactobacillus plantarum of the present invention as a health drink, the health drink composition may contain various flavoring agents or natural carbohydrates as an additional component, like a conventional drink. The natural carbohydrates described above include monosaccharides such as glucose and fructose; Disaccharides such as maltose and sucrose; Polysaccharides such as dextrin and cyclodextrin; It may be a sugar alcohol such as xylitol, sorbitol, and erythritol. Sweeteners include natural sweeteners such as taumatin and stevia extract; Synthetic sweeteners such as saccharin and aspartame can be used. The ratio of the natural carbohydrate is generally about 0.01 to 0.04 g, preferably about 0.02 to 0.03 g per 100 mL of the composition of the present invention.

In addition, the Lactobacillus plantarum Ln4 strain of the present invention may be contained as an active ingredient of a food composition for preventing and improving obesity, and the amount is not particularly limited to an amount effective to achieve the action of preventing and improving obesity. , It is preferably 0.01 to 100% by weight based on the total weight of the total composition. The food composition of the present invention can be prepared by mixing Lactobacillus plantarum Ln4 strain together with other active ingredients known to be effective in a composition for preventing and improving obesity.

In addition to the above, the health food of the present invention includes various nutrients, vitamins, electrolytes, flavoring agents, colorants, pectic acid, salts of pectic acid, alginic acid, salts of alginic acid, organic acids, protective colloid thickeners, pH adjusters, stabilizers, preservatives, It may contain glycerin, alcohol, or a carbonating agent. In addition, the health food of the present invention may contain flesh for the manufacture of natural fruit juice, fruit juice beverage, or vegetable beverage. These ingredients may be used independently or in combination. Although the proportion of these additives is not very important, it is generally selected from 0.01 to 0.1 parts by weight per 100 parts by weight of the composition of the present invention.

Hereinafter, the present invention will be described in more detail through examples. These examples are for illustrative purposes only, and it will be apparent to those of ordinary skill in the art that the scope of the present invention is not construed as being limited by these examples.

Useful from traditional fermented foods Probiotics Isolation and acquisition of strains

<1-1> Origin of traditional fermented food Pure separation of microorganisms

In order to carry out the examples of the present invention, microorganisms were purely isolated from traditional fermented foods.

Specifically, a fermented food sample was prepared by purchasing soybean paste, soy sauce, and gochujang paste purchased in a traditional market, and ^{diluting it in distilled water at a ratio of 10 -1} to 10 ^-7. Then, the fermented food sample was plated on a lactobacilli MRS medium (lactobacilli MRS), a phenylethyl alcohol medium, and a lactobacillus selective agar medium. The smeared medium was cultured at 37° C. to obtain a colony, and the morphology was observed. The smeared medium was plated on a new medium, and the microorganisms were purely separated.

<1-2> Identification of isolated lactic acid bacteria

In order to confirm what strain the microorganisms purely isolated through the lactic acid bacteria selection medium were, the isolated microorganisms were identified.

Specifically, genomic DNA was extracted from the strain isolated in Example <1-1>, and the nucleotide sequence was analyzed (Bionics, commissioned by Korea) to obtain the gene nucleotide sequence of 16S rRNA. The nucleotide sequence of the 16S rRNA was analyzed for homology with other standard strains registered in the database using the BLAST program, and was identified as a strain exhibiting 98% or more homology. In addition, homology was analyzed and a schematic was constructed using Cluster X and Mega 7 programs.

As a result, called the lactic acid bacteria is Lactobacillus Planta room (Lactobacillus plantarum) with Lactobacillus Planta room Ln4 (Lactobacillus plantarum Ln4) showed a homology with the reported strain isolated from a traditional fermented foods, as also it is shown in the first Therefore, it was deposited with the Korea Microbial Conservation Center (KCCM) on September 22, 2016 (accession number KCCM 11897P).

Lactobacillus Planta Room Ln4 Identification of strain characteristics

<2-1> Lactobacillus Plata Room Ln4 Acid resistance And Bile resistance Confirm

To confirm the characteristics of Lactobacillus plantarum Ln4 isolated from traditional fermented foods, acid resistance and bile resistance were confirmed.

Specifically, first, in order to confirm the acid resistance, the Lactobacillus plantarum Ln4 strain was inoculated into an artificial gastric juice medium (pH 2.5) of a 0.3% pepsin aqueous solution, followed by stationary culture for 3 hours. After cultivation, the bacteria were obtained, plated on a plate medium, incubated at 37°C for 48 hours, and then the number of colonies generated was counted to measure the number of viable bacteria to confirm the relative number of viable bacteria to the number of viable bacteria in the solvent control group.

