KR101809172B1 - Composition for preventing, improving or treating metabolic disease comprising Akkermansia muciniphila strain or its culture broth cultivated in medium without mucin as effective component - Google Patents

Abstract

The present invention relates to a pharmaceutical composition and a health functional food composition for preventing or treating a metabolic disease containing an acacia mucinipila strain cultured in a mucin-free medium or a culture thereof as an active ingredient, and the intestinal microorganism Akkermansia < RTI ID = 0.0 > muciniphila ) is useful as a food, medicine or feed for the prevention or treatment of metabolic diseases since it improves metabolic diseases including hyperlipidemia, fatty liver, obesity and diabetes mellitus. Particularly, in the present invention, when a strain of Ackmannia mycaminifera cultured in a mucin-free medium was orally administered (in vivo) in the case of culturing in a medium containing mucin, It was confirmed that the effect of improving hyperlipemia, fatty liver, obesity and hyperglycemia occurred remarkably. Thus, by developing a specific culture medium composition and optimized absolute anaerobic culture method for maximizing the efficacy of Ackermansia mucinipila strain through the present invention , The present invention is very useful for related industries.

Description

TECHNICAL FIELD [0001] The present invention relates to a composition for preventing, ameliorating or treating a metabolic disease comprising an acacia mucinipila strain cultured in a mucin-free medium or a culture solution thereof as an active ingredient. broth cultivated in medium without mucin as effective component}

The present invention relates to a pharmaceutical composition and a health functional food composition for preventing or treating a metabolic disease containing an acacia mucinipila strain cultured in a mucin-free medium or a culture thereof as an active ingredient.

Metabolic disease is a disease caused by metabolic disorders in vivo. Generally, it is caused by imbalance such as carbohydrate, lipid, protein, vitamin, electrolyte and moisture. Examples thereof include obesity, diabetes, hyperlipidemia, arteriosclerosis and hypertension. Among them, type 2 diabetes is characterized by an increased resistance to insulin, which is a predominant type of diabetes mellitus in adulthood. If the insulin receptor itself is reduced, or its sensitivity is lowered, or if there is a problem with the secondary messenger that causes intracellular glycogen synthesis, the insulin sensitivity will be reduced. Thus, type 2 diabetes is called non-insulin dependent diabetes mellitus and accounts for 85% to 90% of diabetes.

Particularly, although obesity is a social issue in appearance, it is the most serious problem of obesity that it can cause serious health risks such as complications of metabolic diseases such as diabetes and hypertension. Symptoms related to the pathological condition of obesity are systemic chronic inflammation in obese individuals. Inflammation is one of the immune mechanisms that occur in the body, and when it occurs locally, it is an important reaction to defend the body against invasion of germs or viruses from the outside. However, such an inflammatory reaction causes systemic and chronic hyperactivity due to the balance collapse of the body 's immune response, which leads to a disorder in the metabolism in the body. Chronic inflammation caused by obesity is known to be a cause of various metabolic diseases such as diabetes, cardiovascular disease and arteriosclerosis, and it is also the most important factor that defines obesity as a disease. Obesity is simply a cosmetic problem without the onset of secondary metabolic diseases caused by chronic inflammatory reactions. Recently, the World Health Organization has been conducting research on obesity as a cause of chronic inflammation, which can lead to secondary metabolic diseases such as diabetes, .

Currently, a steady increase in food intake with high energy loads and social changes associated with low exercise are increasing the incidence of metabolic diseases caused by obesity and obesity. Conventional treatments based on low-calorie diets and exercises have little effect on obesity control and only result in temporary weight loss. The development of safe and effective drugs to induce weight loss has been going on for decades. However, to date, efficacious drugs have had serious side effects or are not as effective in clinical trials. Therefore, a new approach is needed to improve metabolic diseases caused by obesity and obesity and to prevent such pathological conditions.

