KR20190068078A - Lactobacillus paracasei AO356 strain with Anti-Obesity Ability and Composition for treatment or improvement or preventing of obesity comprising the same - Google Patents

Abstract

A novel lactobacillus paracasei AO356 strain of the present invention is separated from a human body, thereby having excellent activity for alleviating, preventing or treating obesity by having high stability, exhibiting adipocyte differentiation suppression and differentiation inducing activity to M2 macrophage from M1 and M0 macrophages in vitro tests, and exhibiting weight loss activity and decrease in blood lipid level through browning of white fat in animal experiments. Therefore, the novel strain of the present invention has low possibility of side effects and can have a diet effect without controlling an amount of food intake, unlike conventional functional diet food or drugs which have a side effect problem, thereby being used for a pharmaceutical composition for treating or preventing obesity or a food composition for alleviating or preventing obesity.

Description

[0001] The present invention relates to a lactobacillus paracase AO356 strain having an obesity-suppressing activity and a composition for improving or treating obesity comprising the same. [0002] Lactobacillus paracasei AO356 strain with anti-obesity ability and composition for treatment,

The present invention relates to a novel Lactobacillus paracase strain AO356, a composition for preventing or treating obesity and a composition for improving obesity.

Obesity is becoming a serious chronic disease as humanity develops into a rich society. It is known that obesity is caused by various causes such as hypertrophy or hyperplasia of body fat cells morphologically, which is caused by imbalance of consumption and consumption of calories.

Obesity is not only psychologically disturbing individuals, but also a major cause of increased national medical expenditure, as it is a direct cause of increasing the risk of various adult diseases.

Specifically, obesity is directly associated with increased prevalence of various adult diseases such as type 2 diabetes, hypertension, hyperlipidemia, and cardiovascular disease, and is referred to as metabolic syndrome or insulin resistance syndrome by binding together diseases associated with obesity . They act as a cause of arteriosclerosis and cardiovascular disease.

Obesity is not only the most common malnutrition disorder in western society, but also in Korea, the frequency of obesity is rapidly increasing due to the improvement of diet and westernization of lifestyle by economic development, and the rate of Korean adult obesity is increased by 3% The problem is becoming more and more serious. Therefore, there is a demand for development of therapeutic agents or treatment methods for treating and preventing 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 are metabolites that use saccharides as an energy source and produce abundant lactic acid. They also produce various organic acids and bacteriocin and other antibacterial substances. However, Amine or ammonia are not produced, so it is a beneficial bacteria that prevents decay, inhibits harmful bacteria, and exhibits physiological activity that is helpful to humans. Among these, the Lactobacillus sp. Strain has been known for a long time as a key member of normal microbial communities in the intestinal tract of the human body and is important for maintaining healthy digestive organs and the 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.

Korean Patent Registration No. 10-1471033 discloses a strain of Weissella sp. F22 (accession number: KACC 91867P) which is excellent in the ability to inhibit obesity. In Korean Patent Registration No. 10-0264361, a cholesterol- , And Lactobacillus plantarum PMO08 (KFCC-11028) strain having an activity to be defoamed against six kinds of conjugated bile acids is disclosed.

However, since the lactic acid bacteria-related technology excellent in an anti-obesity effect does not appear commercially so as to be successful, the inventors of the present invention have conducted research on probiotics which have no side effects in the body and have an excellent effect of treating obesity, and newly found a Lactobacillus paracase strain AO356 And found that the strain exhibits a high level of survival and activity in the body of an animal and that when the cells themselves are directly added to an effective ingredient of food and medicine, the effect of improving, preventing and treating obesity is excellent, thereby completing the present invention .

Patent Document 1: Korean Patent Publication No. 10-1471033 Patent Document 2: Korean Patent Publication No. 10-0264361

DISCLOSURE Technical Problem The present invention has been conceived to solve the above problems, and it is an object of the present invention to provide a Lactobacillus paracasei AO356 strain (KCCM12145P) having an obesity-suppressing activity.

Another object of the present invention is to provide probiotics comprising the Lactobacillus paracasei strain AO356 ( KCCM12145P ) or a culture thereof.

It is still another object of the present invention to provide a pharmaceutical composition for preventing or treating obesity, which comprises the Lactobacillus paracasei strain AO356 (KCCM12145P) or a culture thereof.

Another object of the present invention is to provide a Lactobacillus paracasei strain AO356 (KCCM12145P) Or a culture solution thereof, and a food composition for improving inflammation caused by obesity.

To achieve the above object, the present invention provides a Lactobacillus paracasei AO356 strain (KCCM12145P) having an obesity-suppressing activity.

The present invention provides probiotics comprising the Lactobacillus paracasei strain AO356 (KCCM12145P) or a culture thereof to achieve the above other object.

The present invention provides a pharmaceutical composition for preventing or treating obesity comprising Lactobacillus paracasei strain AO356 (KCCM12145P) or a culture thereof to achieve the above other object.

The Lactobacillus paracasei strain AO356 (KCCM12145P) or a culture thereof may be contained in an amount of 0.01 to 50% by weight based on the total weight of the composition.

The composition may be one that inhibits adipocyte differentiation.

The composition may be one which induces differentiation into macrophages of M2 type.

The composition may be one which reduces body weight.

The present invention provides a food composition for preventing or ameliorating obesity comprising Lactobacillus paracasei strain AO356 (KCCM12145P) or a culture thereof to achieve another object.

Another object of the present invention is to provide a food composition for improving inflammation caused by obesity comprising Lactobacillus paracasei strain AO356 (KCCM12145P) or a culture thereof.

The Lactobacillus paracasei strain AO356 according to the present invention is a strain isolated from a human body and has high stability and has an activity of inhibiting adipocyte differentiation and inducing differentiation of M1 and M0 macrophages into M2 macrophages in vitro In animal experiments, it is excellent in the improvement of obesity, prevention or treatment activity such as weight loss activity and reduction of blood lipid concentration through the browning of white fat. Therefore, the novel strain of the present invention has a low possibility of side effects, so that it can show a diet effect without adjusting the amount of food to be consumed unlike the conventional dietary functional foods or medicines, which have side effects. Therefore, a pharmaceutical composition for obesity treatment or prevention, Or a food composition for prevention.

