KR102077720B1 - Pediococcus acidilactici AO22 strain with Anti-Obesity Ability and Composition for treatment or improvement or preventing of obesity comprising the same - Google Patents

Abstract

The novel Pediococcus esidilactis AO22 strain of the present invention is a strain isolated from the human body, has high stability, inhibits adipocyte differentiation and induces differentiation of M1 and M0 macrophages into M2 macrophages in vitro. Obesity improvement, prevention or treatment activity is excellent, such as weight loss activity is observed in animal experiments. Therefore, since the new strain of the present invention is unlikely to produce side effects, unlike the conventional diet foods or drugs that had side effects, the new strain may exhibit a diet effect without controlling the amount of food, thereby improving or preventing obesity pharmaceutical compositions or obesity Or it may be utilized as a food composition for prevention.

Description

Fediococcus acidilactici AO22 strain with Anti-Obesity Ability and Composition for treatment or improvement or preventing of obesity comprising the same}

The present invention relates to a novel pediococcus esididilacti AO22 strain and a composition for preventing or treating obesity and a composition for improving obesity.

As mankind develops into an affluent society, obesity is becoming one of the most serious chronic diseases. Obesity is caused by a variety of causes, a metabolic disease caused by the imbalance between the intake and consumption of calories, morphologically known to be caused by hypertrophy or hyperplasia of fat cells in the body.

Obesity is a major cause of the increase in national medical expenditures, as it is not only psychologically diminishing individuals but also socially increasing the risk of developing various adult diseases.

Specifically, obesity is directly related to the increase in the prevalence of various adult diseases such as type 2 diabetes, hypertension, hyperlipidemia, and heart disease. . They act as a cause of atherosclerosis and cardiovascular disease.

Obesity is not only the most common malnutrition in western society, but also in Korea, the frequency of obesity is rapidly increasing due to the improvement of diet and economic way of life due to economic development, and the proportion of Korean adult obesity increases by 3% every year. Because of this trend, the problem is getting worse. Therefore, there is a need for development of a therapeutic agent or treatment method for treating and preventing obesity.

Known obesity treatments such as Xenical (Roche Pharmaceuticals, Switzerland), Reductil (Eboth, USA), Exolise (Atopama, France), etc. Classified as inhibitors, most anti-obesity agents are appetite suppressants that suppress appetite by regulating neurotransmitters associated with the hypothalamus. However, conventional treatments have low side effects such as heart disease, respiratory disease, and neurological disease, and thus have low sustainability. Therefore, the development of more improved obesity treatments is needed. Since there are few therapeutic agents, development of new obesity treatments is required.

On the other hand, many efforts have been made to reduce blood cholesterol levels using lactic acid bacteria, which have been considered safe microorganisms. Lactobacillus is a metabolite that uses saccharides as an energy source to produce a large amount of lactic acid and other organic acids and bacteriocins as well as antibacterial substances, but it is harmful to humans and animals, such as indole, sketol, phenol, Amine or ammonia is a beneficial bacterium that does not produce rot, prevents harmful bacteria, and shows physiological activity that is beneficial to humans. Among them, the strain Lactobacillus is a major member of the normal microbial community in the intestine of the human body, which has long been known to be important for maintaining a healthy digestive system and the vaginal environment, and the US Public Health Service (US Public Health Service). guidelines) are currently classified as 'Bio-safty Level 1', where none of the Lactobacillus strains currently deposited with the American Strain Deposit Organization (ATCC) is known about the potential risk of causing disease in humans or animals. have.

Korean Patent Publication No. 10-1471033 discloses a strain of Weissella sp. F22 (Accession Number: KACC 91867P) excellent in inhibiting obesity, and Korean Patent Publication No. 10-0264361 discloses a cholesterol lowering ability. A Lactobacillus plantarum PMO08 (KFCC-11028) strain is disclosed, which has active activity to deflate against six conjugated bile acids.

However, since there are no commercially successful lactic acid bacteria-related technologies with excellent anti-obesity effects, the inventors of the present invention, while conducting research on probiotics that have no side effects in the body and are excellent in the treatment of obesity, newly developed Pediococcus edidilacthysis. Completion of the present invention by finding an AO22 strain and confirming that the strain exhibits high viability and activity in the body of the animal and that the bacterium itself is directly added as an active ingredient of food and medicine, the effect of improving obesity, prevention and treatment is excellent. Was done.

Patent Document 1. Republic of Korea Patent Publication No. 10-1471033 Patent Document 2. Republic of Korea Patent Publication No. 10-0264361

The present invention has been made to solve the above problems, an object of the present invention is to provide a Pediococcus acidilactici AO22 strain (KCCM12146P) having an obesity inhibitory activity.

Another object of the present invention is to provide a probiotics including the Pediococcus acidilactici AO22 strain (KCCM12146P) or a culture thereof.

Still another object of the present invention is to provide a pharmaceutical composition for preventing or treating obesity, including the Pediococcus acidilactici AO22 strain (KCCM12146P) or a culture thereof.

Another object of the present invention to provide a food composition for preventing or improving obesity and a food composition for improving inflammation by obesity, including Pediococcus acidilactici AO22 strain (KCCM12146P) or a culture thereof will be.

In order to achieve the above object, the present invention, pediococcus ecidi lactici ( Pediococcus) having an obesity inhibitory activity acidilactici ) AO22 strain (KCCM12146P) is provided.

