KR102136718B1 - Lactobacillus pentosus AO21-1 strain with Anti-Obesity Ability and Composition for treatment or improvement or preventing of obesity comprising the same - Google Patents

Abstract

The novel Lactobacillus pentosus AO21-1 strain of the present invention has high stability as a strain isolated from the human body, exhibits ability to inhibit adipocyte differentiation in vitro, and improves, prevents or prevents obesity, such as weight loss activity observed in animal experiments. Excellent therapeutic activity. Therefore, since the new strain of the present invention has a low possibility of side effects, unlike conventional diet functional foods or drugs that have side effects, it can exhibit a diet effect without controlling the amount of eating, and thus, a pharmaceutical composition for treating or preventing obesity or improving obesity. Or it can be used as a preventive food composition.

Description

Lactobacillus pentosus AO21-1 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 Lactobacillus pentosus AO21-1 strain and a composition for preventing or treating obesity including the same, and a composition for improving obesity.

As mankind gradually develops into a prosperous society, obesity is emerging as one of the serious chronic diseases. Obesity is caused by a variety of causes, and is a metabolic disease caused by an imbalance between intake and consumption of calories, and it is known that it is caused by an increase in the size (hypertrophy) or an increase in the number (hyperplasia) of fat cells in the body when viewed morphologically.

Obesity is a direct cause of increasing the risk of developing various adult diseases socially as well as psychologically atrophy, and thus is the main cause of the increase in national medical expenditure.

Specifically, obesity is directly related to an increase in the prevalence of various adult diseases such as type 2 diabetes, hypertension, hyperlipidemia, and heart disease, and is called metabolic syndrome or insulin resistance syndrome by binding obesity-related diseases together. . They act as a cause of arteriosclerosis and cardiovascular disease.

Obesity is not only the most common nutritional disorder in Western society, but also in Korea recently, the incidence of obesity is increasing rapidly due to the improvement of dietary life due to economic development and the westernization of lifestyle, and the rate of obesity in Korean adults increases by 3% every year. Because it is a trend, the problem is gradually getting serious. Therefore, there is a need to develop a therapeutic or therapeutic method for treating and preventing obesity.

Obesity treatment agents known to date include Xenical (Roche Pharmaceuticals, Switzerland), Reductil (Ebot, USA), and Exolise (Atopharma, France). Classified as an inhibitor, most obesity treatments are appetite suppressants that suppress appetite by regulating neurotransmitters related to the hypothalamus. However, conventional treatments have side effects such as heart disease, respiratory disease, and nervous system disease, as well as low persistence of their efficacy, and thus further improved obesity treatments need to be developed, and products currently being developed have satisfactory therapeutic effects without side effects. Since eggplant has few treatments, the development of new obesity treatments is required.

On the other hand, many efforts have been made to reduce blood cholesterol levels by 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. It also produces various organic acids and antibacterial substances such as bacteriocin, but indole, sketol, phenol, etc. are harmful to the intestines of humans and animals. As it does not produce amines or ammonia, it is a beneficial bacterium that prevents spoilage, suppresses harmful bacteria, and shows physiological activity that is beneficial to humans. Among them, Lactobacillus strain is a major member of the normal microbial community living in the intestine of the human body, and has long been known to be important in maintaining a healthy digestive system and vaginal environment, and the US Public Health Service guidelines), all Lactobacillus strains currently deposited with the US Strains Depository (ATCC) are classified as'Bio-safty Level 1', which is recognized as having no known potential risk of causing disease to humans or animals. have.

Republic of Korea Patent Publication No. 10-1471033 discloses a Weissella sp. (Weissella sp.) F22 (Accession No.: KACC 91867P) strain having excellent obesity suppression, and Korean Patent Publication No. 10-0264361 discloses cholesterol-lowering ability. A strain of Lactobacillus plantarum PMO08 (KFCC-11028) having an activity to defoaming against six types of conjugated bile acids is disclosed.

However, since no technology related to lactic acid bacteria having excellent anti-obesity effect has emerged so as to be commercially successful, the present inventors have been conducting research on probiotics having no side effects in the body and having excellent anti-obesity treatment effect, while newly Lactobacillus pentosus AO21-1 The present invention was completed by discovering the strain, and confirming that the strain exhibits high viability and activity in the body of the animal, and that the effect of improving, preventing, and treating obesity is excellent even when the microorganism itself is directly added as an active ingredient of food and medicine. Became.

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

The present invention was conceived to solve the above problems, and an object of the present invention is to provide a Lactobacillus pentosus AO21-1 strain (KCCM12144P) having obesity inhibiting activity.