Korean Patent Laid-Open Publication No. 2015-0093711 discloses 'use of acacmansia for treating metabolic disorders', Korean Patent Laid-Open Publication No. 2013-0021920 discloses that an extracellular endoplasmic reticulum from Akkermansia muciniphila or Bacteroides acidifaciens is used as an active ingredient A composition for preventing or treating a metabolic disease containing an acacia mucinipila strain cultured in a mucin-free medium or a culture thereof as an active ingredient, as in the present invention, A therapeutic pharmaceutical composition and a health functional food composition "have not been disclosed.

SUMMARY OF THE INVENTION The present invention has been made in view of the above needs, and it is an object of the present invention to provide a safe, effective, and safe method for effectively inhibiting metabolic diseases including hyperlipemia, fatty liver, obesity and diabetes, As a result of intensive research to develop a new preventive or therapeutic agent for a metabolic disease using a substance which does not show toxicity to human body even when consumed as a drug, the intestinal microorganism Akkermansia muciniphila AK : American strain bank deposit number ATCC BAA-835, the strain is the same strain as deposited at the German Bioresource Bank Deposit No. DSM 22959).

Particularly, in the present invention, when a strain of Ackmannia mycaminifera cultured in a mucin-free medium was orally administered (in vivo) in the case of culturing in a medium containing mucin, The present inventors have found that the present invention has the effect of improving hyperlipemia, fatty liver, obesity and hyperglycemia.

In order to solve the above problems, the present invention is only a cyano mu shinny pillar (Akkermansia a marker cultured in the absence of mucin (mucin) medium muciniphila strain, the strain derived from the strain, a culture of the strain, a concentrate of the culture, a dried product of the culture, and an extract of the culture, for the prevention or treatment of a metabolic disease containing, as an active ingredient, at least one selected from the group consisting of To provide a pharmaceutical composition.

In addition, the present invention only markers were incubated in the absence of mucin (mucin) medium cyano mu shinny pillar (Akkermansia muciniphila strain, the strain derived from the strain, the culture of the strain, the concentrate of the culture, the dried product of the culture, and the extract of the culture, as an active ingredient, for the prevention or amelioration of a metabolic disease Health functional food composition.

In addition, the present invention only markers were incubated in the absence of mucin (mucin) medium cyano mu shinny pillar (Akkermansia muciniphila strain, the strain derived from the strain, the culture of the strain, the concentrate of the culture, the dried product of the culture, and the extract of the culture, as an active ingredient, for the prevention or amelioration of a metabolic disease A feed additive composition is provided.

The intestinal microorganism of the present invention, Akkermansia muciniphila ) is useful as a food, medicine or feed for the prevention or treatment of metabolic diseases since it improves metabolic diseases including hyperlipidemia, fatty liver, obesity and diabetes mellitus.

Particularly, in the present invention, when a strain of Ackmannia mycaminifera cultured in a mucin-free medium was orally administered (in vivo) in the case of culturing in a medium containing mucin, It was confirmed that the effect of improving hyperlipemia, fatty liver, obesity and hyperglycemia occurred remarkably. Thus, by developing a specific culture medium composition and optimized absolute anaerobic culture method for maximizing the efficacy of Ackermansia mucinipila strain through the present invention , The present invention is very useful for related industries.

