KR101981333B1 - Lactobacillus fermentum MG4231 or MG4244 having antiobesity activity derived from human and composition comprising the same - Google Patents

Abstract

The present invention relates to: human-derived Lactobacillus fermentum MG4231 or Lactobacillus fermentum MG4244; and a pharmaceutical composition and a food composition, a quasi-drug composition, and health functional food which are to prevent or treat obesity-related diseases by having the same as active components. The lactobacillus fermentum MG4231 strain or MG4244 strain of the present invention: shows anti-obesity activity which suppresses the activity of lipase enzyme, the differentiation of preadipocytes, and the accumulation of triglyceride; has excellent ability to form colonies on the epithelial cells of digestive tracts due to a good self-aggregating ability; has resistance to acids and bile salts; and thus, can be used for a pharmaceutical composition, a food composition, a quasi-drug composition, and health functional food in various ways to treat and prevent obesity-related diseases.

Description

Lactobacillus perfumolum MG4231 or Lactobacillus permentum MG4244 strain having human anti-obesity activity and a composition comprising the same, wherein the Lactobacillus fermentum MG4231 or MG4244 has an antiobesity activity derived from human and composition comprising the same,

The present invention relates to human-derived Lactobacillus permentum MG4231 or Lactobacillus permentum MG4244 and to pharmaceutical compositions and food compositions, quasi-drug compositions and health functional foods for the prevention or treatment of obesity-related diseases using the same as an active ingredient.

Lactic acid bacteria have a specific physiological activity and are generally regarded as safe strains (GRAS). Lactic acid bacteria are used not only for the production of various fermented foods, but also for dairy products and fermented fruit and vegetable products having functional and probiotic characteristics. In recent years, as consumers' demand for natural adjuvants to replace chemical adjuvants has increased, they have become an alternative. Probiotics are living microorganisms that have a very beneficial effect on the health of host animals, improving the balance of intestinal microorganisms and promoting uptake of nutrients. In addition, probiotics are characteristic of antibiotic action of pathogenic microorganisms in intestinal environment. Probiotics include a variety of microorganisms, but Lactobacillus , Genus Bifidobacterium < RTI ID = 0.0 > The genus occupies the most distribution. In particular, the genus Lactobacillus (Lactobacillus) are generally used in the dairy, meat, and gwachae fermentation of cereal products.

Obesity is known to be a major cause of heart disease, cancer, arthritis, and diabetes, and obesity is a growing trend despite increasing public awareness to address obesity. Obesity is associated with an increase in the number of adipocytes as a result of adipogenesis and an increase in the lipid content of adipocytes. Adipocytes play a major role in the synthesis and storage of excess calories into triglycerides and as a result of fat differentiation, the size and number of adipocytes increase and the accumulation of intracellular lipids accelerates.

In the past, adipose tissue was thought to be an energy storage organ that stores and releases excessive energy in the form of triacylglycerol, but recently, adiponectin, leptin, and resistin It is accepted as an important endocrine organ that regulates energy homeostasis by secreting various adipokines (Trends Endocrinol Metab 13:18, 2002). Therefore, understanding of the proliferation of adipocytes and the substances secreted by adipocytes and their in vivo mechanisms of regulation are considered to serve as a basis for understanding obesity and various diseases caused by it and for developing an effective therapeutic agent. Studies on the regulation of adipocyte differentiation have been actively conducted and it is considered to be the main mechanism that differentiation from preadipocytes in the body is related to the increase of adipocytes in obese patients. The differentiation process of precursor adipocytes into adipocytes has been studied using cells such as 3T3-L1, and several transcription factors, particularly transcription factors known to be involved in localization, C / EBPs (CAAT enhancer binding proteins, PPARs (Peroxisome Proliferator Activated Receptor), and ADD / SREBPs (Adipocyte determination and differentiation dependent element binding proteins), are known to regulate the process and to control the process (Bart A Jessen et al., Gene, 299, pp 95-100, 2002; Darlington et al., J. Biol. Chem., 273, pp30057-30060, 1998; Brun RP et al., Curr Opin. , pp 826-832, 1996). Given the stimulation of hormones such as MDI (isobutylmethylxanthin, dexamethasone and insulin), C / EBP [beta] and [delta] are first transiently expressed and initiate differentiation into adipocytes (Reusch J. E et al., Mol Cell. Biol., 20, pp 1008-1020, 2000). Only the fat cells that have undergone the differentiation process synthesize fatty acids and store triglycerides. Therefore, current research trends are focused on the search for substances that can inhibit metabolic processes related to adipocyte differentiation as a method for preventing or treating obesity-related metabolic diseases. In other words, attempts have been made to treat obesity through the regulation of fat cells based on the mechanism of obesity, and it has been attempted to inhibit fat synthesis, to reduce fat amount by promoting lipolysis and oxidation, The goal is to reduce the number of cells, the transcription factors, proteins and adipokines, which are known to mediate or modulate these processes, as a new therapeutic target for obesity. Currently known herbal remedies include Xenical (Roche Pharmaceuticals, Switzerland), Reductil (Evothe, USA) and Exolise (Atopama, France), which are widely used as appetite suppressants, And most obesity drugs are appetite suppressants that suppress appetite by controlling the neurotransmitters associated with the hypothalamus.