Therefore, the novel strain according to the present invention can be used as a pharmaceutical material useful for preventing, improving and treating obesity.

1 is a diagram showing a series of analysis procedures for selecting strains having an obesity-suppressing activity according to Experimental Example 1 of the present invention.
FIG. 2 is a graph showing lipid accumulation by Oil red O staining of each of 36 strains measured according to Experimental Example 1 of the present invention. FIG.
FIG. 3 is a diagram showing a series of analysis procedures for selecting a strain having an obesity-suppressing activity according to Experimental Example 2 of the present invention.
FIG. 4 is a graph showing the amount of cytokine secretion (%) in macrophages measured when each of 36 strains of dead cells was treated according to Experimental Example 2 of the present invention.
FIG. 5 is a graph showing the ratio of IL / 10 to IL-12 in macrophages when treated with each of 36 strains of dead cells according to Experimental Example 2 of the present invention.
FIG. 6 is a graph showing the amount of cytokine secretion (%) in macrophages measured when each of the 36 strains of live cells was treated according to Experimental Example 2 of the present invention.
FIG. 7 is a graph showing the ratio of IL-10 to IL-12 in macrophages measured when each of the 36 strains of live cells was treated according to Experimental Example 2 of the present invention.
8 is a diagram illustrating a procedure for setting an experimental animal and an experimental group according to Experimental Example 3 of the present invention.
9 is a graph showing a change in body weight in each experimental group according to Experimental Example 3 of the present invention.
FIG. 10 is a graph showing the weight change of each experimental group according to Experimental Example 5 of the present invention measured weekly. FIG.
11 is a graph showing an analysis of weight gain in each experimental group according to Experimental Example 5 of the present invention.
FIG. 12 shows the fat weights of liver (a), epididymal fat (b), retroperitoneal fat (c), cholesteatol fat (d) and shiguk liver fat (e) in each experimental group according to Experimental Example 5 of the present invention Fig.
FIG. 13 is a graph showing the results of measurement of LDL-cholesterol (a), triglyceride (TG) (b), HDL-cholesterol (c), glucose (E), and HOMA-IR (f).
FIG. 14 is a graph showing the mRNA expression level of a porcine metabolism-related gene analyzed by extracting RNA from epididymal white fats isolated from each experimental group according to Experimental Example 5 of the present invention and performing qPCR analysis therefrom.

Hereinafter, various aspects and various embodiments of the present invention will be described in more detail.

One aspect of the present invention relates to a Lactobacillus paracasei AO356 strain (KCCM12145P) having an obesity-suppressing activity.

The Lactobacillus paracasei strain AO356 according to the present invention is a strain isolated from a human body and deposited on November 2, 2017 at the Korean Microorganism Conservation Center (KCCM).

The Lactobacillus paracasei AO356 strain according to the present invention is a 16S rDNA comprising the nucleotide sequence shown in SEQ ID NO: 1, and the Lactobacillus paracasei AO356 strain is a Gram-positive bacterium And glucose as a carbon source, galactose, maltose, D-ribose, sucrose, lactose, trehalose raffinose and the like are used.

In addition, the Lactobacillus paracasei strain AO356 may exhibit an anti-inflammatory activity in addition to obesity activity. Lactobacillus paracasei strain AO356, when ingested orally, inhibits adipocyte differentiation, induces M2 macrophage differentiation, inhibits obesity, and reduces dietary fat absorption of the host On the other hand, dietary fat excretion is increased to reduce the body weight of the host.

In addition, the Lactobacillus paracasei AO356 strain of the present invention is excellent in survival ability against gastric acid or bile, and exhibits high survival ability in the animal body, so that the obesity-suppressing activity can be maintained in the body for a long time.

Another aspect of the present invention relates to probiotics comprising Lactobacillus paracasei strain AO356 (KCCM12145P) or a culture thereof.

The Lactobacillus paracasei strain AO356 according to the present invention can be used in the form of a dry powder or in a cultured state.

The culture medium used in the present invention is a culture medium in which a Lactobacillus paracasei AO356 strain is cultured in a known liquid medium or solid medium capable of supplying nutrients so that it can grow and survive in vitro in the presence of Lactobacillus paracasei ) Refers to whole medium including cultured strain, metabolite and extra nutrient obtained by culturing AO356 strain for a certain period of time, and also includes a culture solution in which the strain is removed after culturing the strain.

The culture broth may be centrifuged or filtered or concentrated, and this step may be carried out according to the needs of a person skilled in the art.

In the present invention, probiotics refers to live microorganisms, that is, live fungi, which provide beneficial health to the intestinal flora, that is, the health of the host. Generally, probiotics are consumed as part of fermented foods such as yogurt or as dietary supplements.

The Lactobacillus paracasei AO356 strain of the present invention is a strain of the genus Lactobacillus known as probiotics, which is excellent in survival ability against gastric acid and bile and has an obesity activity effect on its own. Thus, it can be used as probiotics, .

The Lactobacillus paracasei strain AO356 ( KCCM12145P ) or a culture thereof is preferably contained in an amount of 0.01 to 50% by weight based on the total weight of the composition.

The number of viable cells of the Lactobacillus paracasei AO356 strain in the composition may be adjusted to be less than 5 x 10 < ⁷ > CFU, which should be selected depending on the patient and the situation, The scope of the present invention is not limited by the number.

Another aspect of the present invention relates to a pharmaceutical composition for the prevention or treatment of obesity comprising a Lactobacillus paracasei strain AO356 (KCCM12145P) or a culture thereof, and a food composition for preventing or improving obesity.

The Lactobacillus paracasei strain AO356 according to the present invention can be used in the form of a dry powder or in a cultured state.