In addition, in order to confirm bile resistance, Lactobacillus plantarum Ln4 strain was inoculated into a medium of 0.3% oxgall and cultured for 24 hours. After cultivation, bacteria were obtained and the relative number of viable bacteria was confirmed by the same method.

As a result, it was confirmed that the Lactobacillus plantarum Ln4 strain maintained a survival rate of 99% or more for 3 hours even in an artificial gastric juice medium as shown in [Table 1], thus exhibiting excellent acid resistance (Table 1). In addition, it was confirmed that the number of bacteria was maintained at 80% or more for 24 hours for bile resistance, thereby showing excellent bile resistance (Table 1).

Confirmation of acid resistance and bile resistance of Lactobacillus plantarum Ln4 strain Strain name Acid resistance (%) Bile resistance (%) Lactobacillus plantarum Ln4 bacteria 99.25 84.20

<2-2> Lactobacillus Planta Room Ln4 enzyme Productivity Confirm

In order to confirm the characteristics of Lactobacillus plantarum Ln4 isolated from traditional fermented foods, Lactobacillus plantarum Ln4 strain can produce various enzymes such as β-glucuronidase, which causes carcinogenesis. Whether or not it was checked.

Specifically, the Lactobacillus plantarum Ln4 strain was cultured until the logarithmic growth phase, and then centrifuged at 10,000×g for 10 minutes to remove the supernatant medium. After removing the medium, the obtained strain was washed with a PBS buffer solution, and then the substrate of the API ZYM kit was mixed with the washed strain and incubated for 4 hours. Then, solutions A and B of the kit were added, respectively, and reacted for 5 minutes at a high light source, and then, according to the degree of color development, whether or not enzyme was produced was confirmed.

As a result, it was confirmed that Lactobacillus plantarum Ln4 did not produce β-glucuronidase, which causes carcinogenesis, as shown in Table 2 below (Table 2).

Confirmation of enzyme production ability of Lactobacillus plantarum Ln4 strain Enzyme Enzyme activity (nmole) Lactobacillus plantarum Ln4 Control 0 Alkaline phosphatase 0 Esterase 0 Esterase lipase 0 Lipase 0 Leucine arylamidase 2 Valine arylamidase One Cystine arylamidase 0 Trypsin 0 α-Chymotrypsin 0 Acid phosphatase One Naphthol-AS-BI-phosphohydrolase One α-Galactosidase 0 β-Galactosidase 4 β-Glucuronidase 0 α-Glucosidase 0 β-Glucosidase 3 N-Acetyl-β-glucosaminidase 0 α-Mannosidase 0 α-Fucosidase 0

(In the above table, 0 represents the production of 0 to 5 nmol of enzyme; 1 represents the production of 5 to 10 nmol of enzyme; 2 represents the production of 10 to 20 nmol of enzyme; 3 represents 20 to Represents to produce 30 nmol of enzyme; 4 represents to produce 30 to 40 nmol of enzyme; and 5 represents to produce 40 nmol or more of enzyme.)

<2-3> Lactobacillus Planta Room Ln4 Cytotoxicity check

In order to confirm the characteristics of Lactobacillus plantarum Ln4 isolated from traditional fermented foods, the cytotoxicity of the Lactobacillus plantarum Ln4 strain was confirmed.

Specifically, normal cells, MRC-5 cells, were inoculated into a 96-well plate and then cultured for 24 hours ^{to prepare MRC-5 cells at a concentration of 2×10 5} cells/ml. Then, the Lactobacillus plantarum Ln4 strain was cultured until the logarithmic growth phase, and then centrifuged at 10,000×g for 10 minutes to remove the supernatant medium. After removing the medium, the obtained strain was washed with a PBS buffer solution, and the obtained Lactobacillus plantarum Ln4 strain was inoculated on the prepared MRC-5 culture plate and cultured for 44 hours. After incubation, washing was repeated three times with a PBS buffer solution, and then 50 µl of MTT reagent was treated and reacted for 4 hours. After completion of the reaction, 50 µl of DMSO was treated, absorbance was measured at a wavelength of 570 nm, and cytotoxicity was confirmed through the level of killed MRC-5 cells according to the degree of color development.