However, conventional therapeutic agents have side effects such as heart diseases, respiratory diseases, nervous system diseases and the like, and the persistence of their effects is also low, so that it is necessary to develop a more improved therapeutic agent for obesity. Also, currently developed products have satisfactory therapeutic effects There is little cure for cervical cancer, and development of a new treatment for obesity is required.

On the other hand, efforts have been made to reduce the blood cholesterol level by using lactic acid bacteria which have been regarded as safe microorganisms. Lactic acid bacteria have been reported to exhibit effects such as maintenance of intestinal flora, improvement of intestinal flora, inhibition of carcinogenesis, suppression of colitis, and nonspecific activity of host immune system. Among them, the Lactobacillus sp. Strain is a major member of normal microbial communities in the intestinal tract of the human body and has long been known to be important for maintaining a healthy digestive tract and vaginal environment, and the US Public Health Service According to the guidelines, all of the Lactobacillus strains deposited at the American Depositary Organization (ATCC) are now classified as "Bio-safty Level 1", which is considered to have no known potential risk of causing diseases in humans or animals have. However, the lactic acid bacteria have been known to excel in the immune response modulating effect and anticancer and antioxidative effect through the existing studies, but studies on the lipid differentiation inhibiting effect and fat accumulation inhibitory effect of Lactobacillus strains are insufficient.

Accordingly, the inventors of the present invention conducted studies on the lipid differentiation inhibitory effect and the fat accumulation inhibitory effect of Lactobacillus strains, and confirmed that the human-derived Lactobacillus fermentum MG4231 and MG4244 have anti-obesity activity, Completed.

Accordingly, the object of the present invention is to provide pharmaceutical composition and food composition, quasi-drug composition and health functional food for prevention or treatment of obesity-related diseases, using Lactobacillus permentum MG4231 or Lactobacillus permentum MG4244 isolated from human body and its active ingredient .

Provides: (KCTC 13594BP deposit number) strain: In order to achieve the above object, the present invention is human-derived Lactobacillus buffer lactofermentum (Lactobacillus fermentum) MG4231 strain (Accession No. KCTC 13593BP) or Lactobacillus buffer lactofermentum (Lactobacillus fermentum) MG4244 .

The present invention also provides a pharmaceutical composition for preventing or treating obesity-related diseases comprising the above-mentioned strains.

The present invention also provides a food composition for preventing or ameliorating an obesity-related disease comprising the strain.

The present invention also provides a health functional food for preventing or ameliorating an obesity-related disease comprising the strain.

The present invention also provides a quasi-drug composition for preventing or ameliorating an obesity-related disease comprising the above-mentioned strain.

The Lactobacillus permentum MG4231 strain or MG4244 strain of the present invention exhibits anti-obesity activity inhibiting lipase enzyme activity, inhibiting lipid progenitor differentiation inhibition and neutral fat accumulation, and exhibiting excellent self-cohesive ability to colonize epithelial cells of the digestive tract And is resistant to acid and bile, and can be used as a pharmaceutical composition, a food composition or a quasi-drug composition and a health functional food for the purpose of treating and preventing obesity-related diseases.

FIG. 1 shows the results of the viability test of Lactobacillus permentum MG4231 strain or MG4244 strain at pH 2 to 4, which is a gastric juice environment, in order to examine the viability of Lactobacillus permentum MG4231 strain or MG4244 strain in a gastrointestinal environment Fig.
FIG. 2 shows the viability test of Lactobacillus permentum MG4231 strain or MG4244 strain against intestinal environments pH 7 and 8, in order to examine the viability of Lactobacillus permentum MG4231 strain or MG4244 strain in gastric environment, Fig.
FIG. 3 is a graph showing the results of experiments confirming the self-aggregating ability of Lactobacillus permentum MG4231 strain or MG4244 strain.
4 is a graph showing the results of experiments for confirming the effect of inhibiting the accumulation of triglycerides in Lactobacillus permentum MG4231 strain or MG4244 strain.

The present invention provides a strain of Lactobacillus fermentum MG4231 (accession number: KCTC 13593BP) or Lactobacillus fermentum MG4244 (accession number: KCTC 13594BP) derived from human body.

Hereinafter, the present invention will be described in more detail.