The culture medium used in the present invention is a culture medium in which a Lactobacillus paracasei AO356 strain is cultured in a known liquid medium or solid medium capable of supplying nutrients so that it can grow and survive in vitro in the presence of Lactobacillus paracasei ) Refers to whole medium including cultured strain, metabolite and extra nutrient obtained by culturing AO356 strain for a certain period of time, and also includes a culture solution in which the strain is removed after culturing the strain. The culture medium from which the strain has been removed may be sterilized to include dead cells, filtered or centrifuged to remove cells or centrifuged supernatant.

The culture broth may be centrifuged or filtered or concentrated, and this step may be carried out according to the needs of a person skilled in the art.

The Lactobacillus paracasei AO356 strain (KCCM12145P) or a culture thereof is contained in an amount of 0.01 to 50% by weight based on the total weight of the composition, and may be directly used, concentrated or used, concentrated or dried, Can be used.

Improve, treat or prevent the effect of the invention Obesity is Lactobacillus para casei (Lactobacillus paracasei), and AO356 strains proliferate while inhibiting the differentiation of mast cells, and inducing differentiation of a macrophage of the M2 type of host dietary fat It is expected to decrease the body weight of the host by reducing absorption, while increasing dietary fat excretion, and the composition may be used as the liquid itself or it may be dried and powdered.

Means that the Lactobacillus paracasei AO356 strain or a culture thereof contains an amount sufficient to achieve an obesity improvement, treatment or prophylactic efficacy or activity of the Lactobacillus paracasei strain.

Refers to all the actions of inhibiting or delaying the development, spread and recurrence of obesity by administration of the composition, and the treatment means all the actions of improving or improving the symptoms of obesity by administration of the composition.

The food composition may be formulated in the form of a capsule, tablet, powder, granule, liquid, ring, flake, paste, syrup, gel, jelly or bar form, Gum, confectionery, etc., and it is produced in the form of general food. If it is taken, it means bringing about a certain effect on health. However, unlike general medicine, it may occur when taking food for a long time There is no side effect.

The food composition is very useful because it can be ingested routinely. The amount of the Lactobacillus paracasei AO356 strain or the culture liquid added to such a food composition can not be uniformly determined depending on the type of food to be the subject, but if it is added within the range that does not impair the original taste of the food, And is usually in the range of 0.01 to 50% by weight, preferably 0.1 to 20% by weight based on the target food. In the case of food compositions in the form of capsules, tablets, powders, granules, liquids, rings, flakes, pastes, syrups, gels, jellies or bar, the content is usually 0.1 to 100% by weight, preferably 0.5 to 80% Of the total weight of the composition.

The food composition may contain, as an active ingredient, Lactobacillus paracasei strain AO356 or a culture thereof, as well as a component which is ordinarily added in food production, for example, a protein, a carbohydrate, a fat, a nutrient , Seasoning agents, and flavoring agents. Examples of the above-mentioned carbohydrates are monosaccharides such as glucose, fructose, and the like; Disaccharides such as maltose, sucrose, oligosaccharides and the like; And polysaccharides such as dextrin, cyclodextrin and the like, and sugar alcohols such as xylitol, sorbitol and erythritol.

As a flavoring agent, a natural flavoring agent [tau Martin, stevia extract (e.g., rebaudioside A, glycyrrhizin, etc.) and synthetic flavor (saccharin, aspartame, etc.) can be used.

For example, when the food composition of the present invention is prepared from a drink and a beverage, it may be added to the Lactobacillus paracasei AO356 strain of the present invention or a culture thereof in addition to citric acid, liquid fructose, sugar, glucose, acetic acid, Various plant extracts, and the like.

Also, in a pharmaceutical composition for preventing or treating obesity comprising Lactobacillus paracasei strain AO356 or a culture thereof as an active ingredient, the number of viable cells of Lactobacillus paracasei AO356 strain is 5 × 10 ⁷ CFU , Preferably from 5 x 10 < ³ > to 5 x 10 < ⁷ > CFU, but this should be selected depending on the patient and the circumstances, and the scope of the present invention is not limited by the number of viable bacteria in the composition.

In addition, in the above composition, the culture medium may be, for example, 0.001 mg / kg or more, preferably 0.1 mg / kg or more, more preferably 10 mg / kg or more, still more preferably 100 mg / Preferably 250 mg / kg or more, and most preferably 0.1 g / kg or more. The Lactobacillus paracasei strain AO356 is a strain isolated from a human body, and even if it is overdosed as a live organism, there is no adverse effect on the human body. Therefore, the quantitative upper limit of the Lactobacillus paracasei AO356 strain contained in the composition of the present invention May be selected and carried out by a person skilled in the art within a suitable range.

The pharmaceutical composition may be prepared by using pharmaceutically acceptable and physiologically acceptable adjuvants in addition to the above-mentioned active ingredients. Examples of the adjuvants include excipients, disintegrants, sweeteners, binders, coating agents, swelling agents, lubricants, A flavoring agent and the like can be used.

The pharmaceutical composition may be formulated to contain at least one pharmaceutically acceptable carrier in addition to the above-described effective ingredients for administration.

The pharmaceutical form of the pharmaceutical composition may be a granule, a powder, a tablet, a coated tablet, a capsule, a suppository, a liquid, a syrup, a juice, a suspension, an emulsion, a drip agent or an injectable liquid agent. For example, for formulation into tablets or capsules, the active ingredient may be combined with an oral, non-toxic pharmaceutically acceptable inert carrier such as ethanol, glycerol, water, and the like. Also, if desired or necessary, suitable binders, lubricants, disintegrants and coloring agents may also be included as a mixture. Suitable binders include, but are not limited to, natural sugars such as starch, gelatin, glucose or beta-lactose, natural and synthetic gums such as corn sweeteners, acacia, tracker candles or sodium oleate, sodium stearate, magnesium stearate, sodium Benzoate, sodium acetate, sodium chloride, and the like. Disintegrants include, but are not limited to, starch, methyl cellulose, agar, bentonite, xanthan gum and the like.

Acceptable pharmaceutical carriers for compositions that are formulated into a liquid solution include sterile water and sterile water suitable for the living body such as saline, sterile water, Ringer's solution, buffered saline, albumin injection solution, dextrose solution, maltodextrin solution, glycerol, And other conventional additives such as an antioxidant, a buffer, and a bacteriostatic agent may be added as needed. In addition, diluents, dispersants, surfactants, binders, and lubricants may be additionally added to formulate into injectable solutions, pills, capsules, granules or tablets such as aqueous solutions, suspensions, emulsions and the like.