FIG. 1 shows the effect of inhibiting hyperlipidemia by administration of strains AK (+) and AK (-) in hyperlipidemic model mice induced by high fat diet by the changes in blood cholesterol content (A) and blood triglyceride content. * p <0.01 vs Student's t- test
FIG. 2 shows the inhibitory effects of the AK (+) and AK (-) strains on liver fat model mice induced by administration of high fat diet by liver fat staining (left) and liver triglyceride contents (right) to be. * p <0.01 vs Student's t- test
FIG. 3 shows the results of comparing the effects of inhibiting weight gain by administration of AK (+) and AK (-) strains in a high fat diet-induced obesity model mouse. * p <0.01 vs Student's t- test
FIG. 4 shows the blood glucose lowering effect of the AK (+) and AK (-) strains in the hyperglycemic model mice induced by the high fat diet system as a result of confirming the fasting glucose change (A) and fasting glucose change (B). * p <0.01 vs Student's t- test
FIG. 5 shows the results of confirming the glucose tolerance (A) and AUC (B) effects of improving the glucose tolerance by administration of AK (+) and AK (-) strains in a mouse model of glucose tolerance induced by high fat diet. AUC: area under curve, * p <0.01 vs Student's t- test
FIG. 6 shows the results of confirming the glucose tolerance (A) and blood glucose change (B) effects on the improvement of the glucose tolerance by administration of AK (+) and AK (-) strains in the insulin sensitivity-lowering model mice induced by high fat diet. * p <0.01 vs Student's t- test
FIG. 7 shows the results of confirming the insulin sensitivity improvement effect of the AK (+) and AK (-) strains in insulin sensitivity-lowering model mice induced by high fat diet by blood insulin concentration. HFD + AK (-): Experimental group in which AK (+) strain grown in medium containing HFD + AK (+): mucin was administered. Experimental group treated with AK (-) strain grown in mucin-free medium. * p <0.01 vs Student's t- test
8 shows the effect of improving the glucose tolerance and insulin sensitivity by the administration of AK (+) and AK (-) strains in the insulin sensitivity-lowering model mice induced by the high-fat diet method as compared with the HOMA-IR (Homeostasis The results of the model assessment of insulin resistance index, [fasting insulin (μIU / mL) fasting plasma glucose (mmol / L)] / 22.5) were obtained. HFD + AK (-): Experimental group in which AK (+) strain grown in medium containing HFD + AK (+): mucin was administered. Experimental group treated with AK (-) strain grown in mucin-free medium. * p <0.01 vs Student's t- test
9 shows photographs of changes in abdominal fat (Gonadal fat) caused by administration of AK (+) and AK (-) in a high fat diet-induced obesity-induced model mouse, . The results of the present study showed that the high-fat diet significantly inhibited the proliferation of adipocytes. HFD + AK (-): a group treated with strain AKT (+), which was grown in medium containing HFD + AK (+): -) strain.
10 shows photographs (A) of changes in subcutaneous fat (A) by administration of AK (+) and AK (-) in obese-derived model mice induced by high fat diet, And the AK (-) strain effectively inhibited adipocyte proliferation by the high-fat diet. Vehicle: Vehicle: The experimental group, the AK (+) strain, the AK (+) strain, the AK (+) strain, the AK One experimental group.
FIG. 11 shows the change of brown fat by the administration of AK (+) and AK (-) strains in obese-derived model mice induced by high fat diet through H & E staining, and AK (- It was confirmed that the decrease of brown fat was suppressed and well preserved similar to that of normal mice. HFD + AK (-): Experimental group in which AK (+) strain grown in medium containing HFD + AK (+): mucin was administered. Experimental group treated with AK (-) strain grown in mucin-free medium.

According to an aspect of the invention there is only a cyano mu shinny pillar (Akkermansia a marker cultured in the absence of mucin (mucin) medium muciniphila strain, the strain derived from the strain, a culture of the strain, a concentrate of the culture, a dried product of the culture, and an extract of the culture, for the prevention or treatment of a metabolic disease containing, as an active ingredient, at least one selected from the group consisting of To provide a pharmaceutical composition.

The strain of the present invention, Akkermansia muciniphila , was purchased from the American Type Culture Collection (ATCC) and used in the present invention (US strain Bank Deposit Number ATCC BAA-835, The same strain as Resource Bank Deposit No. DSM 22959).

In the present invention, in order to cultivate the strain of Ackmannia mucenipila, any medium may be used as long as it is a medium used for culturing a strain of Ackermansy mucinemi pylorus and free of mucin, , BHI medium, or BTTM medium can be used as long as the addition composition of the culture medium of the Ackmannia mucenipila strain used in the art is a medium with no mucin in the modified medium.