The Lactobacillus fermentum MG4231 strain or the Lactobacillus fermentum MG4244 strain is a novel strain having an anti-obesity effect. In the present invention, the term " lactobacillus " refers to a bacterium that is fermented by a widely distributed saccharide in nature and produces energy to produce a large amount of lactic acid. The microorganism is morphologically polymorphic in the form of Gram-positive spore bacillus, L. fermentum , L. plantarum , L. brevis , and the like. Identifying the inventors as to the lipase (lipase) enzyme activity inhibition, preadipocytes differentiation inhibiting or Lactobacillus having the triglyceride accumulation inhibitory activity buffer lactofermentum (Lactobacillus fermentum) MG4231 strain or Lactobacillus buffer lactofermentum (Lactobacillus fermentum) MG4244 strain Respectively.

In order to isolate the Lactobacillus strain according to the present invention, a strain was isolated from a healthy Korean female vagina. There are numerous lactic acid bacteria in healthy vagina, and their distribution is known to vary with race, age and environment. Among the isolated strains, two strains having the highest anti-obesity activity were selected and identified to be Lactobacillus fermentum .

The strain was designated as Lactobacillus fermentum MG4231 and deposited with the Korean Society for Microbiological Protection and assigned accession number KCTC 13593BP. Another strain was named Lactobacillus fermentum MG4244 and deposited with Accession No. KCTC 13594BP.

In the examples of the present invention, it was confirmed that the newly isolated Lactobacillus fermentum strain MG4231 or Lactobacillus fermentum MG4244 exhibited excellent anti-obesity effect.

The Lactobacillus fermentum MG4231 strain or the Lactobacillus fermentum MG4244 strain has at least one activity selected from the group consisting of inhibition of lipase enzyme activity, inhibition of lipid progenitor differentiation inhibition and neutral fat accumulation inhibition, .

Further, the strain is characterized in that it has acid resistance and bile resistance. In one embodiment of the present invention, it was confirmed that Lactobacillus fermentum MG4231 strain or Lactobacillus fermentum MG4244 strain maintained high survival rate at pH 2 to 4, which is a strong acid condition containing pepsin. The Lactobacillus fermentum strain MG4231 or the Lactobacillus fermentum strain MG4244 is also useful for the treatment of bile salts containing pancreatin at pH 7 and pH 8, .

Lactobacillus spread lactofermentum (Lactobacillus fermentum) MG4231 strain or Lactobacillus spread lactofermentum (Lactobacillus fermentum) MG4244 strain of the present invention is an excellent self prevent the removal of probiotics by coagulation ability (autoaggregation ability) appeared chapter twitching movements effectively digestive tract in Chapter Lt; RTI ID = 0.0 > epithelial < / RTI >

The present invention also provides a pharmaceutical composition for preventing or treating obesity-related diseases comprising the above-mentioned strains.

The pharmaceutical composition comprises a Lactobacillus buffer lactofermentum (Lactobacillus fermentum) or MG4231 strain Lactobacillus buffer lactofermentum (Lactobacillus fermentum) MG4244 strain of the present invention as an active ingredient. The amount of the strain is preferably 0.01 to 95 parts by weight, more preferably 1 to 80 parts by weight, based on 100 parts by weight of the total amount of the pharmaceutical composition. When the content is less than 0.01 part by weight, the efficiency of taking may be lowered, and when it is more than 95 parts by weight, it may be difficult to formulate.

The pharmaceutical composition comprising Lactobacillus fermentum MG4231 strain or Lactobacillus fermentum MG4244 strain as an active ingredient according to the present invention can be used for inhibiting lipase enzyme activity, It can be directly used as a composition itself having an effect of preventing, ameliorating or treating obesity-related diseases caused by fat accumulation-inhibiting effects, and can also be used as an adjuvant.

The pharmaceutical composition according to the present invention may contain a pharmacologically effective amount of Lactobacillus fermentum MG4231 strain or Lactobacillus fermentum MG4244 strain alone or in combination with one or more pharmaceutically acceptable carriers, Or a diluent. The term " pharmaceutically effective amount " as used herein refers to an amount sufficient to exert the preventive, ameliorative, or therapeutic effect of an obesity-related disease caused by lipase enzyme activity inhibition, lipid precursor differentiation inhibition, It says. The phrase " pharmaceutically acceptable " as used herein refers to a composition that is physiologically acceptable and does not normally cause an allergic reaction such as gastrointestinal disorder, dizziness, or the like when administered to a human.

The pharmaceutical composition according to the present invention may be formulated in the form of powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols and the like, oral preparations, suppositories and sterilized injection solutions according to a conventional method . Suitable formulations known in the art are preferably those as disclosed in Remington ' s Pharmaceutical Science, recently, Mack Publishing Company, Easton PA. Examples of carriers, excipients and diluents which may be included include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, Cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, mineral oil and the like. When the pharmaceutical composition is formulated, it is prepared using a diluent such as a filler, an extender, a binder, a wetting agent, a disintegrant, a surfactant, or an excipient usually used. Solid formulations for oral administration include tablets, pills, powders, granules, capsules and the like, which may contain at least one excipient such as starch, calcium carbonate, sucrose, lactose, Gelatin and the like. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used. Examples of the liquid preparation for oral use include suspensions, solutions, emulsions, and syrups. In addition to water and liquid paraffin, simple diluents commonly used, various excipients such as wetting agents, sweeteners, fragrances, preservatives and the like may be included . Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, suppositories, and the like. Examples of the suspending agent include propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate, and the like. Examples of the suppository base include witepsol, macrogol, tween 61, cacao butter, laurin, glycerogelatin and the like.