Further, it can be suitably formulated according to each disease or ingredient, using the method disclosed in Remington's Pharmaceutical Science, Mack Publishing Company, Easton PA as an appropriate method in the field.

The pharmaceutical composition may be administered orally or parenterally. In the case of parenteral administration, the pharmaceutical composition may be administered by intravenous injection, subcutaneous injection, muscle injection, intraperitoneal injection, transdermal administration or the like, preferably oral administration.

The appropriate dosage of the pharmaceutical composition will vary depending on factors such as the formulation method, the manner of administration, the age, weight, sex, pathological condition, food, administration time, route of administration, excretion rate and responsiveness of the patient, The physician can easily determine and prescribe dosages effective for the desired treatment or prophylaxis. According to a preferred embodiment, the daily dosage of the pharmaceutical composition is 0.001-10 g / kg.

The pharmaceutical composition may be prepared in a unit dose form by formulating it using a pharmaceutically acceptable carrier and / or excipient according to a method which can be easily carried out by a person having ordinary skill in the art to which the present invention belongs. Into a capacity container. The formulations may be in the form of solutions, suspensions or emulsions in oils or aqueous media, or in the form of excipients, powders, granules, tablets or capsules, and may additionally contain dispersing or stabilizing agents.

Hereinafter, the present invention will be described in more detail with reference to Examples and the like, but the scope and content of the present invention can not be construed to be limited or limited by the following Examples. It will be apparent to those skilled in the art that various modifications and variations can be made in the present invention without departing from the spirit and scope of the present invention as set forth in the following claims. It is natural that it belongs to the claims.

In addition, the experimental results presented below only show representative experimental results of the embodiments and the comparative examples, and the respective effects of various embodiments of the present invention which are not explicitly described below will be specifically described in the corresponding part.

Example  1. 36 strains

In order to select candidates for probiotics having an activity of inhibiting obesity, 36 strains derived from human body (Table 1) were collected, and each strain was isolated and identified and shown in Table 1. Each strain was incubated with MRS medium (Difco, 288110) at 37 ° C for 18-24 hours.

number Strain name One Lactobacillus sakei subsp. Sakei 2 Streptococcus infantarius subsp. Coli 3 Bacillus licheniformis 4 Bacillus siamensis 5 Lactobacillus sakei subsp. Carnosus 6 Enterococcus faecalis 7 Enterococcus hirae 8 Carnobacterium maltaromaticum BA 9 Lactobacillus reuteri 10 Enterococcus faecium 11 Enterococcus faecium 12 Enterococcus faecium 13 Lactobacillus fermentum 14 Lactobacillus mucosae 15 Lactobacillus paracasei AO365 16 Lactobacillus acidilactici 17 Lactobacillus zeae 18 Enterococcus faecium 19 Lactobacillus reuteri 20 Lactobacillus johnsonii 21 Lactobacillus pentosus 22 Lactobacillus gasseri 23 Lactobacillus ruminis 24 Lactobacillus mesenteroides subsp. Mesenteroides 25 Obesumbacterium proteus 26 D29 27 I01-7 28 I02-4 29 M40a 30 Lactobacillus acidophilus 31 Lactobacillus rhamnosus 32 pediococcus pentosaceus 33 Bifidobacterium lactis 34 Bifidobacterium breve 35 Lactobacillus acidophilus 36 Lactobacillus intestinalis

Experimental Example  1. Selection of strains with activity to inhibit adipocyte formation

(Example 1) and splenic macrophage isolated from mice (Example 2), to evaluate the anti-obesity efficacy of the above candidate candidates, Were analyzed and compared.

(1) Experimental method

The accumulation of lipids in fat cells is the most representative feature of obesity. Adipocytes are formed by the differentiation of pre-adipocytes derived from stem cells. During the differentiation of 3T3-L1 cells (adipocytes) into adipocytes, morphological, biochemical Through the change, the intracellular fat sphere is formed, and as the differentiation progresses, the size of the fat sphere becomes larger. Since most lipids are composed of proteins such as triglyceride and perilipin A, the level of lipid differentiation can be confirmed by measuring the intracellular triglyceride content.

Therefore, 3T3-L1 cells, which are representative preadipocytes, were treated with high-grade candidate strains, and the activity of inhibiting adipogenesis was evaluated by measuring the triglyceride (TG) content by inducing differentiation.

Cell culture was maintained for at least 8 days for the above TG content measurement experiment. In order to exclude the influence of the higher candidate strains on the growth of 3T3-L1 cells, the upper candidate strains were treated in the form of dead cells.

Prior to the TG experiment, the cytotoxicity of WST (water soluble tetrazolium) was examined in the upper candidate strains. As a result, the cells did not show cytotoxicity at 1 × 10 ⁷ to 1 × 10 ⁶ CFU, In order to measure the inhibitory activity, the concentration of dead cells of the upper candidate strains to be treated was fixed at 1 × 10 ⁷ to 1 × 10 ⁶ CFU / well.

Specifically, Oil Red O staining was carried out according to the method of Negrel and Dani in order to investigate the effect of the candidate strains on the degree of fat accumulation in the lipid differentiation process. FBS + PS + Insulin + Dexamethasone + IBMX + 1 + 10 + CFU / mL were treated with 3 × 10 ⁷ CFU / mL of each of the above 36 candidate strains of the above candidate strains in 100% confluent 3T3- Insulin). After 10 days of differentiation, the culture medium was removed, washed with PBS, and fixed with 10% formaldehyde for 10 minutes. 10% formaldehyde was removed and the cells were fixed for more than 1 hour by adding saturated formaldehyde again. After that, 60% isopropanol was added to the flask. The flask was washed with distilled water, stained with oil red O staining solution for 30 minutes, and washed with distilled water. The intracellular lipid accumulation of stained lipid was observed with a microscope and the effect of 36 kinds of strains on fat accumulation during lipid differentiation was observed visually. To confirm the accumulation of fat, isopropanol was added to the dried oil to elute the oil red O dye, and absorbance was measured at 490 nm using a multi-plate reader. At this time, isopropanol was used as a blank.