The present invention to achieve the above another object, the Pediococcus ecidi lactici ( Pediococcus acidilactici ) A probiotics comprising AO22 strain (KCCM12146P) or a culture thereof are provided.

The present invention to achieve the above another object, Pediococcus ecidi lactici ( Pediococcus acidilactici ) AO22 strain (KCCM12146P) or a culture thereof comprising a pharmaceutical composition for preventing or treating obesity.

Pediococcus acidilactici ) AO22 strain (KCCM12146P) or a culture thereof may be included in an amount of 0.01 to 50% by weight based on the total weight of the composition.

The composition may be to inhibit adipocyte differentiation.

The composition may be to induce differentiation into M2 type macrophages.

The composition may be to reduce weight.

The present invention to achieve the above another object, Pediococcus ecidi lactici ( Pediococcus acidilactici ) AO22 strain (KCCM12146P) or a culture composition thereof to provide a food composition for preventing or improving obesity.

In addition, the present invention in order to achieve the above another object Pediococcus ecidi lactici ( Pediococcus acidilactici ) AO22 strain (KCCM12146P) or a culture comprising a food composition for improving inflammation caused by obesity comprising a culture thereof.

Pediococcus ecidi lacticci according to the present invention acidilactici ) AO22 strain, which is isolated from human body, has high stability, inhibits adipocyte differentiation and induces differentiation of M1 and M0 macrophages into M2 macrophages in vitro, and shows weight loss activity in animal experiments. It is excellent in improving obesity, preventing or treating activity. Therefore, since the new strain of the present invention is unlikely to cause side effects, unlike the conventional diet functional foods or drugs that had side effects, the new strain may have a diet effect without controlling the amount of eating, thereby improving or preventing obesity pharmaceutical compositions or obesity Or it may be utilized as a food composition for prevention.

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

1 is a diagram showing a series of analytical procedures for selecting strains having obesity inhibitory activity according to Experimental Example 1 of the present invention.
Figure 2 is a graph showing the fat accumulation (Lipid accumulation) using Oil red O staining of each of the 36 strains measured according to Experimental Example 1 of the present invention.
3 is a diagram showing a series of analytical procedures for selecting strains having obesity inhibitory activity according to Experimental Example 2 of the present invention.
Figure 4 is a graph showing the measurement of cytokine secretion (%) in macrophages when each of the 36 strains of the dead type 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 each of 36 strains of dead cell type according to Experimental Example 2 of the present invention was treated.
Figure 6 is a graph showing the cytokine secretion (%) measured in macrophages when each of the 36 strains of the live cell type 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 when each of 36 strains of live bacteria according to Experimental Example 2 of the present invention was treated.
8 is a view showing a process of setting the experimental animals and the experimental group according to Experimental Example 3 of the present invention.
9 is a graph showing the measured weight change in each experimental group according to Experimental Example 3 of the present invention.
10 is a graph showing the weight change in each experimental group according to Experimental Example 5 of the present invention measured weekly.
Figure 11 is a graph showing the analysis of the weight gain in each experimental group according to Experimental Example 5 of the present invention, feed intake (g / day), drinking water (g / day), calorie intake (kcal / day) in Table 4 Indicated.

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

One aspect of the present invention is pediococcus Ecidi lactici ( Pediococcus) having an anti-obesity activity acidilactici ) AO22 strain (KCCM12146P).

Pediococcus ecidi lacticci according to the present invention acidilactici ) AO22 strain is a strain isolated from the human body, which was deposited on November 2, 2017 by the Korea Microorganism Conservation Center (KCCM).

Depositary Name: Korea Microorganism Conservation Center (overseas)

Accession number: KCCM12146P

Deposit date: 20171102

Pediococcus esidiractis according to the present invention acidilactici ) AO22 strain is 16S rDNA and contains the nucleotide sequence shown in SEQ ID NO: 1, Pediococcus acidilactici AO22 strain is a Gram-positive cocci that shows purple when Gram stain, glucose, galactose, Xylose, ribose, mannose, fructose, tagatose, mannitol and the like are used.

In addition, the Pediococcus ecidi lactici ( Pediococcus acidilactici ) AO22 strain, in addition to the activity of obesity, may further show the anti-inflammatory activity caused by obesity. Pediococcus acidilactici AO22 strain, when ingested through oral administration, inhibits the differentiation of adipocytes, induces differentiation into M2 macrophages, inhibits obesity, and reduces the absorption of dietary fat by the host. While reducing the weight of the host by increasing dietary fat excretion.

In addition, Pediococcus ecidi lactici of the present invention ( Pediococcus) acidilactici ) AO22 strain has excellent viability against gastric acid or bile, and shows a high viability in the body of the animal, it can maintain the obesity inhibitory activity in the body for a long time.

In addition, another aspect of the present invention relates to a probiotics comprising a Pediococcus acidilactici AO22 strain (KCCM12146P) or a culture thereof.

Pediococcus ecidi lacticci according to the present invention acidilactici ) AO22 strain can be used as is or in the form of dry powder.

In the present invention, the culture medium is, in the known liquid medium or solid medium which can supply nutrients such as Pediococcus acidilactici AO22 strain to grow and survive in vitro, the pediococcus ecidi lacti Pediococcus acidilactici refers to the entire medium including the cultured strain obtained by culturing the AO22 strain for a certain period of time, its metabolites, extra nutrients, and the like, and the culture solution from which the strain is removed after the strain culture.