The pharmaceutical composition of the present invention is useful as an effective ingredient or amount of a pharmaceutical composition that induces a biological or medical response in a tissue system, an animal or a human, as considered by a researcher, veterinarian, physician or other clinician, Or a therapeutically effective amount which is sufficient to induce relief. It will be apparent to those skilled in the art that the therapeutically effective dose and the frequency of administration for the pharmaceutical compositions of the present invention will vary with the desired effect. Thus, the optimal dosage to be administered can be readily determined by those skilled in the art and will vary with the nature of the disease, the severity of the disease, the amount of active and other ingredients contained in the composition, the type of formulation, and the age, The age, body weight, sex, diet, time of administration, route of administration and fraction of the composition, duration of treatment, concurrent medication, and the like. For a desired effect, the pharmaceutical composition of the present invention may be administered in an amount of 1 to 10,000 mg / kg / day, preferably 1 to 200 mg / kg / day, or may be administered once a day, . ≪ / RTI >

The pharmaceutical composition of the present invention may be administered to a subject in various routes. All modes of administration may be expected, for example, by oral, rectal or intravenous, intramuscular, subcutaneous, intra-uterine dural or intracerebral injection.

The content of Lactobacillus fermentum MG4231 strain or Lactobacillus fermentum MG4244 strain in the pharmaceutical composition according to the present invention is determined by the absorbance of the active ingredient in the body, the excretion rate, the age, sex, State, and the like.

The pharmaceutical composition according to the present invention may be used alone, but it may be administered to a patient in need of other appropriate treatment methods (for example, surgery, radiation therapy, hormone therapy, chemotherapy, A method of using a biological reaction modifier, etc.).

The obesity-related disease of the present invention may be one selected from the group consisting of fatty liver, type 2 diabetes, hyperlipidemia, cardiovascular disease, arteriosclerosis and lipid-related metabolic syndrome, It is not.

The term " prevention " as used herein in the present invention means all actions that inhibit or delay the onset of obesity-related diseases by administering to a subject a composition for the prevention or treatment of obesity-related diseases according to the present invention.

In the present invention, the term " treatment " may mean all the actions that cause the symptoms of obesity-related diseases to be improved or benefited by administering the composition according to the present invention to suspect individuals with obesity-related diseases.

As used herein, the term " improvement " may mean any action that at least reduces the degree of symptom associated with the condition being treated.

In the present invention, the term " individual " may mean all animals including humans who have developed or are likely to develop an obesity-related disease.

The present invention also provides a food composition for preventing or ameliorating an obesity-related disease comprising the strain.

In the food composition according to the present invention, the type of the food is not particularly limited, and may include foods in a conventional sense. Non-limiting examples of the food to which the substance can be added include meat, sausage, bread, chocolate, candy, snacks, confectionery, pizza, ramen, other noodles, gums, dairy products including ice cream, , A drink, an alcoholic beverage, and a vitamin complex. When the composition is used as a food additive, the composition may be added as it is or may be used together with other food or food ingredients, and may be suitably used according to a conventional method.

The term " food " as used herein means a natural product or a processed product containing one or more nutrients. Preferably, the term " food " The food composition is intended to include food, food additives, health functional foods and beverages.

Foods to which the composition of the present invention can be added include, for example, various foods, beverages, gums, candies, tea, vitamin complexes, and functional foods. In addition, in the present invention, the food may contain special nutritional foods (e.g., crude oil, spirits, infant food, etc.), meat products, fish products, tofu, jelly, noodles (Such as soy sauce, soybean paste, hot pepper paste, mixed sauce), sauces, confectionery (eg snacks), dairy products (eg fermented milk, cheese), other processed foods, kimchi, pickled foods But are not limited to, natural flavors (eg, ramen soup, etc.), vitamin complexes, alcoholic beverages, alcoholic beverages and other health supplement foods. The food, beverage or food additive may be prepared by a conventional production method.

In particular, the inventors of the present invention found that Lactobacillus fermentum MG4231 strain or Lactobacillus fermentum MG4244 strain inhibits lipase enzyme activity, inhibits lipid progenitor differentiation, or inhibits the accumulation of triglycerides. It is possible to directly use the above food composition for all foods including fermented food.

The present invention also provides a health functional food for preventing or ameliorating an obesity-related disease comprising the strain.

The term " health functional food " in the present invention refers to a food prepared by processing a specific ingredient as a raw material for the purpose of health assisting or by extracting, concentrating, refining, or mixing a specific ingredient contained in a food raw material, Refers to a food designed and processed so that the body control function such as bio-defense, regulation of biorhythm, prevention and recovery of disease, etc. can be sufficiently exhibited to the living body, and the function related to prevention of disease or recovery of health It can be said.