Another object of the present invention is to provide probiotics comprising the Lactobacillus pentosus AO21-1 strain ( KCCM12144P ) or a culture solution thereof.

Another object of the present invention is to provide a pharmaceutical composition for preventing or treating obesity, comprising the Lactobacillus pentosus AO21-1 strain ( KCCM12144P ) or a culture solution thereof.

Another object of the present invention is to provide a food composition for preventing or improving obesity and a food composition for improving inflammation due to obesity, including Lactobacillus pentosus AO21-1 strain ( KCCM12144P ) or a culture solution thereof. .

In order to achieve the above object, the present invention provides a Lactobacillus pentosus AO21-1 strain (KCCM12144P) having obesity inhibiting activity.

The present invention provides probiotics comprising the Lactobacillus pentosus AO21-1 strain ( KCCM12144P ) or a culture solution thereof in order to achieve the other object.

The present invention provides a pharmaceutical composition for preventing or treating obesity, including Lactobacillus pentosus AO21-1 strain ( KCCM12144P ) or a culture solution thereof in order to achieve the above other object.

The Lactobacillus pentosus ( Lactobacillus pentosus ) AO21-1 strain ( KCCM12144P ) or a culture solution 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 one that inhibits adipocyte differentiation.

The composition may be to reduce weight.

The present invention provides a food composition for preventing or improving obesity, including Lactobacillus pentosus AO21-1 strain ( KCCM12144P ) or a culture solution thereof in order to achieve the another object.

In addition, the present invention provides a food composition for improving inflammation due to obesity, including Lactobacillus pentosus AO21-1 strain ( KCCM12144P ) or a culture solution thereof to achieve the another object.

Lactobacillus pentosus according to the present invention ( Lactobacillus pentosus ) AO21-1 strain is a strain isolated from the human body, has high stability, shows adipocyte differentiation inhibitory ability in in vitro tests, weight loss activity is observed in animal experiments, etc., obesity It has excellent improvement, prevention or treatment activity. Therefore, since the new strain of the present invention has a low possibility of side effects, unlike conventional diet functional foods or drugs that have side effects, it can exhibit a diet effect without controlling the amount of eating, and thus, a pharmaceutical composition for treating or preventing obesity or improving obesity Or it can be used as a preventive food composition.

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

1 is a diagram showing a series of analysis procedures for selecting a strain having obesity inhibitory activity according to Experimental Example 1 of the present invention.
2 is a graph showing lipid accumulation using Oil red O staining for each of 36 strains measured according to Experimental Example 1 of the present invention.
3 is a diagram showing a process of setting an experimental animal and an experimental group according to Experimental Example 2 of the present invention.
4 is a graph showing the amount of change in weight in each experimental group according to Experimental Example 2 of the present invention.
5 is a graph showing a weekly measurement of weight change in each experimental group according to Experimental Example 4 of the present invention.
6 is a graph showing the weight gain in each experimental group according to Experimental Example 4 of the present invention, and the feed intake (g/day), drinking water (g/day), and calorie intake (kcal/day) are shown 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 relates to a Lactobacillus pentosus AO21-1 strain (KCCM12144P) having obesity inhibitory activity.

Lactobacillus pentosus according to the present invention ( Lactobacillus pentosus ) AO21-1 strain is a strain isolated from the human body, a strain deposited as of November 2, 2017 in the Korea Microbiological Conservation Center (KCCM).

Lactobacillus pentosus according to the present invention ( Lactobacillus pentosus ) AO21-1 strain contains the nucleotide sequence shown in SEQ ID NO: 1 as 16S rDNA, Lactobacillus pentosus ( Lactobacillus pentosus ) AO21-1 strain exhibits purple when gram staining It is a gram-positive bacillus, and uses glucose, galactose, lactose, maltose, xylose, trehalose, sucrose, raffinose, and aribinose as a carbon source.

In addition, the Lactobacillus pentosus ( Lactobacillus pentosus ) AO21-1 strain may further exhibit anti-inflammatory activity due to obesity in addition to obesity. Lactobacillus pentosus AO21-1 strain, when ingested via oral administration, inhibits the differentiation of adipocytes and reduces host's dietary fat absorption, while increasing dietary fat excretion, thereby reducing the host's weight. .

In addition, the Lactobacillus pentosus AO21-1 strain of the present invention has excellent viability against gastric acid or bile, and thus exhibits high viability in the body of an animal, so that the obesity inhibitory activity can be maintained in the body for a long time.