The culture solution may be subjected to centrifugation or filtration, or may be concentrated, and this step may be performed according to the needs of those skilled in the art.

Probiotics in the present invention refers to the intestinal bacterial flora that is beneficial to health, ie, live microorganisms, or live fungi, which provide benefits to the health of the host. Probiotics are generally consumed as part of a fermented food such as yoghurt or as a dietary supplement.

Pediococcus ecidi lactici of the present invention acidilactici ) AO22 strain is a strain of the genus Pediococcus known as probiotics, and has excellent viability against gastric acid and bile and has an obesity activity effect in itself, and thus can be used as a probiotic, that is, a probiotic.

Pediococcus acidilactici ) AO22 strain ( KCCM12146P ) or a culture thereof is preferably included in an amount of 0.01 to 50% by weight based on the total weight of the composition.

Pediococcus ecidi lactici in the composition acidilactici ) AO22 strain may be administered to contain a viable count of less than 5 × 10 ⁷ CFU, which is to be selected according to the patient and the situation is limited by the number of viable bacteria in the composition is limited no.

Another aspect of the present invention relates to a pharmaceutical composition for preventing or treating obesity and a food composition for preventing or improving obesity, including a Pediococcus acidilactici AO22 strain ( KCCM1214 6 P ) or a culture thereof.

Pediococcus ecidi lacticci according to the present invention acidilactici ) AO22 strain can be used as is or in the form of dry powder.

In the present invention, the culture medium is, in the known liquid medium or solid medium which can supply nutrients such as Pediococcus acidilactici AO22 strain to grow and survive in vitro, the pediococcus ecidi lacti Pediococcus acidilactici refers to the entire medium including the cultured strain obtained by culturing the AO22 strain for a certain period of time, its metabolites, extra nutrients, and the like, and the culture solution from which the strain is removed after the strain culture. The culture medium from which the strain has been removed may be sterilized by the culture medium to include the microbial cells, or may be filtrate or centrifugal supernatant from which the microbial cells are removed by filtration or centrifugation.

The culture solution may be subjected to centrifugation or filtration, or may be concentrated, and this step may be performed according to the needs of those skilled in the art.

Pediococcus acidilactici ) AO22 strain (KCCM12146P) or its culture solution may be included in an amount of 0.01 to 50% by weight based on the total weight of the composition, and may be used directly, may be used in concentration, or diluted after concentration or drying.

The improvement, treatment or prevention effect of obesity of the present invention is pediococcus acidilactici ) Proliferation of AO22 strains inhibits the differentiation of adipocytes, induces differentiation into M2 type macrophages, and reduces host body weight by reducing dietary fat absorption while increasing host fat release. The composition may be used as a liquid itself, or may be dried and powdered.

'Including as an active ingredient' is the pediococcus ecidi lactici ( Pediococcus acidilactici ) is meant to comprise an amount sufficient to achieve an obesity improvement, treatment or prophylactic efficacy or activity of the AO22 strain or culture thereof.

By administration of the composition is meant any action that inhibits or delays the development, spread and recurrence of obesity, and treatment means any action in which the symptoms of obesity are improved or beneficially altered by administration of the composition.

The food composition may be a formulation of the composition in the form of capsules, tablets, powders, granules, liquids, pills, flakes, pastes, syrups, gels, jelly or bars, or beverages, teas, spices, It is manufactured in the form of general food by adding it to food materials such as chewing gum and confectionery, and when it is ingested, it means that it has a specific effect on health. There are no side effects, etc.

The food composition is very useful because it can be consumed on a daily basis. The amount of Pediococcus acidilactici AO22 strain or its culture solution added in such a food composition cannot be uniformly defined depending on the type of food, but does not impair the original taste of the food. What is necessary is just to add, and it is the range of 0.01-50 weight% normally with respect to target foodstuff, Preferably it is 0.1-20 weight%. In addition, in the case of a food composition in the form of capsules, tablets, powders, granules, liquids, rings, flakes, pastes, syrups, gels, jelly or bars, usually 0.1 to 100% by weight, preferably 0.5 to 80% by weight What is necessary is just to add in the range of.

The food composition may include, as an active ingredient, a Pediococcus acidilactici AO22 strain or a culture thereof, as well as components commonly added to foods, for example, proteins, carbohydrates, and fats. Contains nutrients, seasonings and flavorings. Examples of the above carbohydrates include monosaccharides such as glucose, fructose and the like; Disaccharides such as maltose, sucrose, oligosaccharides and the like; And sugars such as conventional sugars such as polysaccharides such as dextrin, cyclodextrin and the like and xylitol, sorbitol, erythritol.

As the flavoring agent, natural flavoring agents [tauumatin, stevia extract (for example, rebaudioside A, glycyrrhizin, etc.) and synthetic flavoring agents (saccharin, aspartame, etc.) can be used.

For example, when the food composition of the present invention is prepared with a drink and a beverage, pediococcus edidilactici of the present invention (Pediococcus acidilacticiIn addition to the AO22 strain or its culture, citric acid, liquid fructose, sugar, glucose, acetic acid, malic acid, fruit juice, and various plant extracts may be further included.