May be used when using a Lactobacillus buffer lactofermentum (Lactobacillus fermentum) MG4231 strain or Lactobacillus buffer lactofermentum (Lactobacillus fermentum) MG4244 strain according to the present invention as health food, as addition of this or in conjunction with other foods or food ingredients, which And can be appropriately selected and used as needed. Also, the amount of Lactobacillus fermentum MG4231 or Lactobacillus fermentum MG4244 to be added may be appropriately determined depending on the purpose of use. Lactobacillus buffer lactofermentum (Lactobacillus fermentum) MG4231 strain or Lactobacillus buffer lactofermentum (Lactobacillus fermentum) MG4244 strain according to the invention exhibits an increased activity effect and ensure the biological safety, the concentration is proportional, the appropriate amount, without limited to a specific range It is obvious that it can be used as.

In addition, Lactobacillus buffer lactofermentum (Lactobacillus fermentum) or MG4231 strain Lactobacillus buffer lactofermentum (Lactobacillus fermentum) in MG4244 strain can be used dietary kinds of food according to the present invention, there is no particular limitation. Examples include dairy products including noodles, other noodles, beverages, tea, drinks, alcoholic beverages, various soups, meats, sausages, breads, chocolates, candies, confectionery, pizza, gums and ice cream, or vitamin complexes. In particular, the Lactobacillus perfumolum MG901 or Lactobacillus plantarum MG989 according to the present invention is useful as a medicament for improving the viability, intracellular adhesion, and inhibition of lipase enzyme activity in gastrointestinal or bile acid in gastrointestinal tract, It is suitable for producing a variety of lactic acid bacteria fermented milk or a fermented product, because it is excellent in prevention, improvement or therapeutic effect of obesity-related diseases caused by the accumulation inhibitory effect. Examples of the fermented milk health functional foods include yogurt, calpis, cheese, butter and the like. Examples of fermented products include tofu, miso, chungkukjang, jelly, and kimchi. The fermented milk or the fermented product can be easily produced by replacing only the strain with Lactobacillus fermentum MG4231 strain or Lactobacillus fermentum MG4244 strain according to the present invention in a conventional manufacturing method.

In addition, Lactobacillus buffer lactofermentum (Lactobacillus fermentum) MG4231 strain or Lactobacillus buffer lactofermentum (Lactobacillus fermentum) MG4244 strain suitable other auxiliary components which typically can be included in the dietary supplement according to the selection of ordinary skill according to the invention and Known additives can be mixed. These known additives also include other microorganisms which can be used in combination with the strain according to the present invention.

The present invention also provides a quasi-drug composition for preventing or ameliorating an obesity-related disease comprising the above-mentioned strain.

The strain of the present invention may be added to a quasi-drug composition for the purpose of preventing or ameliorating an obesity-related disease. Lactobacillus buffer lactofermentum (Lactobacillus fermentum) of the present invention when using the MG4231 strain or Lactobacillus buffer lactofermentum (Lactobacillus fermentum) MG4244 strain as a quasi-drug additives, it is possible as it was added to the strain, or be used in combination with another quasi-drug components, with a conventional method Can be appropriately used. The amount of the active ingredient to be mixed can be suitably determined according to the intended use (prevention, health or therapeutic treatment).

Hereinafter, preferred embodiments and examples of the present invention will be described in order to facilitate understanding of the present invention. However, the following examples and preparation examples are provided only for the purpose of easier understanding of the present invention, and thus the content of the present invention is not limited thereto.

Example  1. Separation of used strains and preparation of samples for use

In Ewha Womans University Mokdong Hospital Obstetrics and Gynecology Department, a sample of healthy women 's vaginal wall was taken from the swab and transferred to the laboratory in a refrigerated state. The kit with the swab samples was directly streaked on Rogosa SL (Difco, USA) and incubated at 37 ° C for 48 hours in anaerobic condition. When the colonies were formed in the medium, each colony was streaked on Rogosa SL medium and purified colonies were finally screened through Gram stain (positive), morphological (bacterium) observation and biochemical characteristics (catalase negative) The final strain frozen at 70 ° C in 10% (v / v) glycerol was used in the following examples.

The culture was then incubated at 37 ° C for 18 hours. The culture was centrifuged at 1,100 × g for 3 minutes at 4 ° C and then washed twice with phosphate-buffered saline (PBS, pH7). The washed lactic acid bacteria were lyophilized, resuspended at a concentration of 10 mg / mL in PBS, and homogenized using a sonicator (VCX 400, Sonics & Materials Inc., CT, USA). The homogenized lactic acid bacteria were centrifuged at 1,100 x g for 15 minutes at 4 ° C, and then used as a sample in the following Examples.