In addition, another aspect of the present invention relates to probiotics including Lactobacillus pentosus AO21-1 strain ( KCCM12144P ) or a culture solution thereof.

Lactobacillus pentosus according to the present invention ( Lactobacillus pentosus ) AO21-1 strain can be used as it is, or can be used in the form of a dry powder.

In the present invention, the culture medium is a known liquid medium or solid medium capable of supplying nutrients so that the Lactobacillus pentosus AO21-1 strain can grow and survive in vitro, the Lactobacillus pentosus. ) It means the whole medium containing the cultured strain obtained by culturing the AO21-1 strain for a certain period of time, its metabolites, and extra nutrients, and the culture solution from which the strain was removed after the strain culture was also included.

The culture solution may have been 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.

In the present invention, probiotics refer to the intestinal flora that is beneficial to health, that is, living microorganisms that provide benefits to the health of the host, that is, live fungi. In general, probiotics are consumed as part of fermented foods such as yogurt or as a dietary supplement.

Lactobacillus pentosus ( Lactobacillus pentosus ) AO21-1 strain of the present invention is a strain of the genus Lactobacillus known as probiotics, has excellent viability against gastric acid and bile, and has an obesity activity effect by itself, as a probiotic, that is, as a probiotic. Can be used.

The Lactobacillus pentosus ( Lactobacillus pentosus ) AO21-1 strain ( KCCM12144P ) or a culture solution 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 pentosus AO21-1 strain in the composition may be administered in a concentration of less than 5×10 ⁷ CFU, but this is a matter to be selected according to the patient and the situation. The scope of the present invention is not limited by the number of viable cells.

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 Lactobacillus pentosus AO21-1 strain ( KCCM12144P ) or a culture solution thereof.

Lactobacillus pentosus according to the present invention ( Lactobacillus pentosus ) AO21-1 strain can be used as it is, or can be used in the form of a dry powder.

In the present invention, the culture medium is a known liquid medium or solid medium capable of supplying nutrients so that the Lactobacillus pentosus AO21-1 strain can grow and survive in vitro, the Lactobacillus pentosus. ) It means the whole medium containing the cultured strain obtained by culturing the AO21-1 strain for a certain period of time, its metabolites, and extra nutrients, and the culture solution from which the strain was removed after the strain culture was also included. The culture solution from which the strain has been removed may be sterilized to contain dead cells, or may be a filtrate or a centrifugation supernatant from which cells are removed through filtration or centrifugation.

The culture solution may have been 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.

The Lactobacillus pentosus (Lactobacillus pentosus ) AO21-1 strain (KCCM12144P) or a culture solution thereof is contained in an amount of 0.01 to 50% by weight based on the total weight of the composition, and it may be used directly or concentrated and used, after concentration or drying. It can be diluted and used.

Increasing the dietary fat emissions while improving, the treatment or prevention of obesity of the present invention, while proliferation is Lactobacillus pento Versus (Lactobacillus pentosus) AO21-1 strain to inhibit the differentiation of mast cells, and reduces the absorption of dietary fats by the host It is expected to reduce the weight of the host, and the composition may be used as a liquid or may be dried and powdered.

The term'comprised as an active ingredient' means that the Lactobacillus pentosus ( Lactobacillus pentosus ) AO21-1 strain or its culture medium contains an amount sufficient to achieve obesity improvement, treatment or prevention efficacy or activity.

The administration of the composition means all actions that inhibit or delay the occurrence, spread, and recurrence of obesity, and the treatment means any action in which the symptoms of obesity are improved or beneficially changed by the administration of the composition.

The food composition may be formulated in the form of a capsule, tablet, powder, granule, liquid, pill, flake, paste, syrup, gel, jelly or bar, or beverage, tea, spices, It is added to food materials such as chewing gum and confectionery, and is manufactured in the form of general food, which means that it has a specific effect on health when ingested.However, unlike general drugs, it may occur when taking the drug for a long time using food as a raw material. It has the advantage of no side effects.

The food composition is very useful because it can be consumed on a daily basis. The amount of Lactobacillus pentosus AO21-1 strain or its culture solution added in such a food composition varies depending on the type of target food and cannot be uniformly defined, but is added within a range that does not impair the original taste of the food. It is sufficient, 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 addition, in the case of a food composition in the form of capsules, tablets, powders, granules, liquids, pills, flakes, pastes, syrups, gels, jelly or bars, usually 0.1 to 100% by weight, preferably 0.5 to 80% by weight It can be added within the range of.