As a result, as shown in the following [Table 3], it was confirmed that the Lactobacillus plantarum Ln4 strain did not induce death of MRC-5 cells even after culturing with normal cells, and the Lactobacillus plantarum Ln4 strain was a normal cell It was confirmed that it did not have cytotoxicity against (Table 3).

Confirmation of cytotoxicity of Lactobacillus plantarum Ln4 Microorganisms Cytotoxicity ( % ) Lactobacillus plantarum Ln4 3.91±1.10

<2-4> Lactobacillus Planta Room Ln4 chapter Adhesion Confirm

In order to confirm the characteristics of Lactobacillus plantarum Ln4 isolated from traditional fermented foods, the intestinal adhesion ability of the Lactobacillus plantarum Ln4 strain was confirmed.

Specifically, colon cancer cells HT-29 cells were inoculated into a 96-well plate and then cultured for 24 hours ^{to prepare MRC-5 cells at a concentration of 1×10 5} cells/ml. Then, the Lactobacillus plantarum Ln4 strain was cultured until the logarithmic growth phase, and then centrifuged at 10,000×g for 10 minutes to remove the supernatant medium. After removing the medium, the obtained strain was washed with a PBS buffer solution, and the obtained Lactobacillus plantarum Ln4 strain was inoculated on the prepared HT-29 culture plate and cultured for 2 hours. After incubation, washing was repeated three times with PBS buffer solution, and then treated for 10 minutes by adding 1 ml of Triton-X100, centrifuged, a supernatant was obtained, plated on lactobacilli MRS agar medium, and incubated for 48 hours at 37°C. The number of colonies generated was checked, and the number of bacteria attached to the colon cancer cell line and the number of bacteria remaining in the medium (the number of colonies generated) were compared to confirm the intestinal adhesion ability.

As a result, it was confirmed that the Lactobacillus plantarum Ln4 strain has high intestinal adhesion to colorectal cancer cells as shown in the following [Table 4] (Table 4).

Confirmation of intestinal adhesion ability of Lactobacillus Plantarum Ln4 Microorganisms Adhesion ability( % ) Lactobacillus plantarum Ln4 7.64±3.25

<2-5> Lactobacillus Planta Room Ln4 Confirmation of antibiotic resistance

In order to confirm the characteristics of Lactobacillus plantarum Ln4 isolated from traditional fermented foods, it was determined whether the Lactobacillus plantarum Ln4 strain exhibits resistance to antibiotics used in the CLSI (Clinical and Laboratory Standards Institute, 2012) criteria. Confirmed.

Specifically, the Lactobacillus plantarum Ln4 strain was cultured until the logarithmic growth phase, and then centrifuged at 10,000×g for 10 minutes to remove the supernatant medium. After removing the medium, the obtained strain was washed with a PBS buffer solution, ^{diluted to a concentration of 10 5} CFU/ml, spread on lactobacilli MRS agar medium, and cultured until the surface of the medium was saturated with Lactobacillus plantarum Ln4. Then, 50 µl of a sterilized paper disc was added to ampicillin, tetracycline, zetamicin, ciprofloxacin, kanamycin, chloramphenicol, and streptomycin. Streptomycin) or doxocycline, respectively, and placed on the surface of the cultured Lactobacillus plantarum Ln4 strain medium. And the degree of antibiotic resistance was determined according to the size of the growth inhibition zone (inhibition zone) produced by incubation at 37°C for 24 hours.

As a result, as shown in the following [Table 5], Lactobacillus plantarum Ln4 is sensitive to ampicillin, tetracycline, chloramphenicol and doxocycline, and shows resistance to zetamicin, ciprofloxacin, kanamycin, and streptomycin. Confirmed (Table 5).

Confirmation of antibiotic resistance of Lactobacillus plantarum Ln4 Antibiotics Lactobacillus plantarum Ln4 Antibiotics Lactobacillus plantarum Ln4 Ampicillin S Tetracycline S Gentamycin R Ciprofloxacin R Kanamycin R Chloramphenicol S Streptomycin R Doxycycline S

(In the above table, R represents antibiotic resistance and S represents antibiotic sensitive.)

Lactobacillus Planta Room Ln4 Live cells And Dead cell Produce

To confirm the pharmaceutical efficacy of Lactobacillus plantarum Ln4 of the present invention in vitro and in vivo , live cells and dead cells of Lactobacillus plantarum Ln4 were prepared.