(2) Conclusion

FIG. 1 is a diagram showing a series of analysis procedures for selecting strains having an obesity-suppressing activity according to Experimental Example 1 of the present invention. Fig. 3 is a graph showing lipid accumulation using oil red O staining method. Fig.

As shown in FIG. 2, the intracellular triglyceride (TG) content of each of 36 strains was compared with that of strains No. 2, 3, 5, 6, 7, 8, 12, 13, 15, 16, 24, 28 and 29 showed that TG content decreased by 10% or more. Among them, strains 15 and 21 showed the most excellent adipogenesis inhibitory activity.

Example  2. Macrophages (splenic macrophages isolated from mice) Control ability  evaluation

In addition to adipocytes, adipose tissue contains macrophages that regulate the inflammatory response. Thus, when obesity occurs, chronic inflammation or diabetes is also accompanied by macrophages present in adipocytes.

Under normal conditions, there are M2 type macrophages in adipocytes, which are anti-inflammatory macrophages that have the activity of relieving inflammation and maintaining and protecting insulin resistance. In contrast, in the case of obesity, macrophages infiltrate into adipocytes and M2 type macrophages switch to M1 type macrophages. M1 type macrophages are inflammatory macrophages that are known to produce chemicals called pro-inflammatory cytokines and chemokines. For example, it induces the production of cytokines such as IL-1beta, TNF-alpha, IL-6 and IL-12 to induce inflammation and promote insulin resistance.

Therefore, in order to prevent disease, inflammation and diabetes associated with obesity, the degree of conversion from M1 type to M2 type macrophage and the induction of M2 type macrophage differentiation in M0 type macrophage were analyzed, The macrophage control ability of the strain was evaluated.

(1) Experimental method

Splenocytes were isolated from mice to isolate macrophage-controllable strains, and various immune cells such as T cells and B cells were present in splenocytes. Therefore, we performed experiments by separating CD11b and macrophages using MACS system. By the separated macrophages 96 well plate divided by 1x10 ⁶ cells / well after more than two hours of incubation, and macrophages are 36 species of 100 ng / ml when the stably attached to the well bottom LPS with 3x10 ⁷ CFU / well (Strain of dead or live cells), respectively. After 48 hours, the amount of secreted IL-12 and IL-10 produced by recovering the supernatant was measured by ELISA. At this time, IL-12 is a confirmation factor of M1 type macrophage, and IL-10 is a confirmation factor of M2 type macrophage.

FIG. 3 is a diagram showing a series of analysis procedures for selecting strains having an obesity-suppressing activity according to Experimental Example 2 of the present invention. FIG. FIG. 5 is a graph showing the amount of cytokine secreted (%) in macrophages when treated with IL-6. FIG. 5 shows that when each of the 36 strains of dead cells was treated according to Experimental Example 2 of the present invention, FIG. 6 is a graph showing the ratio of IL / 10 to -12. FIG. 6 is a graph showing the ratio of the amount of cytokine secretion in macrophages FIG. 7 is a graph showing the ratio of IL-10 to IL-12 in macrophages when each of the 36 strains of live cells was treated according to Experimental Example 2 of the present invention. Fig.

6, 8, 10, 16, 20, 21, 22, and 22, when the dead cells were treated, the ratio of Il-10 / IL- 30, 31, 33).

As shown in FIGS. 6 and 7, 11 strains (strain number: 1, 3, 5, 6, 7, 8, 13, 14, 15 16, and 23) were identified.

Among the strains identified on the basis of the above results, strains showing both activity at the same time, even if the activity of inhibiting lipid differentiation and controlling macrophages were not excellent, were selected first, and a total of four strains were selected. Specifically, strains 3 and 6 were found to inhibit adipocyte formation, and the ratio of Il-10 / IL-12 was found to be the highest. In strain No. 13, the strain inhibited adipocyte formation and macrophages And it was identified as a strain showing all the regulatory activities.

In addition, Lactobacillus paracasei AO356, the 15th strain, was selected because it has the highest lipid differentiation inhibitory activity and has a relatively high IL10 / IL12 ratio.

(3) Comprehensive

A total of four strains (strain number: 3, 6, 13, and 15) having superior adipogenesis inhibitory activity and excellent control activity against macrophages were selected by combining the results of Experimental Examples 1 and 2, Among the four strains selected, in order to secondly select strains exhibiting excellent activity of inhibiting obesity in vivo, the in vivo inhibitory activity in vivo was compared with that of the in vivo test described below.

Experimental Example  3. Identification of effects of obesity inhibitory activity on selected strains by animal experiment

end. Experimental animals and Experimental group

C57BL / 6 6 week old males were purchased from Central laboratory animals and cultured at a temperature of 20 ± 2 ℃ and humidity of 55 ± 5% for 12 hours. Experimental animals were purified for 2 weeks after purchase, and 5 mice per group were placed in the experimental group as shown in Table 2.

division
(N = 5) Experimental group Normal diet Normal control group (ND, 10% fat) fed general diet, PBS 5 times a week, 100 μl / head orally for 8 weeks high fat diet A negative control diet (HFD, 45% fat), which consumed high fat diets for induction of obesity, was administered 5 times per week for 8 weeks with 100 μl / head orally A Consume high fat diets for obesity-induced, and strain number 3 (Bacillus licheniformis) to 0 and then orally administered five times a week 10 ⁸ CFU / head for 4 weeks, 5-8 weeks five times a week 5X10 ⁸ CFU / head during Oral administration B Consume high fat diets for obesity-induced, and strain No. 6 (Enterococcus faecalis) 0 to after oral administration of five times a week 10 ⁸ CFU / head for 4 weeks, 5-8 weeks five times a week 5X10 ⁸ CFU / head during Oral administration C The animals were fed high fat diets for induction of obesity, and the strain No. 13 ( Lactobacillus fermentum) was orally administered 10 ⁸ CFU / head five times a week for 0 to 4 weeks, followed by 5 times 10 ⁸ CFU / Oral administration of head D High-fat diets for obesity induction were obtained and the strain No. 15 ( Lactobacillus paracasei AO356) was orally administered at 10 ⁸ CFU / head five times a week for 0 to 4 weeks, followed by 5 times 10 ⁸ CFU / Oral administration of head

In all experimental groups except for normal diet and negative control (Hihg fat diet), each strain was mixed with feed, followed by oral administration of feed and strain. FIG. 8 shows a procedure of setting an experimental animal and an experimental group according to Experimental Example 3 of the present invention.