The food composition may include, as an active ingredient, Lactobacillus pentosus AO21-1 strain or a culture solution thereof, as well as ingredients commonly added during food production, for example, proteins, carbohydrates, fats, Contains nutrients, seasonings and flavoring agents. Examples of the aforementioned carbohydrates include monosaccharides such as glucose, fructose, and the like; Disaccharides such as maltose, sucrose, oligosaccharides, and the like; And polysaccharides, for example, common sugars such as dextrin and cyclodextrin, and sugar alcohols such as xylitol, sorbitol, and erythritol.

As flavoring agents, natural flavoring agents (taumatin, stevia extract (eg, rebaudioside A, glysirhizin, etc.) and synthetic flavoring agents (saccharin, aspartame, etc.) can be used.

For example, when the food composition of the present invention is made of drinks and beverages, in addition to the Lactobacillus pentosus AO21-1 strain of the present invention or a culture solution thereof, citric acid, liquid fructose, sugar, glucose, acetic acid, malic acid, fruit juice, And it may further include various plant extracts and the like.

In addition, in a pharmaceutical composition for the prevention or treatment of obesity using Lactobacillus pentosus AO21-1 strain or its culture medium as an active ingredient, the number of viable cells of the Lactobacillus pentosus AO21-1 strain is 5×10 Less than ⁷ CFU, preferably 5×10 ³ to It may be administered to be included in a concentration of 5×10 ⁷ CFU, but this is a matter to be selected according to the patient and the situation, and the scope of the present invention is not limited by the number of viable cells in the composition.

In addition, in the composition, the culture medium 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 is included. Lactobacillus pentosus ( Lactobacillus pentosus ) AO21-1 strain is a strain isolated from the human body and does not have side effects on the human body even if it is administered in excess as live bacteria, so the Lactobacillus pentosus AO21-1 strain contained in the composition of the present invention The upper limit of the quantity can be selected and implemented by a person 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 be an excipient, a disintegrant, a sweetener, a binder, a coating agent, an expanding agent, a lubricant, a lubricant, or Flavoring agents and the like can be used.

For administration, the pharmaceutical composition may be formulated by including one or more pharmaceutically acceptable carriers in addition to the above-described active ingredients.

The formulation form 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, non-toxic pharmaceutically acceptable inert carrier such as ethanol, glycerol, water and the like. In addition, if desired or necessary, suitable binders, lubricants, disintegrants and coloring agents may also be included in the mixture. Suitable binders are, but are not limited to, starch, gelatin, natural sugars such as glucose or beta-lactose, corn sweeteners, natural and synthetic gums such as acacia, lacquercanth 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 formulated as liquid solutions are sterilized and suitable for living organisms, such as saline, sterile water, Ringer's solution, buffered saline, albumin injection solution, dextrose solution, maltodextrin solution, glycerol, ethanol, and these One or more of the components may be mixed and used, and other conventional additives such as antioxidants, buffers, and bacteriostatic agents may be added as needed. In addition, diluents, dispersants, surfactants, binders, and lubricants may be additionally added to prepare injectable formulations such as aqueous solutions, suspensions, emulsions, etc., pills, capsules, granules, or tablets.

Furthermore, it can be preferably formulated according to each disease or ingredient using a 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, intramuscular injection, intraperitoneal injection, transdermal administration, and the like, preferably oral administration.

The suitable dosage of the pharmaceutical composition varies depending on factors such as formulation method, mode of administration, patient's age, weight, sex, pathological condition, food, administration time, route of administration, excretion rate and response sensitivity, and is usually skilled. The qualified physician can easily determine and prescribe an effective dosage for the desired treatment or prophylaxis. According to a preferred embodiment, the daily dosage of the pharmaceutical composition is 0.001-10g/kg.

The pharmaceutical composition is prepared in unit dosage form by formulating using a pharmaceutically acceptable carrier and/or excipient according to a method that can be easily carried out by a person having ordinary knowledge in the technical field to which the present invention belongs, or It can be manufactured by enclosing it into a volume container. At this time, 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 an extract, powder, granule, tablet or capsule, and may additionally include a dispersant or a stabilizer.

Hereinafter, the present invention will be described in more detail through examples and the like, but the scope and contents of the present invention are reduced or limited by the examples below and cannot be interpreted. In addition, if it is based on the disclosure of the present invention including the following examples, it is obvious that the present invention for which no specific experimental results are presented can be easily carried out by a person skilled in the art. It is natural to fall within the claims.

In addition, the experimental results presented below are only representative of the experimental results of the above examples and comparative examples, and the effects of each of the various embodiments of the present invention not explicitly presented below will be described in detail in the corresponding section.