Specifically, the Lactobacillus plantarum Ln4 isolated and identified in <Example 1> was inoculated into a lactobacilli MRS liquid medium, incubated at 37°C for 18 hours, and then centrifuged at 5,000×g for 10 minutes to precipitate the cells. And separated into the supernatant of the culture solution. Then, the separated precipitate was lyophilized to obtain it as a live cell, and the live cell was heat-treated at 121° C. for 5 minutes to prepare and obtain a dead cell.

Confirmation of anti-obesity effect of Lactobacillus plantarum Ln4

<4-1> Confirmation of Lactobacillus Plantarum Ln4's ability to inhibit fat accumulation

To determine whether the Lactobacillus plantarum Ln4 of the present invention can exhibit an anti-obesity effect at an in vitro level, the effect of inhibiting fat accumulation in adipocytes was confirmed.

Specifically, 3T3-L1 cells, which are adipocyte progenitor cell lines, were inoculated in DMEM medium, and then cultured in a 5% CO ₂ incubator at 37° C. until the saturation of the plate reached 100%. When the plate was grown to fill the cells, 10% FBS/DMEM differentiation medium containing 5 μg/ml insulin, 115 μg/ml IBMX and 10 μM dexamethasone was exchanged as a cell medium, and further cultured for 2 days. . At this time, 100 μg/ml of Lactobacillus plantarum Ln4 dead cells prepared in Example 3 was added to the differentiation medium. After 2 days of cultivation, Lactobacillus plantarum Ln4 dead cells were exchanged with 10% FBS/DMEM medium containing 100 µg/ml and 5 µg/ml insulin, and cultured for 4 to 6 days to differentiate from adipocytes. Adipocytes were obtained. The obtained adipocytes were fixed by adding a 10% formalin aqueous solution, and treated with an Oil Red O solution to react for about 15 minutes to 2 hours to stain the fat accumulated in the cells. After completion of the dyeing, after observation under a microscope, the Oil Red O solution was dissolved again with isopropyl alcohol, and the absorbance was measured at a wavelength of 518 nm. As a control, a non-differentiated undifferentiated cell control (ND, non-differentiated) and a differentiated cell control (D, differentiated) differentiated in a medium to which dead cells were not added were used. The degree of increase in the absorbance level was calculated using the following [Equation 1].

[Equation 1]

As a result, as shown in FIG. 2, when compared with the differentiated cell control group (D), it was confirmed that the level of fat accumulation in the Lactobacillus plantarum Ln4-treated experimental group of the present invention was reduced, and also stained in red through microscopic observation. It was confirmed that the number and size of fat cells significantly decreased (FIG. 2).

<4-2> Preparation of a diabetes induction animal model by a high fat diet

To determine whether Lactobacillus plantarum Ln4 can exhibit a significant anti-obesity effect in a metabolic disease-induced animal model, a diabetes-inducing mouse model, which is a subject experimental animal, was constructed.

Specifically, the Lactobacillus plantarum Ln4 live cells prepared in Example 3 ^{were suspended in distilled water at a concentration of 2.5×10 9} CFU/ml to prepare a strain sample. Then, 0.2 ml of the prepared strain sample was orally administered daily to 6-week-old C57/BL6 mice while freely feeding high-fat feed, and 5×10 ⁸ CFU was administered daily. The high fat feed was used in the form of a pellet containing 21.0% protein, 24.6% carbohydrates and 54.4% fat based on the caloric value (㎉) of the total feed. Mice after administration of a strain sample and feeding a high-fat diet in the same environment for a total of 5 weeks were used as a diabetes induction experimental group (Ln4) administered with Ln4 Ln4. In order to compare the results of the Ln4 experimental group, the group that was reared in the same environment while administering live cells and self-supplied with normal feed was used as a normal control, and a high-fat feed was fed without administration of live cells while rearing in the same environment. The self-paying group was used as the high fat diet induction group (HFD). Ln4 experimental group, normal control group and HFD induction group checked their respective weights to determine whether diabetes and metabolic diseases were induced by a high fat diet, and at the same time confirmed whether the effect of weight reduction by Lactobacillus plantarum Ln4 appeared. I did.

As a result, as shown in FIG. 3, it was confirmed that the weight gain of the HFD-inducing group was significantly higher from 1 week after breeding compared to the normal control group, compared to the weight gain of the HFD group, in the oral administration group of Ln4 probiotics (Ln4). It was confirmed that the level of weight gain was significantly reduced (FIG. 3).