Pediococcus also known as Pediococcus acidilactici ) A pharmaceutical composition for the prevention or treatment of obesity, comprising AO22 strain or its culture as an active ingredient, Pediococcus acidilactici ) The number of viable cells of AO22 strain is less than 5 × 10 ⁷ CFU, preferably from 5 × 10 ³ to It may be administered to be included in a concentration of 5 × 10 ⁷ CFU, which is to be selected according to the patient and the situation is not limited by the number of live bacteria in the composition.

In addition, the culture medium in the composition is, for example, 0.001 mg / kg or more, preferably 0.1 mg / kg or more, more preferably 10 mg / kg or more, even more preferably 100 mg / kg or more, even more Preferably at least 250 mg / kg, most preferably at least 0.1 g / kg. Pediococcus acidilactici AO22 strain is a strain isolated from the human body, even if excessively administered as a live bacterium, since there is no side effect on the human body Pediococcus acidilactici ( Pediococcus acidilactici ) AO22 The upper limit of the quantity of the strain can be selected and carried out by those skilled in the art within an appropriate range.

The pharmaceutical composition may be prepared using a pharmaceutically suitable and physiologically acceptable adjuvant in addition to the active ingredient, and the adjuvant may include excipients, disintegrants, sweeteners, binders, coatings, swelling agents, lubricants, lubricants or A flavourant etc. can be used.

The pharmaceutical composition may be formulated to include one or more pharmaceutically acceptable carriers in addition to the active ingredients described above for administration.

Formulation forms of the pharmaceutical composition may be granules, powders, tablets, coated tablets, capsules, suppositories, solutions, syrups, juices, suspensions, emulsions, drops or injectable solutions. For example, for formulation in the form of tablets or capsules, the active ingredient may be combined with an oral, nontoxic, pharmaceutically acceptable inert carrier such as ethanol, glycerol, water and the like. In addition, if desired or necessary, suitable binders, lubricants, disintegrating agents and coloring agents may also be included in the mixture. Suitable binders include, but are not limited to, natural and synthetic gums such as starch, gelatin, glucose or beta-lactose, corn sweeteners, acacia, trackercance 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 in compositions formulated in liquid solutions are sterile and physiologically compatible, including saline, sterile water, Ringer's solution, buffered saline, albumin injectable solutions, dextrose solution, maltodextrin solution, glycerol, ethanol and these One or more components may be mixed and used, and other conventional additives such as antioxidants, buffers and bacteriostatic agents may be added as necessary. Diluents, dispersants, surfactants, binders and lubricants may also be added in addition to formulate into injectable formulations, pills, capsules, granules or tablets such as aqueous solutions, suspensions, emulsions and the like.

Furthermore, the method disclosed in Remington's Pharmaceutical Science, Mack Publishing Company, Easton PA can be formulated according to each disease or component according to the appropriate method in the art.

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

Suitable dosages of the pharmaceutical compositions vary depending on factors such as formulation method, mode of administration, patient's age, weight, sex, morbidity, food, time of administration, route of administration, rate of excretion, and reaction sensitivity, and are usually skilled The physician can easily determine and prescribe a dosage 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 unit dosage form by formulating with a pharmaceutically acceptable carrier and / or excipient according to a method which can be easily carried out by those skilled in the art. It may be prepared by incorporation into a dose container. In this case, the formulation may be in the form of a solution, suspension or emulsion in an oil or aqueous medium, or may be in the form of extracts, powders, granules, tablets or capsules, and may further include a dispersant or stabilizer.

Hereinafter, the present invention will be described in more detail with reference to examples and the like, but the scope and contents of the present invention are not limited or interpreted by the following examples. In addition, if it is based on the disclosure of the present invention including the following examples, it will be apparent that those skilled in the art can easily carry out the present invention that does not specifically present the experimental results, these modifications and modifications are attached to the patent It goes without saying that it belongs to the claims.

In addition, the experimental results presented below are only representative of the experimental results of the Examples and Comparative Examples, and the effects of each of the various embodiments of the present invention not explicitly set forth below will be described in detail in the corresponding sections.

Example  1. 36 strains

In order to select a candidate group of probiotics having an obesity inhibitory activity (36 species) derived from the human body was collected (Table 1), each strain was isolated and identified as shown in Table 1. Each strain was incubated in 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 Pediococcus acidilactici AO22 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 inhibitory activity on adipocyte formation

In order to evaluate the anti-obesity efficacy of the top candidate strains, the ability to inhibit the differentiation of fat cells (3T3-L1 cells) into adipocytes (Example 1) and the ability to regulate the macroscopic (splenic macrophage isolated from mice) (Example 2) Were analyzed and compared.

(1) Experiment Method

The accumulation of lipids in adipocytes is the most representative feature of obesity. Adipocytes are formed by the differentiation of pre-adipocytes derived from stem cells, which are morphologically and biochemical during the differentiation of 3T3-L1 cells (preadipocytes) into adipocytes. Intracellular fat globules are formed through the change, and as the differentiation progresses, the size of fat globules increases. Since most fat globules are composed of triglycerides and proteins such as perilipin A, the degree of fat differentiation can be determined by measuring the amount of triglycerides in the cells.

Therefore, we evaluated the activity of inhibiting adipocyte formation (dipogenesis) by treating the top candidate strains to 3T3-L1 cells, which are representative allied cells, and inducing differentiation to measure intracellular triglyceride (TG) content.

Cell culture was maintained for at least 8 days for the above-described TG content measurement experiment. In addition, the top candidate strains were treated in dead form to rule out the effects of the top candidate strains on the growth of 3T3-L1 cells.