Example 1. 36 strains

In order to select a candidate group for probiotics having obesity inhibitory activity, 36 strains derived from humans (Table 1) were collected, and each strain was isolated and identified and shown in Table 1. Each strain was cultured 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 Lactobacillus pentosus AO21-1 16 Lactobacillus acidilactici 17 Lactobacillus zeae 18 Enterococcus faecium 19 Lactobacillus reuteri 20 Lactobacillus johnsonii 21 Lactobacillus acidophilus 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 adipocyte formation inhibitory activity

In order to evaluate the anti-obesity efficacy of the top candidate strains, the ability to inhibit differentiation of fat cells (3T3-L1 cells) into adipocytes (Example 1) was analyzed and compared.

(1) Experiment method

The accumulation of lipids in adipocytes is the most representative characteristic of obesity. Adipocytes are formed by the differentiation of pre-adipocytes derived from stem cells. While 3T3-L1 cells (preadipocytes) are differentiated into adipocytes, morphological and biochemical Through the change, intracellular adipocytes are formed, and as differentiation proceeds, the size of adipocytes increases. Since most of the fat cells are composed of proteins such as triglyceride and perilipin A, the degree of fat differentiation can be confirmed by measuring the intracellular triglyceride content.

Therefore, the activity of inhibiting adipocyte formation (adipogenesis) was evaluated by treating each of the top candidate strains in 3T3-L1 cells, which is a representative provincial cell, and measuring the intracellular triglyceride (TG) content by inducing differentiation.

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

Prior to the TG experiment, as a result of confirming cytotoxicity through water soluble tetrazolium (WST) analysis on the upper candidate strains, it did not show cytotoxicity at 1×10 ⁷ to 1×10 ⁶ CFU, so adipogenesis In order to measure the inhibitory activity, the killing concentration of the upper candidate strain to be treated was 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 examine the effect of the candidate strain on the degree of fat accumulation in the fat differentiation process. In 100% confluent 3T3-L1 cells, each of the 36 top candidate strains was treated with 1×10 ⁷ CFU/mL, and MDI medium (DMEM + FBS + PS + Insulin + Dexamethasone + IBMX + Insulin). After 10 days of differentiation, the culture medium was removed, washed with PBS, and then fixed with 10% formaldehyde for 10 minutes. After removing 10% formaldehyde, saturated formaldehyde was added to fix the cells for at least 1 hour. After that, 60% isopropanol was added and immediately removed, washed with distilled water, and the fat bulbs were dyed with Oil red O dyeing solution for 30 minutes, and washed with distilled water. The intracellular fat accumulation of stained fat cells was observed using a microscope, and the effect of 36 strains on fat accumulation during fat differentiation was visually observed through photographing. To check the accumulated fat, oil red O dye was eluted by adding isopropanol in a well-dried state, and absorbance was measured at 490 nm using a multi-plate reader. At this time, isopropanol was used as a blank.

(2) Conclusion

1 is a diagram showing a series of analysis procedures for selecting strains having obesity inhibitory activity according to Experimental Example 1 of the present invention, and FIG. 2 is a diagram of each of 36 strains measured according to Experimental Example 1 of the present invention. This is a graph showing the lipid accumulation using the Oil red O staining method.

As shown in Figure 2, as a result of comparing the intracellular triglyceride (TG) content of each of the 36 strains, strain numbers 2, 3, 5, 6, 7, 8, 12, 13, 14, 15, 16, It was confirmed that the TG content decreased by 10% or more in the strains 24 and 29, and among them, 4 strains (strain number: 3, 6, 13, 15) that had excellent effects were selected.

Among the four selected strains, in order to secondarily select a strain exhibiting excellent obesity inhibitory activity even in vivo, the obesity inhibitory activity was compared in vivo through an experiment in in vivo, which will be described later.