I. Weight, feed intake and Quantity of water

All experimental groups were examined for changes in body weight (g), feed intake (g), and water volume (㎖) from the time of feeding the feed to sacrificing the experimental animals. The diet and water intake were measured three times a week. The cages were changed twice a week. Body weight was measured once a week and compared.

FIG. 9 is a graph showing the change in body weight in each experimental group. According to this, the decrease in weight gain was clearly confirmed in Lactobacillus paracasei AO356 strain No. 15.

Experimental Example  4. Identification of selected strains

16S rDNA  analysis

The final selected strains were analyzed for 16S rRNA gene (SEQ ID NO: 1), which is a nucleotide conservation sequence of bacteria, using a universal bacterial primer (518F, 800R) for PCR reaction. The results were interpreted using NCBI blast (http://www.ncbi.nlm.nih.gov/).

The present inventors named Lactobacillus paracasei AO356 as " Lactobacillus paracasei AO356 (KCCM12145P :)" and deposited it on November 2, 2017 at the Korean Microorganism Conservation Center.

Name of depository: Korea Microorganism Conservation Center (overseas)

Accession number: KCCM12145P

Checked on: 20171102

Experimental Example  5. Selected final AO356  Effect of inhibiting activity of obesity in animal experiment of strain Sho sign

end. Culture of the strain

To produce a final end Lactobacillus para casei screening AO356 strains (Lactobacillus paracasei AO356), Lactobacillus para casei (Lactobacillus paracasei) the AO356 strain solid medium; inoculation hoekseon to (Lactobacilli MRS Agar BD Difco Co, USA) and , And cultured at 37 ° C for 24 hours. After thoroughly checking whether the cells were contaminated or formed a single colony during the culturing process, the following experiment was conducted. After the cultivation was completed, a colony was taken from each solid medium and inoculated into 5 mL of a liquid medium (Lactobacilli MRS broth; BD Difco Co, USA) and incubated at 37 ° C and 140 rpm for 24 hours. Was inoculated in 200 mL of another liquid medium (Lactobacilli MRS broth; BD Difco Co, USA) in a 1% (v / v) inoculation stepwise. Activated Lactobacillus paracasei strain AO356 was cultured at 37 ° C for 24 hours at 37 ° C and centrifuged at 6000 rpm at 4 ° C for 15 minutes (Avanti JE, Beckman Coulter, USA). The recovered cells were washed twice with sterilized 0.85% physiological saline, and lyophilized (FDCF-12003, OPERON, Korea), powdered and stored at -80 ° C until use.

I. Experimental animals and Experimental group

C57BL / 6 male mice, 6 weeks old, were purchased from a central experimental animal and maintained at a temperature of 20 ± 2 ° C and a humidity of 55 ± 5% for 12 hours. Experimental animals were purified for 2 weeks after purchase, and 10 rats per group were randomly assigned to a complete block design.

During the refinement period, the litter was mixed at intervals of 2 to 3 days to homogenize intestinal microflora in the animal model. After that, 10 rats were placed in each experimental group. Normal diet (ND) group was fed normal diet and the other group was fed high diet (Rodent diet with 45% kcal Fat, Research Diets, USA). The Lactobacillus paracasei strain AO356 was weighed to a concentration of 5 × 10 ⁷ CFU / head, suspended in sterilized PBS, and administered orally at a fixed interval of 5 times per week for 10 weeks.

At the end of the experiment period, the animals were fasted for 12 hours or more. Blood was collected from orbital blood, left at room temperature for 30 minutes or more, and centrifuged at 1,690 × g for 10 minutes. . Liver fat, epididymal fat, retroperitoneal fat, saliva fat and shabbat liver brown fat were extracted, washed with saline to remove water and white fat removed from the brown fat tissue of the scabbard liver was removed and the weight of each was measured.

division
(N = 10) Experimental group ND Oral administration of 100 μl / head for 10 weeks, 5 times a week, a normal control group (ND, 10% fat) HFD The subjects were fed a high-fat diets (Rodent diet with 45% kcal Fat, Research Diets, USA), PBS for 5 weeks, 100 μl / head for 10 weeks L. paracasei AO356 High fat diets for obesity induction were administered and the final selected Lactobacillus paracasei AO356 strain was orally administered 5 x 10 ⁷ CFU / head five times a week for 10 weeks

All. Weight, feed intake and Quantity of water

All experimental groups were examined for body weight change (g), feed intake (g / day), and water volume (g / day) of the experimental animals from the feeding time to the experimental animal sacrifice. Daily diets and water intake were measured, and cages were changed twice a week. Body weight was measured once a week and compared.

FIG. 10 is a graph showing the change in body weight in each experimental group by week, FIG. 11 is a graph showing an analysis of body weight gain in each experimental group, and FIG. 10 is a graph showing feed intake (g / day) The intake (kcal / day) is shown in Table 4.

ND HFD L. paracasei AO356 P value Average of daily food intake
(g / day) 3.56 ± 0.05 2.64 ± 0.04 2.55 ± 0.05 <0.001 Average of daily water intake
(g / day) 3.81 ± 0.08 2.96 ± 0.09 2.99 ± 0.20 0.003 Average of daily calorie intake
(kcal / day) 12.83 + - 0.17 13.36 ± 0.21 12.88 ± 0.23 N.S.