Prior to the TG experiment, the cytotoxicity was confirmed by water soluble tetrazolium (WST) analysis of the top candidate strains, and thus did not show cytotoxicity at 1 × 10 ⁷ to 1 × 10 ⁶ CFU, thus adipogenesis. In order to measure the inhibitory activity, the killing concentrations of the top candidate strains treated were fixed at 1 × 10 ⁷ to 1 × 10 ⁶ CFU / well.

Specifically, Oil Red O staining was performed according to the method of Negrel and Dani in order to determine the effect of the candidate strain on the degree of fat accumulation in the differentiation process.

100% confluent 3T3-L1 cells were treated with 1 × 10 ⁷ CFU / mL of each of the 36 top candidate strains, followed by MDI medium (DMEM + FBS + PS + Insulin + Dexamethasone + IBMX +). Insulin). After 10 days of differentiation, the culture was removed, washed with PBS and fixed for 10 minutes with 10% formaldehyde. 10% formaldehyde was removed and saturated formaldehyde was added to fix the cells for at least 1 hour. Thereafter, 60% isopropanol was added thereto, immediately removed, washed with distilled water, dyed for 30 minutes with oil red O dye solution, and then washed with distilled water. The intracellular fat accumulation of the stained fat globules was observed using a microscope, and the effects of 36 strains on the fat accumulation during fat differentiation were visually observed. To confirm the accumulated fat, oil red O dye was eluted with isopropanol in a well-dried state, and then absorbance was measured at 490 nm using a multi-plate reader. Isopropanol was used as a blank.

(2) conclusion

1 is a diagram showing a series of analytical procedures for selecting strains having obesity inhibitory activity according to Experimental Example 1 of the present invention, Figure 2 is a view of each of the 36 strains measured in accordance with Experimental Example 1 of the present invention A graph showing lipid accumulation using oil red O staining.

As shown in FIG. 2, intracellular triglyceride (TG) content of each of 36 strains was compared. As a result, strain numbers 2, 3, 5, 6, 7, 8, 12, 13, 15, 16, 21, It was confirmed that the TG content was reduced by 10% or more in the strains of 24, 28 and 29, and among them, strains 15 and 21 were found to have the best adipocyte inhibitory activity.

Example  2. macrophage isolated from mice Controllability  evaluation

In adipose tissue, there are macrophages that control the inflammatory response in addition to adipoocytes. Therefore, when obesity occurs, chronic inflammation or diabetes is also accompanied by macrophages present in fat cells.

Under normal conditions, M2 type macrophages are present in adipocytes, which are anti-inflammatory macrophages that have activity to alleviate inflammation and maintain and protect against insulin resistance. In contrast, in obesity, macrophages infiltrate into adipocytes and M2 type macrophages are switched to M1 type macrophages. M1 type macrophages are inflammatory macrophages, known to produce chemicals called pro-inflammatory cytokines and chemokines. For example, the production of cytokines, such as IL-1beta, TNF-alpha, IL-6, IL-12, induces inflammation and promotes insulin resistance.

Therefore, in order to prevent diseases, inflammation, diabetes, and the like caused by obesity, the degree of conversion from M1 type to M2 type macrophages and whether M0 type macrophages induce differentiation from M2 type macrophages are analyzed. The macrophage regulation of the strain was evaluated.

(1) Experiment Method

Splenocytes were isolated from mice to select strains capable of controlling macrophage, and splenocytes were isolated. Splenocytes contain various immune cells such as T cells and B cells. Therefore, only the CD11b and macrophages (macrophages) were separated using the MACS system. The separated macrophages were divided into 96 well plates at 1x10 ⁶ cells / well and incubated for more than 2 hours. When macrophages adhered stably to the bottom of the well, 36 species of 100 ng / ml LPS and 3x10 ⁷ CFU / well Add strains (killing or live bacteria) of respectively. After 48 hours, the supernatant was recovered and the amount of IL-12 and IL-10 produced was measured by ELISA. IL-12 is an identifying factor of M1 type macrophages and IL-10 is an identifying factor of M2 type macrophages.

FIG. 3 is a diagram illustrating a series of analytical procedures for selecting strains having an obesity inhibitory activity according to Experimental Example 2 of the present invention, and FIG. 4 is a diagram showing 36 strains of each of the dead forms according to Experimental Example 2 of the present invention. When treated with, it is a graph showing the cytokine secretion (%) in the macrophages, Figure 5 is treated with each of the 36 strains of the dead type according to Experimental Example 2 of the present invention, IL in macrophages Figure 6 is a graph showing the ratio of the ratio of IL / 10 to -12, Figure 6 shows the cytokine secretion in macrophages when each of the 36 strains of the live type according to Experimental Example 2 of the present invention ( %) Is a graph showing the measurement, Figure 7 shows the ratio of IL-10 to IL-12 in the macrophages when each of the 36 strains of the live cell type according to Experimental Example 2 of the present invention It is a graph measured and shown.

Referring to Figures 4 and 5, 19 strains (1, 2, 3, 6, 7, 8, 9, 10, 11, 1, 2, 3, 6, 7, 8, 9, 10, 1, 5) increased the ratio of Il-10 / IL-12 when the bacterium was treated. 15, 16, 19, 20, 21, 22, 23, 24, 30, 31, 33).