<4-3> Analysis of body fat reduction efficacy of Lactobacillus plantarum Ln4

To determine whether Lactobacillus plantarum Ln4 can exhibit a significant anti-obesity effect in a metabolic disease-induced animal model, the effect of reducing body fat in an animal model inducing metabolic disease was confirmed.

Specifically, after analyzing the weight control efficacy in the Ln4 probiotic administration group (Ln4) and the control group that induced metabolic disease for 5 weeks by the method of Example <4-2>, the epididymis fat, which is a representative visceral fat, was separated and the weight was measured. It was measured. Then, compared with the weight of epididymis fat before administration of the high fat diet, the level of increase in fat weight due to induction of the high fat diet for 5 weeks was calculated relatively.

As a result, as shown in FIG. 4, the control group (HFD) inducing a high-fat diet increased the weight of epididymis fat to about twice the level when compared to the control group fed with a normal diet, but in the experimental group (Ln4), the HFD group It was confirmed that the level of increase in the weight of epididymis fat was significantly decreased compared to (FIG. 4 ).

Korea Microorganism Conservation Center (overseas) KCCM11897P 20160922

<110> Konkuk University Industrial Cooperation Corp KOREA FOOD RESEARCH INSTITUTE <120> Novel Lactobacillus plantarum Ln4 strain and compositions for the prevention and treatment of obesity containing the same <130> 1061538 <160> 1 <170> KopatentIn 2.0 <210> 1 <211> 943 <212> RNA <213> Lactobacillus plantarum <400> 1 tgctaataca tgcagtcgaa cgaactctgg tattgattgg tgcttgcatc atgatttaca 60 tttgagtgag tggcgaactg gtgagtaaca cgtgggaaac ctgcccagaa gcgggggata 120 acacctggaa acagatgcta ataccgcata acaacttgga ccgcatggtc cgagcttgaa 180 agatggcttc ggctatcact tttggatggt cccgcggcgt attagctaga tggtggggta 240 acggctcacc atggcaatga tacgtagccg acctgagagg gtaatcggcc acattgggac 300 tgagacacgg cccaaactcc tacgggaggc agcagtaggg aatcttccac aatggacgaa 360 agtctgatgg agcaacgccg cgtgagtgaa gaagggtttc ggctcgtaaa actctgttgt 420 taaagaagaa catatctgag agtaactgtt caggtattga cggtatttaa ccagaaagcc 480 acggctaact acgtgccagc agccgcggta atacgtaggt ggcaagcgtt gtccggattt 540 attgggcgta aagcgagcgc aggcggtttt ttaagtctga tgtgaaagcc ttcggctcaa 600 ccgaagaagt gcatcggaaa ctgggaaact tgagtgcaga agaggacagt ggaactccat 660 gtgtagcggt gaaatgcgta gtaatatgga agaacaccag tggcgaaggc ggctgtctgg 720 tctgtaactg acgctgaggc tcgaaagtat gggtagcaaa caggattaga taccctggta 780 gtccataccg taaacgatga atgctaagtg ttggagggtt tccgcccttc agtgctgcag 840 ctaacgcatt aagcattccg cctgggggag tacggccgcc aggctgaaac tcaaaggaat 900 ttgacggggg cccgcacaag cggtcggagc atgtggtttc att 943

Claims (6)

A pharmaceutical composition for the prevention and treatment of obesity comprising Lactobacillus plantarum Ln4 strain deposited with Accession No. KCCM 11897P, a culture thereof or a mixture thereof as an active ingredient.
The pharmaceutical composition for preventing and treating obesity according to claim 1, wherein the strain has the 16S rRNA base sequence set forth in SEQ ID NO: 1.
The pharmaceutical composition for preventing and treating obesity according to claim 1, wherein the strain has acid resistance, biliary properties, intestinal adhesiveness and antibiotic resistance.
The pharmaceutical composition for the prevention and treatment of obesity according to claim 1, wherein the Lactobacillus plantarum Ln4 strain exhibits a fat accumulation inhibitory effect and a body fat reducing effect.
Lactobacillus plantarum Ln4 deposited with accession number KCCM 11897P, a culture solution thereof, or a mixture thereof as an active ingredient.
The health functional food according to claim 5, wherein the Lactobacillus plantarum Ln4 strain is characterized by an effect of inhibiting fat accumulation and reducing body fat.

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