Example  2. Identification of strains that have the effect of preventing and treating obesity

2.1 Lipase Enzyme Activity Inhibition  Identification of strains

The inhibitory activity of lipase enzyme activity was measured in order to select a strain having excellent effect of preventing and treating obesity among the 221 strains obtained in Example 1 above. In particular, when the lipase enzyme activity inhibiting ability is excellent, absorption of fat in the intestines can be suppressed, and thus, it is effective for prevention of obesity. First, 14 strains with high lipase activity were selected first. In order to prevent the obesity among the 14 strains and to select the strains having the best therapeutic effect, experiments were conducted to measure lipase activity inhibition by porcine pancreatic lipase (Sigma, USA). Samples were diluted to a concentration of 0.1 mg / mL and then placed on a plate with 0.167 mM p- nitrophenylpalmitate (PNP; Sigma, USA) solution, 0.061 M Tris-HCl buffer (pH 8.5) and 0.3 mg / mL lipase The reaction was carried out at 25 DEG C for 10 minutes. After the reaction, the absorbance was measured at 405 nm, and the control was performed by replacing the sample with a solvent. The lipase inhibitory activity was then calculated by the following equation, and the measurement results are shown in Table 1.

Lipase inhibitory activity (%) = {1- (absorbance of sample / absorbance of control)} x 100

Strains Anti-lipase activity ( % ) MG4227 12.24 MG4231 12.24 MG4244 11.22 MG4261 10.53 MG4270 11.84 MG5008 10.53 MG5013 12.24 MG5029 11.84 MG5033 10.53 MG5040 10.53 MG5055 10.53 MG5087 11.84 MG5098 14.47 MG5105 11.84

As shown in Table 1, it was confirmed that lipase inhibitory activity appears in the order of MG4227, MG4231 and MG4244 among the strains. Thus, the lipase inhibitory activity of the above four strains was found to be 10% or more, and it was confirmed that the absorption of fat in the intestines was inhibited, and the treatment and prevention effect on obesity was excellent.

2.2 Lipocytic cells  differentiation Inhibition  Identification of strains

Experiments were conducted to confirm the ability of lipid precursor cells to inhibit differentiation into adipocytes in order to select a strain having excellent effect for preventing and treating obesity in the strains obtained in Example 1 above. To induce the differentiation of 3T3-L1 adipose precursor cells, the adipose precursor cells were divided into 1.25 × 10 ⁵ cells / well on each 96-well plate, and after 2 days, the cells were completely exchanged and the cells were completely fused on the fourth day . MDI (0.5 mM 3-isobutyl-1-methylxanthine, 1 μM dexamethasone and 1 μg / mL insulin solution was treated with 10% FBS in DMEM medium and then in the medium The culture supernatant of the strain obtained in Example 1 was added to the medium at a concentration of 1,000 μg / mL and 100 μM of baicalin, which is known to be excellent in inhibition of lipid differentiation, was added as a positive control. After the differentiation was completed 6 days later, the accumulation amount of lipid produced in the cells was measured using Oil Red O (Sigma, USA) which specifically reacts with intracellular lipids. After the lipolysis, the medium was removed and PBS And fixed with 10% formalin for 1 hour at 4 ° C. The fixed cells were washed twice with 60% isopropanol (in PBS) and stained with 0.5% Oil Red O solution for 30 minutes at room temperature. After staining, remove the staining solution After washing with distilled water, isopropyl alcohol was added to completely dry wells, and the absorbance was measured at 520 nm. Then, lipid droplets ) Was compared with the MDI-treated group, and the degree of inhibition of the differentiation ability of adipocytes was analyzed. The results are shown in Table 2.

Strains Lipid accumulation
( %  of control) MG4227 84.46 MG4231 80.04 MG4244 71.90 MG4261 75.52 MG4270 73.14 MG5008 83.15 MG5013 81.50 MG5029 83.45 MG5033 77.02 MG5040 82.94 MG5055 75.56 MG5087 78.74 MG5098 80.71 MG5105 82.18

As shown in Table 2, MG4224, MG4261, MG5055, MG5033, MG5087 and MG4231 had the highest relative lipid content of 81.40% And it was confirmed that the treatment of 7 kinds of lactic acid bacteria effectively inhibited the differentiation from adipocyte precursor cells into adipocytes. In particular, considering that the positive control group, baicalin treated group, exhibited a relative lipid content of 81.40%, it was confirmed that the seven strains described above exhibited a lower content than that of the above-mentioned strains, indicating excellent adipocyte differentiation ability.