According to Figures 10, 11 and Table 4, Lactobacillus paracasei strain AO356 was administered to mice fed a high fat diet for 10 weeks, showing a significant inhibition of weight gain compared with the HFD group fed only with high fat diets . &Lt; / RTI &gt;

The Lactobacillus paracasei strain AO356 showed a weight gain of 31.33% compared with the HFD group, despite the high fat feed intake, as it was confirmed that the body weight gain was 10.63% less than the HFD group consuming high fat diets , Lactobacillus paracasei strain AO356 was found to have an effect of inhibiting weight gain.

The calorie intake was calculated from the amount of feed consumed. Mean daily dietary intake and water intake were significantly different between ND and HFD groups. It is expected that this is due to differences in the constituents and properties of feed, and ND group consumes more feed and water intake than HFD group.

HFD group and L. paracasei The AO356 group was found to have no difference in feed, water and caloric intake, and it was confirmed that the body weight gain inhibitory activity of Lactobacillus paracasei AO356, It can be seen that the effect of

la. Liver, epididymal fat, Posterior peritoneum  Fat, Cry  Fat, Shoulder bag  Local analysis

At the end of the experimental period, liver, epididymal fat, retroperitoneal fat, follicular fat and shabu - shabu brown fat were extracted, washed with saline to remove water, and white fat attached to brown fat tissue After removal, the weight to weight ratio was measured and shown in Table 5 and Figures 12a-e. In Table 5 and Figs. 12a-e, liver is liver fat, EFT is epididymis fat, RFT is retroperitoneal fat, IFT is fat and iBAT is interscapular brown adipose tissue.

Tissue weight
(mg) ND HFD HFD +
L. paracasei AO356 P value Liver 834.6 ± 15.48 916.16 + 28.37 838.88 ± 17.99 N.S. EFT 270.49 + - 19.51 ^a 1741.68 + - 188.37 ^c 1149.36 + - 104.31 ^b <0.001 RFT 82.00 ± 22.96 ^a 642.58 + - 57.26 ^c 414.01 ± 50.25 ^b <0.001 IFT 159.19 ± 14.20 ^a 873.74 + - 84.50 ^c 567.89 + 54.80 ^b <0.001 iBAT 54.27 ± 2.59 ^a 88.10 ± 5.91 ^c 64.05 ± 5.43 ^b 0.001 Serum leptin
(pg / ml) 0.296 + 0.034 ^a 7.200 + - 1.482 ^c 3.435 + 0.756 ^b <0.001

According to FIG. 12 and Table 5, the HFD group consuming the high fat diet showed a slight increase in liver tissue weight compared with the ND group and the Lactobacillus paracasei AO356 group.

In addition, the weight of the epididymal white fat tissue (EFT), the retroperitoneal white fat tissue (RFT), the white fat tissue (IFT), and the brown fat tissue of the scabbit liver (iBAT) On the contrary, when Lactobacillus paracasei AO356 was administered, it was confirmed that the fat weight in each tissue was significantly decreased.

On the other hand, leptin is a cytokine secreted from adipose tissue, which is known to increase in proportion to the increase of adipose tissue, and is one of the representative indicators of obesity. As a result of the changes, it was found that the high fat diet was increased in the HFD group ingested, and when the Lactobacillus paracasei AO356 was administered, it was significantly decreased.

hemp. Biochemical analysis of serum

After the experiment, the animal model was fasted for 12 hours or more. Blood was collected from the orbital blood, left at room temperature for 30 minutes or more, and centrifuged at 1,690 × g for 10 minutes.

Using the above serum, the concentration of insulin and leptin in serum was measured by an ELISA based assay kit. Homeostasis model assessment for insulin resistance (HOMA-IR) was calculated by the following equation using measured serum glucose and serum insulin concentrations. Serum triglyceride, HDL-cholesterol and LDL-cholesterol concentrations were measured by colorimetry and serum glucose concentrations were measured by UV spectrophotometry.

[Formula 1]

HOMA-IR = fasting glucose (mg / dL) fasting insulin (μU / mL) / 2430

FIG. 13 is a graph showing the results of measurement of LDL-cholesterol (a), triglyceride (TG) (b), HDL-cholesterol (c), glucose (E), and HOMA-IR (f).

As shown in FIG. 13, when repeated high fat diet (HFD group) is consumed, the blood glucose and insulin concentrations increase and hyperglycaemia and hyperinsulinaemia are induced to promote insulin resistance. This was confirmed through the experimental example of the present invention.

Specifically, the HFD group showed significantly higher blood glucose and insulin concentrations than the ND group. Furthermore, it was confirmed that LDL-cholesterol, triglyceride (TG), HDL-cholesterol, glucose and insulin were totally decreased when Lactobacillus paracasei AO356 strain ( L. paracasei AO356 group) was administered.

HOMA-IR, which is used as an indicator of insulin resistance, was also increased in the HFD group but not in the L. lactis paracasei AO356 strain. paracasei The AO356 group showed a significant decrease in the level.

Insulin has the function of inhibiting lipolysis of adipocytes and transferring fatty acids and glucose into the cells and accumulating them in the form of triglycerides. As in the case of the HFD group, insulin resistance does not work well, Free, and lipoprotein lipase (LPL) activity is reduced, resulting in the production of dyslipidaemia, which increases serum LDL cholesterol and triglycerides. In the HFD group, dyslipidaemia such as an increase in serum triglyceride and LDL cholesterol level occurred. However, in the L. paracasei AO356 group in which Lactobacillus paracasei AO356 strain was administered, It is confirmed that the same phenomenon can be significantly reduced.

bar. RNA precipitation and qPCR  analysis

The total RNA was extracted using RNeasy lipid tissue kit, and oligo-dT primer was used to synthesize cDNA. qPCR was performed using a SYBR green qPCR (Qiagen) kit.

FIG. 14 is a graph showing RNA extracted from epididymal white fats isolated from each test group according to Experimental Example 5 of the present invention, and qPCR analysis is performed thereon to analyze mRNA expression level of a fat metabolism-related gene.

In general, brown fat among the fat tissues in the body functions to consume energy through heat generation, while white fat mainly functions to store energy in the form of fat. Therefore, in the present invention, it was tried to determine whether the finally selected Lactobacillus paracasei strain AO356 transforms white fat into brown fat and has the effect of treating diseases such as obesity.