In the pharmaceutical composition for preventing or treating a metabolic disease according to an embodiment of the present invention, the metabolic disease may be selected from the group consisting of hyperlipidemia, fatty liver, obesity, diabetes, arteriosclerosis and hypertension, but is not limited thereto.

The pharmaceutical composition for preventing or treating metabolic diseases of the present invention may comprise 0.02 to 80% by weight, preferably 0.02 to 50% by weight, of the above-mentioned dry powder of the strain or the culture medium of the strain, based on the total weight of the composition.

The composition comprising the strain dry powder or the culture medium of the present invention may further comprise suitable carriers, excipients and diluents conventionally used in the production of pharmaceutical compositions.

The pharmaceutical dosage form of the strain dry powder or strain culture of the present invention may be used alone or in combination with other pharmaceutically active compounds as well as in a suitable combination.

The composition containing the strain dry powder or the culture broth according to the present invention may be formulated into tablets or capsules by conventional methods such as oral, granule, tablet, capsule, suspension, emulsion, syrup, aerosol or the like, oral preparation, May be formulated in the form of injection solutions. Examples of carriers, excipients and diluents that can be contained in the composition including the strain dry powder or the culture medium of the strain include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin , Calcium phosphate, calcium silicate, cellulose, methylcellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil, etc. . In the case of formulation, a diluent or excipient such as a filler, an extender, a binder, a wetting agent, a disintegrant, or a surfactant is usually used.

The solid preparations for oral administration include tablets, pills, powders, granules, capsules and the like. These solid preparations may contain at least one excipient such as starch, calcium carbonate, , Oligosaccharide, sucrose or lactose, gelatin and the like. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used. Examples of the liquid preparation for oral use include suspensions, solutions, emulsions, and syrups. In addition to water and liquid paraffin, simple diluents commonly used, various excipients such as wetting agents, sweeteners, fragrances, preservatives and the like may be included . Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, and suppositories. Examples of the suspending agent include propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate, and the like. Examples of the suppository base include witepsol, macrogol, tween 61, cacao butter, laurin, glycerogelatin and the like.

The preferred dose of the strain dry powder or strain culture of the present invention varies depending on the condition and the weight of the patient, the degree of disease, the drug form, the administration route and the period, but can be appropriately selected by those skilled in the art. However, for the desired effect, it is preferable to administer the strain dry powder or the culture medium of the present invention at a dose of 0.0001 to 100 mg / kg, preferably 0.001 to 100 mg / kg per day. The administration may be carried out once a day or divided into several times. The dose is not intended to limit the scope of the invention in any way.

The strain dry powder or strain culture of the present invention can be administered to mammals such as rats, mice, livestock, humans, and the like in various routes. All modes of administration may be expected, for example, by oral, rectal or intravenous, intramuscular, subcutaneous or intra-dermal injection.

In addition, the present invention only markers were incubated in the absence of mucin (mucin) medium cyano mu shinny pillar (Akkermansia muciniphila strain, the strain derived from the strain, the culture of the strain, the concentrate of the culture, the dried product of the culture, and the extract of the culture, as an active ingredient, for the prevention or amelioration of a metabolic disease Health functional food composition.

In the health functional food composition for preventing or ameliorating a metabolic disease according to an embodiment of the present invention, the metabolic disease may be selected from the group consisting of hyperlipidemia, fatty liver, obesity, diabetes, arteriosclerosis and hypertension, but is not limited thereto .

The health functional food composition for preventing or ameliorating a metabolic disease according to an embodiment of the present invention may further contain nutrients, vitamins, electrolytes, flavors, colorants, enhancers, pectic acid and salts thereof, alginic acid and its salts , Organic acids, protective colloid thickeners, pH adjusting agents, stabilizers, preservatives, glycerin, alcohols, and carbonating agents used in carbonated beverages.

The strain may be added to a health functional food for the purpose of preventing or improving a metabolic disease. When the above-mentioned strain of the present invention or a culture solution thereof or the like is used as a health functional food composition, the strain or a culture thereof may be directly added or used together with other foods or food ingredients, . The amount of the active ingredient to be mixed can be suitably determined according to the intended use (prevention, health or therapeutic treatment).