Experimental example 2. Confirmation of the effect of inhibiting obesity through animal testing according to the selected 4 strains

end. Laboratory animals and Experimental group

Experimental animals were C57BL/6 6-week-old males purchased from Central Experimental Animal Co., Ltd., and were reared under a temperature of 20±2 ℃, a humidity of 55±5%, and a light and dark condition for 12 hours. Experimental animals were purified for 2 weeks after purchase, and then 5 animals were placed for each group to set the experimental group as shown in Table 2.

division
(N=5) Experimental group Diet control Normal diet Normal control (ND, 10% fat) ingesting general feed, PBS administered 5 times a week, 100 µl/head orally for 8 weeks high fat diet Negative control group (HFD, 45% fat) ingesting high fat feed for obesity induction, PBS administered 5 times a week, 100 µl/head orally for 8 weeks A Ingest a high-fat feed for obesity induction, and oral administration of strain number 3 (Bacillus licheniformis) 5 times a week for 0-4 weeks 10 ⁸ CFU/head 5 times a week for 5-8 weeks 5X10 ⁸ CFU/head Oral administration B After ingesting high fat feed for obesity induction, strain number 6 (Enterococcus faecalis) was orally administered 10 ⁸ CFU/head 5 times a week for 0-4 weeks, and 5X10 ⁸ CFU/head 5 times a week for 5-8 weeks. Oral administration C After ingesting high fat feed for obesity induction, strain number 13 (Lactobacillus fermentum) was orally administered 10 ⁸ CFU/head 5 times a week for 0-4 weeks, and 5X10 ⁸ CFU/head 5 times a week for 5-8 weeks. Oral administration D Ingest a high-fat feed for obesity induction, and administer strain number 15 (Lactobacillus pentosus AO21-1) 5 times a week for 0-4 weeks 10 ⁸ CFU/head orally 5 times a week for 5-8 weeks 5X10 ⁸ Oral administration of CFU/head

All experimental groups, except for the normal control and negative control (Hihg fat diet), mixed each strain with feed, and then orally administered the feed and strain together. 3 shows a process of setting an experimental animal and an experimental group according to Experimental Example 2 of the present invention.

I. Weight, feed intake, and Drinking water

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

Figure 4 is a graph showing the amount of change in weight in each experimental group, according to this, it was clearly confirmed the decrease in the amount of weight gain in the 15th strain Lactobacillus pentosus ( Lactobacillus pentosus ) AO21-1.

Experimental example 3. Identification of the selected strain

16S rDNA analysis

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

The present inventors named Lactobacillus pentosus AO21-1 as " Lactobacillus pentosus AO21-1 (accession number: KCCM12144P)", respectively, and deposited it with the Korea Microbiological Conservation Center on November 2, 2017.

Name of deposit institution: Korea Microorganism Conservation Center (overseas)

Accession number: KCCM12144P

Consignment date: 20171102

Experimental example 4. Selected final AO21 -Affect of inhibition of obesity in animal experiments of 1 strain Check sign

end. Strain culture

In order to generate the final selected final Lactobacillus pentosus AO21-1 strain ( Lactobacillus pentosus AO21-1), the Lactobacillus pentosus AO21-1 strain was stroked and inoculated in a solid medium (Lactobacilli MRS Agar; BD Difco Co, USA). , 37 ℃, cultured for 24 hours. The next experiment was carried out after meticulously checking whether contamination occurred on the cells during the culture process or whether a single colony was formed. After the cultivation was completed, colonies were taken from each solid medium, and then inoculated into 5 mL of a liquid medium (Lactobacilli MRS broth; BD Difco Co, USA), and cultured at 37° C., 140 rpm, for 24 hours, the prepared Another liquid medium (Lactobacilli MRS broth; BD Difco Co, USA) was inoculated in 200 mL of 1% (v/v), followed by stepwise enrichment. Activated Lactobacillus pentosus AO21-1 strain was cultured at 2.4L-5L, cultured at 37℃ for 140rpm for 24 hours, and then centrifuged at 6000 rpm for 15 minutes at 4℃ (Avanti JE, Beckman Coulter, USA ) To recover the cells. The recovered cells were washed twice with sterilized 0.85% physiological saline, then freeze-dried (FDCF-12003, OPERON, Korea), powdered, and stored at -80°C until use.

I. Laboratory animals and Experimental group

Experimental animals were 6 weeks old C57BL/6 male mice were purchased and used from Central Experimental Animal Co., Ltd., and were reared under conditions of temperature 20±2°C, humidity 55±5%, and 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 a randomized complete block design, and the experimental group was set as shown in Table 3.

During the acclimatization period, litter was mixed at intervals of 2-3 days in order to homogenize the intestinal microflora in the animal model. Thereafter, 10 animals were placed per experimental group, and the normal diet (ND) group was fed a normal diet and the rest group was fed a high-fat diet (Rodent diet with 45% kcal Fat, Research Diets, USA). The Lactobacillus pentosus AO21-1 strain was weighed at a concentration of 5×10 ⁷ CFU/head, suspended in sterilized PBS, and administered 5 times per week for 10 weeks through oral administration at a constant time.