When the live bacteria were treated as shown in FIGS. 6 and 7, a total of 17 strains in which the ratio of Il-10 / IL-12 increased by 2.5 times or more (strain numbers: 1, 3, 5, 6, 7, 8, 13, 14, 15) , 16, 18, 21, 22, 23, 34).

Among the strains identified on the basis of the above results, even if the ability to inhibit adifferentiation or macrophage control was not excellent, strains showing both activities were selected first, and a total of four strains were selected. Specifically, the 3, 6 strains were selected to have an inhibitory activity on adipocyte formation on in vitro, and the ratio of Il-10 / IL-12 was the highest, and strain 13 was selected to inhibit adipocyte formation and macrophages on in vitro. It was confirmed that the strain showing all regulatory activity was selected.

In addition, strain 15 ( Pediococcus acidilactici AO22) was selected because it was found to have the best anti-differentiation activity and has a relatively high IL10 / IL12 ratio.

(3) synthesis

By combining the results of Experimental Examples 1 and 2, four strains (strain number: 3, 6, 13, 15) which were excellent in inhibiting adipogenesis and excellent in regulating activity on macrophages were selected. Among the four strains selected, strains showing excellent obesity inhibitory activity in vivo were secondarily selected, and the obesity inhibitory activity was compared in vivo through experiments on in vivo compensation described below.

Experimental Example  3. Confirmation of the effect of obesity inhibitory activity through animal experiments according to the selected four strains

end. Laboratory animals Experimental group

C57BL / 6 6-week-old males were purchased from the central laboratory animals, and were bred at 20 ± 2 ° C., 55 ± 5% humidity, and 12 hours of contrast. Experimental animals were purified for 2 weeks after purchase, and then placed 5 animals in each group to set the experimental group as shown in Table 2.

division
(N = 5) Experimental group Diet Normal diet Normal control diet (ND, 10% fat), PBS 5 times a week, 100 μl / head oral administration for 8 weeks high fat diet Negative control (HFD, 45% fat) fed high fat diet for obesity induction, oral administration of 100 μl / head PBS 5 times a week for 8 weeks A After eating high-fat diet for obesity, strain No. 3 ( Bacillus licheniformis ) orally administered 10 ⁸ CFU / head 5 times a week for 0-4 weeks, and then 5X10 ⁸ CFU / head 5 times a week for 5-8 weeks. Oral administration B After eating high-fat diet for obesity, strain No. 6 ( Enterococcus faecalis ) orally administered 10 ⁸ CFU / head 5 times a week for 0-4 weeks, and then 5X10 ⁸ CFU / head 5 times a week for 5-8 weeks. Oral administration C After eating high-fat diet for obesity, strain No. 13 ( Lactobacillus fermentum ) orally administered 10 ⁸ CFU / head 5 times a week for 0-4 weeks, and then 5X10 ⁸ CFU / head 5 times a week for 5-8 weeks. Oral administration D Ingestion of high-fat diet for obesity induction, oral administration of strain No. 15 ( Pediococcus acidilactici AO22 ) 5 times weekly for 10 weeks to 10 ⁸ CFU / head, and 5 times weekly for 5 to 8 weeks 5X10 ⁸ CFU / oral administration of head

All experimental groups except the normal diet and the negative control diet were mixed with each strain and then orally fed the feed and the strain together. 8 shows a process of setting the experimental animals and experimental groups according to Experimental Example 3 of the present invention.

I. Body weight, feed intake, Negative water

All experimental groups confirmed the change in body weight (g), feed intake (g), drinking water (ml) of the animals from the time of feeding to sacrifice the animals. Feed and water intake were measured three times a week, cages were replaced twice a week, and body weight was measured once a week and compared.

Figure 9 is a graph showing the measurement of the weight change in each experimental group, according to this, strain 15 Pediococcus ecytolactici AO22 ( Pediococcus acidilactici AO22 ) clearly showed a decrease in weight gain.

Experimental Example  4. Identification of Selected Strains

16S rDNA  analysis

The final selected strains were subjected to 16S rRNA gene analysis (SEQ ID NO: 1), which is a bacterial base preservation sequence using Universal bacterial primers (518F, 800R) for PCR reaction. The results were interpreted using NCBI blast (http://www.ncbi.nlm.nih.gov/).

The present inventors named Pediococcus acidilactici AO22 as " Pediococcus acidilactici AO22 (KCCM12146P :)" and deposited it on November 2, 2017 at the Korea Microbiological Conservation Center.

Experimental Example  5. Selected Final AO22  Identification of anti-obesity activity in animal experiments of strains

end. Strain culture

Final screened final Pediococcus edidiractisci ( Pediococcus acidilactici ) In order to generate AO22 strain, Pediococcus esididilacti AO22 strain was inoculated in solid medium (Lactobacilli MRS Agar; BD Difco Co, USA) and incubated at 37 ° C. for 24 hours. During the cultivation process, whether the contamination occurred or formed a single colony was carefully checked before proceeding to the next experiment. After the incubation was completed, colonies were taken from each solid medium, and then inoculated into 5 mL of liquid medium (Lactobacilli MRS broth; BD Difco Co, USA) and incubated at 37 ° C., 140 rpm, for 24 hours, and then prepared. 200% of other liquid medium (Lactobacilli MRS broth; BD Difco Co, USA) was incubated in steps of 1% (v / v) inoculation. Activated Pediococcus Escidilactis AO22 strain was incubated at 2.4 rpm for 5 hours at 37 rpm for 24 hours, followed by centrifugation at 6000 rpm and 4 ℃ for 15 minutes (Avanti JE, Beckman Coulter, USA) cells were recovered. The recovered cells were washed twice with sterile 0.85% saline solution, lyophilized (FDCF-12003, OPERON, Korea) and powdered and stored at -80 ° C until use.