Increased expression of UCP1 in white fat promotes heat-producing ability of mitochondria in white fat, promotes browning / beige fat through β3-adrenergic agonist or cold exposure, increased expression of UCP1 gene through exercise , And decreased body fat.

UCP1 is a UCP1 gene expression of which was reduced in the HFD group plays an important feature, Referring to FIG. 14 in the browning of the white of visceral fat L. paracasei AO356 group.

In addition, PGC1α promotes the expression of the protein Fndc5, which secretes the irisin, a hormone that promotes the expression of UCP1. Thus, PGC1a expression is decreased in the HFD group and L. paracasei AO356 The PGC1a expression rate was significantly recovered in the control group.

In addition, the administration of Lactobacillus paracasei AO356 increased the expression levels of PRDM16, Cidea and PPARγ genes, which are transcription factors controlling gene programming for the whitening of white adipose tissue.

On the other hand, in FIG. 14C, it was confirmed that the CD36 decreased in the HFD group was significantly increased by the administration of Lactobacillus paracasei AO356 strain, which is necessary for the normal function of the brown fat, and fatty acids Especially, it plays a role of transporting polyunsaturated fatty acid into cells, and polyunsaturated fatty acid acts as ligands with the highest affinity of PPARγ and activates PPARγ.

Taken together, the administration of Lactobacillus paracasei AO356 strain in a high fat diet-induced obesity animal model not only has an anti-obesity activity of body fat reduction and weight gain inhibition by high fat diet, And that it has an effect of increasing the expression of genes related to the browning.

<110> KOREA FOOD RESEARCH INSTITUTE <120> Lactobacillus paracasei AO356 strain with Anti-Obesity Ability          and Composition for treatment or prevention          obesity comprising the same <130> HPC-7705 <160> 1 <170> KoPatentin 3.0 <210> 1 <211> 1452 <212> DNA <213> Artificial Sequence <220> <223> 16s rDNA of Lactobacillus paracasei AO356 <400> 1 ggatgaacgc tggcggcgtg cctaatacat gcaagtcgaa cgagttctcg ttgatgatcg 60 gtgcttgcac cgagattcaa catggaacga gtggcggacg ggtgagtaac acgtgggtaa 120 cctgccctta agtgggggat aacatttgga aacagatgct aataccgcat agatccaaga 180 accgcatggt tcttggctga aagatggcgt aagctatcgc ttttggatgg acccgcggcg 240 tattagctag ttggtgaggt aatggctcac caaggcgatg atacgtagcc gaactgagag 300 gttgatcggc cacattggga ctgagacacg gcccaaactc ctacgggagg cagcagtagg 360 gaatcttcca caatggacgc aagtctgatg gagcaacgcc gcgtgagtga agaaggcttt 420 cgggtcgtaa aactctgttg ttggagaaga atggtcggca gagtaactgt tgtcggcgtg 480 acggtatcca accagaaagc cacggctaac tacgtgccag cagccgcggt aatacgtagg 540 tggcaagcgt tatccggatt tattgggcgt aaagcgagcg caggcggttt tttaagtctg 600 atgtgaaagc cctcggctta accgaggaag cgcatcggaa actgggaaac ttgagtgcag 660 aagaggacag tggaactcca tgtgtagcgg tgaaatgcgt agatatatgg aagaacacca 720 gtggcgaagg cggctgtctg gtctgtaact gacgctgagg ctcgaaagca tgggtagcga 780 acaggattag ataccctggt agtccatgcc gtaaacgatg aatgctaggt gttggagggt 840 ttccgccctt cagtgccgca gctaacgcat taagcattcc gcctggggag tacgaccgca 900 aggttgaaac tcaaaggaat tgacgggggc ccgcacaagc ggtggagcat gtggtttaat 960 tcgaagcaac gcgaagaacc ttaccaggtc ttgacatctt ttgatcacct gagagatcag 1020 gtttccctt cgggggcaaa atgacaggtg gtgcatggtt gtcgtcagct cgtgtcgtga 1080 gatgttgggt taagtcccgc aacgagcgca acccttatga ctagttgcca gcatttagtt 1140 gggcactcta gtaagactgc cggtgacaaa ccggaggaag gtggggatga cgtcaaatca 1200 tcatgcccct tatgacctgg gctacacacg tgctacaatg gatggtacaa cgagttgcga 1260 gaccgcgagg tcaagctaat ctcttaaagc cattctcagt tcggactgta ggctgcaact 1320 cgcctacacg aagtcggaat cgctagtaat cgcggatcag cacgccgcgg tgaatacgtt 1380 cccgggcctt gtacacaccg cccgtcacac catgagagtt tgtaacaccc gaagccggtg 1440 gcgtaaccct tt 1452

Claims (9)

Lactobacillus paracasei strain AO356 ( KCCM12145P ) with obesity inhibitory activity . A probiotics comprising the Lactobacillus paracasei strain AO356 ( KCCM12145P ) of claim 1 or a culture thereof. A pharmaceutical composition for preventing or treating obesity comprising the Lactobacillus paracasei strain AO356 ( KCCM12145P ) or a culture thereof according to claim 1. The pharmaceutical composition for preventing or treating obesity according to claim 3, wherein the Lactobacillus paracasei strain AO356 ( KCCM12145P ) or a culture thereof is contained in an amount of 0.01 to 50% by weight based on the total weight of the composition . The pharmaceutical composition for preventing or treating obesity according to claim 3, wherein the composition inhibits adipocyte differentiation. The pharmaceutical composition for preventing or treating obesity according to claim 3, wherein said composition induces differentiation into M2 type macrophages. The pharmaceutical composition for preventing or treating obesity according to claim 3, wherein the composition reduces weight. A food composition for prevention or improvement of obesity comprising the Lactobacillus paracasei strain AO356 ( KCCM12145P ) or a culture thereof according to claim 1. A food composition for improving inflammation caused by obesity comprising the Lactobacillus paracasei strain AO356 ( KCCM12145P ) of claim 1 or a culture thereof.

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