At the end of the experiment period, the experimental animals were fasted for at least 12 hours, blood was collected through orbital blood collection, left at room temperature for at least 30 minutes, and then centrifuged at 1,690×g for 10 minutes to use the separated serum. . Liver, epididymal fat, retroperitoneal fat, inguinal fat, and scapular brown fat were extracted, washed with saline, dried, and white fat adhering to the brown adipose tissue between the shoulder blades was removed, and the weight of each was measured.

division
(N=10) Experimental group ND Normal control (ND, 10% fat) ingesting general feed, PBS administered 5 times a week, 100 µl/head orally for 10 weeks HFD Negative control (Rodent diet with 45%kcal Fat, Research Diets, USA) ingesting high fat feed for obesity induction, PBS administered 5 times a week, 100 µl/head orally for 10 weeks Lactobacillus pentosus AO21-1 Ingest high fat feed for obesity induction, and administer 5×10 ⁷ CFU/head orally 5 times a week for 10 weeks with the finally selected Lactobacillus pentosus AO21-1 strain

All. Weight, feed intake, and Drinking water

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

5 is a graph showing the amount of weight change in each experimental group measured by week, and FIG. 6 is a graph showing the amount of weight gain in each experimental group, and feed intake (g/day), drinking water (g/day), calories The intake amount (kcal/day) is shown in Table 4.

ND HFD Lactobacillus pentosus AO21-1 P value Average of daily food intake
(g/day) 3.56±0.05 2.64±0.04 2.57±0.04 <0.001 Average of daily water intake
(g/day) 3.81±0.08 2.96±0.09 2.95±0.10 <0.001 Average of daily calorie intake
(kcal/day) 12.83±0.17 13.36±0.21 13.00±0.04 N.S.

According to Figures 5, 6 and Table 4, Lactobacillus pentosus ( Lactobacillus pentosus ) AO21-1 strain was administered to mice fed high fat feed for 10 weeks, as a result, significantly inhibiting weight gain compared to the HFD group fed only high fat feed It can be seen that is expressed.

That is, it was confirmed that less weight was obtained than the HFD group that consumed high fat feed, so Lactobacillus pentosus AO21-1 strain was confirmed that weight gain was suppressed compared to the HFD group, despite ingesting high fat feed. Bar, Lactobacillus pentosus ( Lactobacillus pentosus ) It can be seen that the AO21-1 strain has a weight gain inhibitory effect.

The amount of calories consumed was calculated through the amount of feed consumed. The mean daily feed intake and water intake were significantly different between the ND and HFD groups. This is expected to be due to the difference in the composition and properties of the feed, and it was confirmed that the ND group consumed more feed than the HFD group while the water intake also increased.

HFD group and Lactobacillus pentosus AO21 -1 group confirmed that there was no difference in feed, water, and calorie intake from each other.Through this, Lactobacillus pentosus proved through the previous experiment. It can be seen that it is not the effect of decreased calorie intake.

la. Liver, epididymis fat, Retroperitoneum Fat, Seohye Fat, Shoulder blade Fat analysis

At the end of the experiment period, after fasting for more than 12 hours, the liver, epididymis fat, retroperitoneal fat, groin fat, and brown fat between the shoulder blades were removed, washed with saline, and dried to remove the white fat attached to the brown fat tissue between the shoulder blades. After removal, the weight relative to the body 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 Lactobacillus pentosus AO21-1 P value Liver 834.6±15.48 916.16±28.37 842.60±15.88 N.S. (0.055) EFT 270.49±19.51 ^a 1741.68±188.37 ^b 1550.53±134.21 ^b <0.001 RFT 82.00±22.96 ^a 642.58±57.26 ^b 579.81±45.60 ^b <0.001 IFT 159.19±14.20 ^a 873.74±84.50 ^b 758.48±49.47 ^b <0.001 iBAT 54.27±2.59 ^a 88.10±5.91 ^c 72.98±6.94 ^b 0.003

According to Table 5, it was confirmed that the HFD group fed a high fat diet slightly increased the weight of liver tissue compared to the ND group and the group administered Lactobacillus pentosus AO21-1.

In addition, the HFD group who ingested a high fat diet significantly increased the weight of epididymal white adipose tissue (EFT), retroperitoneal white adipose tissue (RFT), inguinal white adipose tissue (IFT), and interscapular brown adipose tissue (iBAT). On the other hand, when Lactobacillus pentosus ( Lactobacillus pentosus ) AO21-1 strain is administered, it can be seen that the fat weight in each tissue is significantly reduced.