I. Laboratory animals Experimental group

The experimental animals were purchased from 6 weeks-old C57BL / 6 male mice from the center experimental animals, and were bred at a temperature of 20 ± 2 ℃, a humidity of 55 ± 5%, light and dark conditions for 12 hours. Experimental animals were purified for 2 weeks after purchase, and then 10 animals were placed in each group by randomized complete block design to set up experimental groups as shown in Table 3.

During the acclimation period, litter was mixed every 2-3 days for homogenization of the intestinal microflora in the animal model. Thereafter, 10 animals were placed in each group, and the normal diet (ND) group fed a normal diet and the other group fed a high-fat diet (Rodent diet with 45% kcal Fat, Research Diets, USA). Pediococcus ecidi lactisi AO22 strain was weighed at a concentration of 5 × 10 ⁷ CFU / head, suspended in sterile PBS and administered orally administered five times per week for 10 weeks.

At the end of the experimental period, the animals were fasted for at least 12 hours, and blood was collected through orbital blood collection, left at room temperature for at least 30 minutes, and then serum was separated by centrifugation at 1,690 × g for 10 minutes. . Liver, epididymal fat, retroperitoneal fat, inguinal fat, and scapula brown fat were extracted, washed with saline to remove water, and white fat adhered to scapula brown adipose tissue.

division
(N = 10) Experimental group ND Normal control diet (ND, 10% fat), PBS 5 times a week, 100 μl / head oral administration for 10 weeks HFD Negative control with high-fat diet for obesity induction (Rodent diet with 45% kcal Fat, Research Diets, USA), 100 μl / head orally administered PBS five times a week for 10 weeks Pediococcus acidilactici AO22 Consume high fat diets for obesity-induced, and for 10 weeks during the AO22 strain CD rakti the Phedi O Lactococcus final sorting five times a week administration of 5 × 10 ⁷ CFU / head oral

All. Body weight, feed intake, Negative water

All experimental groups confirmed the change in body weight (g), feed intake (g / day) and drinking water (g / day) of the animals from the time of feeding to sacrifice of the animals. Feed and water intake were measured three times a week, cages were replaced twice a week, and body weight was measured once a week and compared.

Figure 10 is a graph showing the weight change in each experimental group measured by week, Figure 11 is a graph showing the analysis of the weight gain in each experimental group, feed intake (g / day), drinking water (g / day), calories Intake (kcal / day) is shown in Table 4.

ND HFD Pediococcus acidilactici AO22 P value Average of daily food intake
(g / day) 3.56 ± 0.05 2.64 ± 0.04 2.55 ± 0.04 <0.001 Average of daily water intake
(g / day) 3.81 ± 0.08 2.96 ± 0.09 2.92 ± 0.06 <0.001 Average of daily calorie intake
(kcal / day) 12.83 ± 0.17 13.36 ± 0.21 12.93 ± 0.22 N.S.

According to FIGS. 10, 11 and Table 4, when Pediococcus acidilactici AO22 strain was administered to mice fed high fat diet for 10 weeks, significant weight gain was suppressed compared to the HFD group fed only high fat feed. It can be seen that the phenomenon is expressed.

That is, since it was confirmed that the body weight gained less than the HFD group fed high fat feed, Pediococcus ecidi lactici ( Pediococcus acidilactici ) AO22 strain was found to be suppressed weight gain compared to the HFD group despite high-fat diet, Pediococcus acidilactici AO22 strain has a weight-inhibiting effect .

The amount of calories ingested was calculated from the amount of feed consumed. Mean daily feed intake and water intake were significantly different between ND and HFD groups. It is expected that this is due to the difference in the composition and properties of the feed, and compared to the HFD group, the ND group consumed more feed, and the water intake increased.

HFD group and Pediococcus pedicoccus acidilactici ) AO22 group showed no difference in feed, water and calorie intake, so that the weight-inhibiting activity of Pediococcus acidilactici AO22 strains proved through the previous experiment Or it can be seen that the effect is not due to reduced calorie intake.

la. Liver, epididymal fat, Posterior peritoneum  Fat, Groin  Fat, Scapula  Analysis of fat

At the end of the experiment period, after fasting for at least 12 hours, liver, epididymal fat, posterior peritoneal fat, inguinal fat, and scapula brown fat were extracted, washed with saline to remove water, and white fat adhered to scapula brown adipose tissue. After removal, the weight to weight was measured and shown in Table 5 below. In Table 5, Liver is liver fat, EFT is epididymal fat, RFT is retroperitoneal fat, IFT is inguinal fat, and iBAT is interscapular brown adipose tissue.