The food (or the health functional food) of the present invention may further comprise ingredients that are conventionally added at the time of food production and which are pharmaceutically acceptable. For example, in the case of beverage preparation, one or more components may be further contained in citric acid, liquid fructose, sugar, glucose, acetic acid, malic acid, juice, etc. in addition to the strain of the present invention.

The amount of the food (or health functional food) according to the present invention that can be included as an active ingredient can be appropriately selected according to the age, sex, weight, condition, and symptom of a desired human for food for preventing or ameliorating a metabolic disease, Preferably about 0.01 g to 10.0 g on an adult basis per day. By ingesting foods having such a content, prevention or amelioration of a metabolic disease can be obtained.

In addition, the present invention only markers were incubated in the absence of mucin (mucin) medium cyano mu shinny pillar (Akkermansia muciniphila strain, the strain derived from the strain, the culture of the strain, the concentrate of the culture, the dried product of the culture, and the extract of the culture, as an active ingredient, for the prevention or amelioration of a metabolic disease A feed additive composition is provided.

The above-mentioned strains are as described above, and may be added as a feed additive composition for the purpose of preventing or improving metabolic diseases. The feed additive of the present invention corresponds to an auxiliary feed in the feed control method.

The feed additive composition of the present invention may be added to the feed and the term "feed" refers to any natural or artificial diet, meal, or the like ingredients for eating, ingesting, digesting, can do. The kind of the feed is not particularly limited, and feeds conventionally used in the art can be used. Non-limiting examples of such feeds include vegetable feeds such as cereals, muscle roots, food processing busines logistics, algae, fibers, pharmaceutical buses, oils, fats, pastes or grain by-products; Animal feeds such as proteins, inorganic substances, fats, oils, fats, oils, monocellular proteins, animal plankton or foods.

The present invention also provides a method for producing a microorganism preparation for preventing or treating a metabolic disease comprising culturing the strain.

The method for culturing the strain of the present invention can be carried out according to a method commonly used in the art.

The microorganism preparation for the prevention or treatment of metabolic diseases of the present invention can be produced using a strain of Akkermansia muciniphila cultured in a medium free of mucin as an active ingredient. The microorganism preparation for preventing or treating metabolic diseases according to the present invention may be prepared by a solution, powder, suspension, dispersion, emulsion, oil dispersion, paste, dust, injecting agent or granule, but is not limited thereto.

Hereinafter, the present invention will be described in detail with reference to examples. However, the following examples are illustrative of the present invention, and the present invention is not limited to the following examples.

Example  One. Highlander  Induced hyperlipidemia in animal models Ackermansia Mucinipila ( Akkermansia muciniphila , AK) for the treatment of hyperlipidemia

Five mice per group were fed a high fat diet for 8 weeks in male C57BL / 6 mice at 8 weeks of age to induce dietary obesity, and a vehicle (25% glycerol / PBS) was administered to the control group, In the experimental group, strains AK (+) grown on mucin-supplemented medium and AK (-) grown on mucin-free media were treated with 2.0 x 10 ⁸ CFU and 1.0 x 10 ⁷ CFU, . After fasting for 16 hours at 5 weeks after the administration, blood was collected and the concentrations of fasting cholesterol and triglyceride in the blood were measured and compared. The AK strain is an intestinal microorganism known as a standard strain, Akkermansia muciniphila (AK: American strain bank deposit number ATCC BAA-835, the strain is the same strain as the deposit number DSM 22959, Strain, purchased from ATCC and used in the present invention.

Blood samples collected from each animal after fasting for 16 hours were analyzed for blood cholesterol and triglyceride levels by fasting blood chemistry analyzer. In the control group, fasting blood cholesterol and triglyceride (191 ± 12 mg / dl) and 198 ± 12 mg / dl (183 ± 6 mg / dl) and AK (- And 156 ± 9 mg / dl and 145 ± 6 mg / dl, respectively, in the experimental group, which showed that blood cholesterol and triglyceride tended to be lower than the control group according to the AK strain administration. Between the AK (+) and AK (-) strains, blood cholesterol and neutral lipids were significantly decreased in the experimental group treated with AK (-) strain, and the effect of improving hyperlipemia was more evident ( p <0.01, Student's t- test) (Fig. 1).