2.3 Identification of Cells without Cytotoxicity

In order to select strains which are excellent in the prevention and treatment of obesity and lack cytotoxicity among the strains obtained in Example 1, 14 strains excellent in the preventive and therapeutic effects of obesity in Examples 2.1 and 2.2 were selected as 3T3-L1 MTT (3- (4,5-dimethylthiazol-2-yl) -2,5-diphenyltetrazolium bromide) assay was performed to determine whether the cells were cytotoxic. 3T3-L1 cells were purchased from the American Type Culture Collection (ATCC, MD, USA). The cells were cultured in Dulbecco's modified Eagle's medium (DMEM, Invitrogen, NY, USA) containing 10% fetal bovine serum (FBS, Invitrogen, NY, USA) and 1% penicillin- streptomycin , USA) medium at 37 ° C in the presence of 5% CO ₂ /95% air. 3T3-L1 cells cultured under the above conditions were dispensed into a 96-well plate at a concentration of 16 × 10 ⁴ cells / well, and after 24 hours of culture, the medium was removed. After incubation for 24 hours, 20 μL of MTT (Sigma, USA) solution (5 mg / mL) was added and the mixture was incubated at 37 ° C for 4 h. Lt; / RTI > After incubation, the supernatant was removed and 200 μL of dimethylsulfoxide (DMSO) was added and the absorbance was measured at 546 nm using a microplate reader (Bio-Rad Model 550, Hercules, CA, USA) Table 3 shows the results of the analysis.

Strains MTT  assay
(1,000 [mu] g / mL) MG4227 92.52 MG4231 99.63 MG4244 108.42 MG4261 98.85 MG4270 88.60 MG5008 80.49 MG5013 97.00 MG5029 88.07 MG5033 92.75 MG5040 81.15 MG5055 84.06 MG5087 96.32 MG5098 88.07 MG5105 82.28

As shown in Table 3 above, the MTT assay results of all 14 kinds of lactic acid bacteria were found to be 80 or more. Therefore, even when treated at a concentration of 1,000 μg / mL, 14 kinds of lactic acid bacteria It was confirmed that there was no cytotoxicity. Thus, the lactic acid bacteria showing excellent ability to inhibit obesity as described above were not cytotoxic, and thus they were verified to be highly utilized for various purposes.

Example  3. Identification of strains that have the effect of preventing and treating obesity

MG4231 and MG4244 strains, which have high inhibitory activity on lipase enzyme activity and lipid precursor differentiation inhibitory activity, which have been proved in Example 2 and have excellent anti-obesity and therapeutic effects, And an experiment was conducted to identify them. The selected strains were cultured in MRS liquid medium at 37 ° C for 24 hours, and then subjected to Gram staining, and morphological characteristics were observed using a phase contrast microscope. In order to identify the selected lactic acid bacteria, 16S rRNA gene sequencing was performed. The universal rRNA gene primers (27F and 1492R) were used for gene amplification. (Daejeon, Korea). The results of 16S rRNA sequencing were analyzed using the GenBank database through the Basic Local Alignment Search Tool (BLAST) analysis (http://blast.ncbi.nlm.nih.gov/Blast.cgi) of the National Center for Biotechnology Institute (NCBI) Respectively. The sequence was aligned through PHYDIT 3.1, and then the tree was created using the neighborjoining method using MEGA 5.1 software. The results of identification of the above two strains are summarized in Table 4.

Isolates Strain Homologous microorganism Human vagina MG4231 Lactobacillus fermentum MG4244 Lactobacillus fermentum

As shown in Table 4, it was confirmed that MG4231 and MG4244 were Lactobacillus fermentum .

Example  4. Selected Lactic acid bacteria Acid resistance  And My biliary  Confirm

Since resistance to acidic and bile salts is important for its use as a probiotic, an experiment was conducted to investigate resistance to acid and bile salts of Lactobacillus permentum MG4231 and MG4244 at low pH (pH 2.0 to 4.0) and bile Respectively. Experiments were carried out by the method of Maragkoudakis to confirm tolerance to artificial gastric acid and artificial bile such as intestinal environment. The selected lactic acid bacteria were cultured for 18 hours, then centrifuged at 2,000 × g for 5 minutes at 4 ° C., washed twice with PBS, and suspended in a concentration of 10 ⁸ CFU / mL. To confirm resistance to artificial gastric acid, the final concentration of pepsin (Sigmae-aldrich, USA) was added to PBS (pH 2, 3, 4) so that the final concentration was 3 g / L. After incubation for the time, viable cell count was confirmed. The artificial bile salt tolerance was determined by adding pancreatin (Sigma-aldrich, USA) to a final concentration of 1 g / L in PBS (pH 7, 8), adding a suspension of lactic acid bacteria for 4 hours at 37 ° C, The results are shown in Fig. 1, and the results of the biliary test are shown in Fig.

As shown in FIG. 1, MG4231 and MG4244 showed high survival rate with almost no decrease in the survival rate of the strain even at pH 3 to 4, which is a strong acid condition containing pepsin, in the acid test. In particular, it was confirmed that there was no rapid decrease in survival rate compared to the survival rate at pH 3 to 4 even in the pH 2 condition. Thus, it was confirmed that both strains at pH 2 to 4 exhibited excellent acid resistance without a rapid decrease in the survival rate of the strain.

As shown in Fig. 2, MG4231 and MG4244 also confirmed that the survival rate did not substantially decrease even after the treatment of bile salts containing pancreatin at pH 7 and pH 8.