Tissue weight
(mg) ND HFD Pediococcus acidilactici AO22 P value Liver 834.6 ± 15.48 916.16 ± 28.37 860.21 ㅁ 15.55 N.S. EFT 270.49 ± 19.51 ^a 1741.68 ㅁ 188.37 ^b 1414.95 ± 111.35 ^b <0.001 RFT 82.00 ± 22.96 ^a 642.58 ± 57.26 ^c 523.01 ± 32.40 ^b <0.001 IFT 159.19 ± 14.20 ^a 873.74 ± 84.50 ^b 682.12 ± 52.22 ^b <0.001 BAT 54.27 ± 2.59 ^a 88.10 ± 5.91 ^b 79.38 ± 5.41 ^b <0.001

According to Table 5 HFD group consumed the high fat was confirmed that it has a slightly increased weight of the liver as compared to the treated group, during CD rakti (Pediococcus acidilactici) AO22 strain in ND group and Phedi O Rhodococcus.

In addition, the HFD group fed high fat diet significantly increased the weight of epididymal white adipose tissue (EFT), peritoneal white adipose tissue (RFT), inguinal white adipose tissue (IFT), and scapula brown adipose tissue (iBAT). On the contrary, when the Pediococcus acidilactici AO22 strain is administered, it can be seen that the fat weight in each tissue is significantly reduced.

That the fat is increased in the HFD group consumed, when administered during a CD rakti (Pediococcus acidilactici) AO22 strain Lactococcus Phedi O, it is understood that it has significantly decreased.

<110> KOREA FOOD RESEARCH INSTITUTE <120> Pediococcus acidilactici AO22 strain with Anti-Obesity Ability          and Composition for treatment or improvement or preventing of          obesity configuring the same <130> HPC7703 <160> 1 <170> KoPatentIn 3.0 <210> 1 <211> 1489 <212> DNA <213> Artificial Sequence <220> <223> 16S rDNA of Pediococcus acidilactici AO22 <400> 1 gaacgctggc ggcgtgccta atacatgcaa gtcgaacgaa cttccgttaa ttgatcagga 60 cgtgcttgca ctgaatgaga ttttaacacg aagtgagtgg cggacgggtg agtaacacgt 120 gggtaacctg cccagaagca ggggataaca cctggaaaca gatgctaata ccgtataaca 180 gagaaaaccg cctggttttc ttttaaaaga tggctctgct atcacttctg gatggacccg 240 cggcgcatta gctagttggt gaggtaacgg ctcaccaagg cgatgatgcg tagccgacct 300 gagagggtaa tcggccacat tgggactgag acacggccca gactcctacg ggaggcagca 360 gtagggaatc ttccacaatg gacgcaagtc tgatggagca acgccgcgtg agtgaagaag 420 ggtttcggct cgtaaagctc tgttgttaaa gaagaacgtg ggtgagagta actgttcacc 480 cagtgacggt atttaaccag aaagccacgg ctaactacgt gccagcagcc gcggtaatac 540 gtaggtggca agcgttatcc ggatttattg ggcgtaaagc gagcgcaggc ggtcttttaa 600 gtctaatgtg aaagccttcg gctcaaccga agaagtgcat tggaaactgg gagacttgag 660 tgcagaagag gacagtggaa ctccatgtgt agcggtgaaa tgcgtagata tatggaagaa 720 caccagtggc gaaggcggct gtctggtctg taactgacgc tgaggctcga aagcatgggt 780 agcgaacagg attagatacc ctggtagtcc atgccgtaaa cgatgattac taagtgttgg 840 agggtttccg cccttcagtg ctgcagctaa cgcattaagt aatccgcctg gggagtacga 900 ccgcaaggtt gaaactcaaa agaattgacg ggggcccgca caagcggtgg agcatgtggt 960 ttaattcgaa gctacgcgaa gaaccttacc aggtcttgac atcttctgcc aacctaagag 1020 attaggcgtt cccttcgggg acagaatgac aggtggtgca tggttgtcgt cagctcgtgt 1080 cgtgagatgt tgggttaagt cccgcaacga gcgcaaccct tattactagt tgccagcatt 1140 cagttgggca ctctagtgag actgccggtg acaaaccgga ggaaggtggg gacgacgtca 1200 aatcatcatg ccccttatga cctgggctac acacgtgcta caatggatgg tacaacgagt 1260 tgcgaaaccg cgaggtttag ctaatctctt aaaaccattc tcagttcgga ctgtaggctg 1320 caactcgcct acacgaagtc ggaatcgcta gtaatcgcgg atcagcatgc cgcggtgaat 1380 acgttcccgg gccttgtaca caccgcccgt cacaccatga agagtttgta acacccaaag 1440 ccggtggggt aaccttttag gagctagccg tctaaggtgg gacagatga 1489

Claims (9)

Pediococcus acidilactici AO22 strain (KCCM12146P) having obesity inhibitory activity. Claim 1 Phedi O Lactococcus when the CD rakti (Pediococcus acidilactici) probiotics (probiotics), including strains AO22 (KCCM12146P) or culture medium thereof. Pediococcus of claim 1 Pediococcus acidilactici ) AO22 strain (KCCM12146P) or a pharmaceutical composition for preventing or treating obesity comprising a culture thereof. The method of claim 3, wherein the Pediococcus acidilactici AO22 strain (KCCM12146P) or its culture is contained in an amount of 0.01 to 50% by weight based on the total weight of the composition, for preventing or treating obesity. Pharmaceutical 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 the 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 preventing or improving obesity comprising a Pediococcus acidilactici AO22 strain (KCCM12146P) or a culture thereof. Pediococcus of claim 1 Pediococcus acidilactici ) AO22 strain (KCCM12146P) or a food composition for improving inflammation due to obesity comprising a culture thereof.

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