These results show that the AK (-) strain cultured under mucin-free conditions is more effective in improving hyperlipidemia induced by the high-fat diet than the AK (+) strain grown in the medium containing mucin (Fig. 1).

Example  2. Highland and With alcohol  Induced fatty liver model Ackermansia  Mucinipila ( Akkermansia muciniphila , AK) by oral administration

The animals were fed a high fat diet for 8 weeks in male C57BL / 6 mice at 8 weeks of age. Five mice were fed diets for dietary obesity. Animals were continuously fed high fat diets and vehicle (25% glycerol / PBS) In the experimental group, strains AK (-) grown on mucin-supplemented medium and strains AK (-) grown on mucin-free medium were cultured in 2.0 × 10 ⁸ CFU and 1.0 × 10 ⁷ CFU for 1 week Liver transplantation was performed at 5 weeks after the administration for 16 hours. Liver tissues were collected and frozen sections were prepared and oil red-O (ORD) staining was performed. Liver tissue was directly measured and measured for the concentration of triglyceride in liver tissue using a triglyceride measurement kit.

As a result of analysis of fatty liver, there was no difference between red (A) and fatty acid (A) in the group treated with AK (+) strain compared to the control group. However, ( P <0.01, Student's t- test) ( p <0.01). In the analysis of triglyceride content of liver, it was confirmed that AK 2).

From these results, we could not observe fatty liver inhibition in AK (+) strain cultured in mucin-containing culture medium, but AK (-) strain grown in mucin-free culture medium induced fatty liver induced by high fat diet (FIG. 2).

Example  3. Highlander  Induced obesity in animal models Ackermansia  Mucinipila ( Akkermansia muciniphila , AK) for the treatment of obesity

The animals were fed a high fat diet for 8 weeks in male C57BL / 6 mice at 8 weeks of age. Five mice were fed diets for dietary obesity. Animals were continuously fed high fat diets and vehicle (25% glycerol / PBS) In the experimental group, strains AK (-) grown on mucin-supplemented medium and strain AK (-) grown on mucin-free medium were cultured in 2.0 × 10 ⁸ CFU and 1.0 × 10 ⁷ CFU for 1 week The mice were weighed at 0 and 4 weeks, and the body weight gain was calculated.

Both the AK (+) strain treated with the AK strain cultured in the mucin-supplemented medium and the AK (-) treated strain, which was cultured in the mucin-free medium, The body weight gain of the control group was 115 ± 5.9% in the AK (+) group and 109 ± 2.0% in the AK (-) group compared to 119 ± 1.4% ( P <0.01, Student's t- test) (Fig. 3).

From these results, it was confirmed that AK strain inhibits weight gain. Especially, AK (-) strain cultured under mucin-free condition is more resistant than AK (+) strain grown in mucin- (Fig. 3). The results are shown in Fig.

Example  4. Highland method in  Induced hyperglycemic animal model Ackermansia  Mucinipila ( Akkermansia muciniphila , AK) by oral administration

The animals were fed a high fat diet for 8 weeks in male C57BL / 6 mice at 8 weeks of age. Five mice were fed diets for dietary obesity. Animals were continuously fed high fat diets and vehicle (25% glycerol / PBS) In the experimental group, strains AK (-) grown on mucin-supplemented medium and strain AK (-) grown on mucin-free medium were cultured in 2.0 × 10 ⁸ CFU and 1.0 × 10 ⁷ CFU for 1 week And 4 weeks after the administration, non-fasting blood glucose levels without dietary restriction and fasting blood glucose levels fasted for 16 hours were directly measured from the blood using a glucometer and compared and analyzed .