In conclusion, it was confirmed that Lactobacillus permentum MG4231 and MG4244 have high resistance to low pH and bile salts, which is one of the most important requirements of probiotics.

Example  5. Selective Lactic acid bacteria  Self-aggregating ability autoaggregation  ability

* If the self-aggregating ability of the strain is good, it can prevent the removal of probiotics by the spasm of the intestines and effectively form colonies in the epithelial cells of the digestive tract in the intestines, which is useful as probiotics. Experiments were conducted to confirm the self-aggregating ability of the selected Lactobacillus permentum MG4231 and MG4244. The selected strains were incubated at 37 ° C for 18 hours, centrifuged at 4,000 × g for 4 minutes at 4 ° C for 5 minutes, and then washed twice with PBS. After the suspension was resuspended to a final concentration of OD ₆₀₀ of 1.0, 5 mL of the suspension was shaken for 10 seconds and allowed to stand for 5 hours to confirm the self-aggregating ability. Immediately after the start of the experiment (A0) and after 5 hours (A), 0.1 mL of the supernatant was mixed with 0.9 mL PBS, and the absorbance was measured at 600 nm (A0, A) The results are shown in FIG.

(%) = (A0-A) / A0 x 100

As shown in FIG. 3, it was confirmed that the Lactobacillus permentum MG4231 had a self-aggregating ability of 65.3 ± 0.9% and a Lactobacillus permentum MG4244 of 68.6 ± 0.9% after 5 hours of the experiment, And that it can act as an effective probiotics.

Example  6. Selected Lactic acid bacteria  Fat accumulation Low performance  Confirm

Experiments were conducted to confirm that the selected strains, Lactobacillus fermentum MG4231 and MG4244, could inhibit the accumulation of triglyceride, a triglyceride. Lactobacillus perthrum MG4231 and MG4244 were cultured and then centrifuged and lyophilized. 10 mg of the lyophilized powdery strain (1 x 10 ⁸ CFU) was quantified and dissolved in 1 ml of distilled water, followed by filtration. (Control), and treated with MDI (0.5 mM 3-isobutyl-1-methyl-xanthine only treated group, which can activate lipid differentiation, and MDI and the filtered strain LFS) 50 and 100 를 were treated with differentiated 3T3-L1 adipocytes. TG assay was performed to measure the amount of triglyceride production. The results were compared with the MDI-treated group to determine the degree of triglyceride inhibition The results of the analysis are shown in FIG.

As shown in Fig. 4, inhibition of the accumulation of triglycerides by 77% was observed in Lactobacillus permentum MG4231 versus MDI-treated group. In addition, Lactobacillus permentum MG4244 showed about 75% inhibition of accumulation of triglycerides. In particular, it is generally known that when the accumulation of triglycerides is inhibited by 50% or more, the effect of inhibiting the accumulation of triglycerides is generally recognized, and Lactobacillus permentum MG4231 and MG4244 have an excellent neutral fat accumulation inhibitory effect.

Korea Biotechnology Research Institute KCTC13593BP 20180720 Korea Biotechnology Research Institute KCTC13594BP 20180720

Claims (9)

delete delete delete delete Lactobacillus fermentum MG4244 (accession number: KCTC) having lipase enzyme inhibitory activity, lipid precursor differentiation inhibitory activity, neutral fat accumulation inhibitory activity, acid resistance, biliary and self-aggregating ability, vaginal wall-derived Lactobacillus fermentum 13594BP) as an effective ingredient for the prophylaxis or treatment of obesity-related diseases.
The method according to claim 5, wherein the obesity-related disease is at least one selected from the group consisting of fatty liver, type 2 diabetes, hyperlipidemia, cardiovascular disease, arteriosclerosis and lipid-related metabolic syndrome. A pharmaceutical composition.
Lactobacillus fermentum MG4244 (accession number: KCTC) having lipase enzyme inhibitory activity, lipid precursor differentiation inhibitory activity, neutral fat accumulation inhibitory activity, acid resistance, biliary and self-aggregating ability, vaginal wall-derived Lactobacillus fermentum 13594BP) as an active ingredient for preventing or ameliorating an obesity-related disease. Lactobacillus fermentum MG4244 (accession number: KCTC) having lipase enzyme inhibitory activity, lipid precursor differentiation inhibitory activity, neutral fat accumulation inhibitory activity, acid resistance, biliary and self-aggregating ability, vaginal wall-derived Lactobacillus fermentum 13594BP) as an active ingredient for the prevention or amelioration of obesity-related diseases.
Lactobacillus fermentum MG4244 (accession number: KCTC) having lipase enzyme inhibitory activity, lipid precursor differentiation inhibitory activity, neutral fat accumulation inhibitory activity, acid resistance, biliary and self-aggregating ability, vaginal wall-derived Lactobacillus fermentum 13594BP) as an effective ingredient for the prevention or amelioration of obesity-related diseases.

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