It can be seen that the increase was increased in the HFD group fed a high fat diet, and then significantly decreased when the Lactobacillus pentosus AO21-1 strain was administered.

<110> KOREA FOOD RESEARCH INSTITUTE <120> Lactobacillus pentosus AO21-1 strain with Anti-Obesity Ability and Composition for treatment or improvement or preventing of obesity comprising the same <130> HPC-7704 <160> 1 <170> KoPatentIn 3.0 <210> 1 <211> 1454 <212> DNA <213> Artificial Sequence <220> <223> 16s rDNA of Lactobacillus pentosus AO21-1 <400> 1 gctggcggcg tgcctaatac atgcaagtcg aacgaactct ggtattgatt ggtgcttgca 60 tcatgattta catttgagtg agtggcgaac tggtgagtaa cacgtgggaa acctgcccag 120 aagcggggga taacacctgg aaacagatgc taataccgca taacaacttg gaccgcatgg 180 tccgagtttg aaagatggct tcggctatca cttttggatg gtcccgcggc gtattagcta 240 gatggtgggg taacggctca ccatggcaat gatacgtagc cgacctgaga gggtaatcgg 300 ccacattggg actgagacac ggcccaaact cctacgggag gcagcagtag ggaatcttcc 360 acaatggacg aaagtctgat ggagcaacgc cgcgtgagtg aagaagggtt tcggctcgta 420 aaactctgtt gttaaagaag aacatatctg agagtaactg ttcaggtatt gacggtattt 480 aaccagaaag ccacggctaa ctacgtgcca gcagccgcgg taatacgtag gtggcaagcg 540 ttgtccggat ttattgggcg taaagcgagc gcaggcggtt ttttaagtct gatgtgaaag 600 ccttcggctc aaccgaagaa gtgcatcgga aactgggaaa cttgagtgca gaagaggaca 660 gtggaactcc atgtgtagcg gtgaaatgcg tagatatatg gaagaacacc agtggcgaag 720 gcggctgtct ggtctgtaac tgacgctgag gctcgaaagt atgggtagca aacaggatta 780 gataccctgg tagtccatac cgtaaacgat gaatgctaag tgttggaggg tttccgccct 840 tcagtgctgc agctaacgca ttaagcattc cgcctgggga gtacggccgc aaggctgaaa 900 ctcaaaggaa ttgacggggg cccgcacaag cggtggagca tgtggtttaa ttcgaagcta 960 cgcgaagaac cttaccaggt cttgacatac tatgcaaatc taagagatta gacgttccct 1020 tcggggacat ggatacaggt ggtgcatggt tgtcgtcagc tcgtgtcgtg agatgttggg 1080 ttaagtcccg caacgagcgc aacccttatt atcagttgcc agcattaagt tgggcactct 1140 ggtgagactg ccggtgacaa accggaggaa ggtggggatg acgtcaaatc atcatgcccc 1200 ttatgacctg ggctacacac gtgctacaat ggatggtaca acgagttgcg aactcgcgag 1260 agtaagctaa tctcttaaag ccattctcag ttcggattgt aggctgcaac tcgcctacat 1320 gaagtcggaa tcgctagtaa tcgcggatca gcatgccgcg gtgaatacgt tcccgggcct 1380 tgtacacacc gcccgtcaca ccatgagagt ttgtaacacc caaagtcggt ggggtaacct 1440 tttaggaacc agcc 1454

Claims (8)

Lactobacillus pentosus having an obesity inhibitory activity AO21-1 strain ( KCCM12144P ). Claim 1 Lactobacillus pentosus ( Lactobacillus pentosus ) AO21-1 strain ( KCCM12144P ) or probiotics comprising a culture medium thereof (probiotics). Claim 1 Lactobacillus pentosus ( Lactobacillus pentosus ) AO21-1 strain ( KCCM12144P ) or a pharmaceutical composition for obesity prevention or treatment comprising a culture solution thereof. The pharmaceutical for preventing or treating obesity according to claim 3, wherein the Lactobacillus pentosus AO21-1 strain ( KCCM12144P ) or a culture solution thereof is contained in an amount of 0.01 to 50% by weight based on the total weight of the composition. Composition. The pharmaceutical composition for preventing or treating obesity according to claim 3, wherein the composition inhibits mast cell differentiation. The pharmaceutical composition for preventing or treating obesity according to claim 3, wherein the composition reduces weight. Claim 1 Lactobacillus pentosus ( Lactobacillus pentosus ) AO21-1 strain ( KCCM12144P ) or a food composition for preventing or improving obesity comprising a culture solution thereof. delete

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