After 4 weeks of administration of the AK strain, the fasting blood glucose levels in the unrestricted state and the fasted state for 16 hours were 206 ± 6 mg / dl and 105 ± 6 mg / dl in the control group, respectively. (+) Strains were found to be 206 ± 6 mg / dl and 113 ± 8 mg / dl, whereas 166 ± 8 mg / dl and 88 ± 3 mg in the AK (-) treated group, which was cultured in mucin- / dl. In comparison with the control group, no significant difference was observed in the group treated with AK (+) compared with the control group. However, in the group treated with AK (-), blood glucose and fasting blood glucose ( P < 0.01, Student's t- test) (Fig. 4). It was observed that the blood glucose lowering effect was superior to the AK (+) strain cultured in the mucin-supplemented medium.

These results demonstrate that the AK (-) strain cultured under mucin-free conditions is highly effective in inhibiting high-glucose-induced hyperglycemia, which was not observed in AK (+) strains grown in mucin-containing medium (Fig. 4).

Example  5. The highland method in  Induced impaired glucose tolerance in animal models Ackermansia  Mucinipila ( Akkermansia muciniphila , AK) by oral administration

The animals were fed a high fat diet for 8 weeks in male C57BL / 6 mice at 8 weeks of age. Five mice were fed diets for dietary obesity. Animals were continuously fed high fat diets and vehicle (25% glycerol / PBS) In the experimental group, strains AK (-) grown on mucin-supplemented medium and strain AK (-) grown on mucin-free medium were cultured in 2.0 × 10 ⁸ CFU and 1.0 × 10 ⁷ CFU for 1 week Glucose tolerance test, which was performed at 4, 6, and 12 hours after glucose administration (2 g / kg), and glucose tolerance was measured at 0, 30, 60, test was used to compare the improvement of IGT.

In order to confirm the improvement of impaired glucose tolerance induced by high fat diet, 4 (4) weeks of AK (+) and AK (-) were injected and glucose was injected into the peritoneal cavity, (+) Strain was lower than that of the control group at 30 and 60 minutes after glucose administration, whereas the AK (-) strain group showed a decrease of glucose uptake to 120 mg / ( P <0.01, Student's t- test) (Fig. 5A) in both groups at 30, 60, 90 and 120 minutes. The area under curve (AUC) was measured by measuring the area of the area between the line graph and the X-axis, which was obtained by measuring the blood glucose level at the time of administration of glucose. As a result, And the effect of improving the glucose tolerance was very clear (FIG. 5B).

These results indicate that the AK (-) strain cultured under mucin-free conditions is superior to the AK (+) strain grown in the mucin-containing medium, (Fig. 5).

Example  6. Highland method in  Reduced insulin sensitivity in animal models Ackermansia  Mucinipila ( Akkermansia muciniphila , AK) by oral administration of insulin sensitizer

The animals were fed a high fat diet for 8 weeks in male C57BL / 6 mice at 8 weeks of age. Five mice were fed diets for dietary obesity. Animals were continuously fed high fat diets and vehicle (25% glycerol / PBS) In the experimental group, strains AK (-) grown on mucin-supplemented medium and strain AK (-) grown on mucin-free medium were cultured in 2.0 × 10 ⁸ CFU and 1.0 × 10 ⁷ CFU for 1 week The insulin tolerance test, which measures insulin resistance and blood glucose levels at 0, 30, 60, 90 and 120 minutes, was used to improve insulin sensitivity. The effect was compared and analyzed. In addition, fasting blood glucose and insulin concentrations of mice in each group were measured after fasting for 16 hours at 4 weeks of administration, and blood samples were collected. Based on the results, HOMA-IR (HOMA-IR , Fasting insulin (μIU / mL) fasting plasma glucose (mmol / L)] / 22.5) were calculated and compared.

In order to confirm the improvement of low insulin sensitivity induced by high fat diet, AK (+) and AK (-) strains were administered for 4 weeks, fasted for 4 hours and insulin was administered. As a result, in the control group, 60% of the insulin was reduced to 60% of the blood glucose level at 60 minutes, and the blood glucose level was restored to 85% at 120 minutes. However, AK (+) and AK In the experimental group, it was significantly decreased to about 40% of 0 minute until 90 minutes after administration, but the AT (+) strain group and AK (-) strain group recovered to the level of 60% ( p < 0.01, Student's t- test) (Fig. 6). Between the AK (+) and AK (-) strains, the experimental group treated with the strain AK (-) showed a more pronounced effect for improving the insulin sensitivity reduction and maintained for a long time (FIG. 6). The fasting plasma insulin concentration (1.1 ± 0.1 ng / ml) was the highest in the control group and 0.7 ± 0.1 ng / ml and 0.6 ± 0.1 ng / ml in the AK (+) and AK (Fig. 7). As shown in Fig. In addition, the HOMA-IR levels were lower in the AK group than in the control group (9.8 ± 1.9), especially in the AK (-) strain (4.9 ± 0.7) (6.3 ± 1.6), and it was confirmed that the AK strain administration improved the insulin resistance in the high fat diet-induced obesity and hyperglycemic mouse model. The AK (-) strain grown in the mucin-free medium (Fig. 8).

These results demonstrate that the AK (-) strain cultured under mucin-free conditions is more effective in improving insulin sensitivity deterioration induced by the high-fat diet than the AK (+) strain grown in mucin-containing medium (Fig. 6, Fig. 7 and Fig. 8).

Example  7: Highlander  In an induced obesity animal model Ackermansia Mucinipila  ( Akkermansia muciniphila , AK) by the size of white adipose tissue and pathological changes of brown adipose tissue

The animals were fed a high fat diet for 8 weeks in male C57BL / 6 mice at 8 weeks of age. Five mice were fed diets for dietary obesity. Animals were continuously fed high fat diets and vehicle (25% glycerol / PBS) In the experimental group, strains AK (+) grown on mucin-supplemented medium and strains AK (-) grown on mucin-free media were treated with 2.0 × 10 ⁸ CFU and 1.0 × 10 ⁷ CFU for 4 weeks (Abdominal fat and subcutaneous fat) and brown adipose tissue were collected, and H & E staining was performed and the size of adipocytes was observed. In the case of white adipose tissue, the size of each adipocyte was calculated using an image analysis program, and the distribution of adipocytes according to the size was calculated Respectively. As a result of analysis, adipose tissue with abdominal fat and subcutaneous adipose tissue size of ≥2500 ㎛ ² was significantly lower in the test group treated with AK (-) strain grown in mucin-free media than in the control group, that exhibited a small 2500 ㎛ ² ≤ the adipose tissue are highly distribution (Figs. 9 and 10). In addition, the observation of brown adipose tissue through H & E staining revealed that the high fat diet method (HFD), which did not treat the strain, was large in size due to the whitening of most of the adipose tissue. However, In the experimental group (HFD + AK (-)) treated with the AK (-) strain grown in the mucin-free medium, the AK (+) strain cultivated in the control and mucin- It was confirmed that brown adipocytes were more conserved than the experimental group (HFD + AK (+)) (FIG. 11). From these results, it was confirmed that the AK (-) strain cultured in mucin-free medium effectively inhibited the proliferation of adipocytes induced by the high fat diet.

Claims (7)

The deposit number of which is DSM 22959, a strain of Akkermansia muciniphila cultivated in a mucin-free medium, the strain derived from the strain, a culture of the strain, a concentrate of the culture, a dried product of the culture, Wherein the composition comprises at least one selected from the group consisting of an extract of the above culture as an active ingredient, and a pharmaceutical composition for preventing or treating hyperlipidemia, fatty liver, obesity, diabetes or arteriosclerosis. delete The pharmaceutical composition for preventing or treating hyperlipidemia, fatty liver, obesity, diabetes or arteriosclerosis according to claim 1, further comprising a carrier, an excipient or a diluent in addition to the active ingredient. The deposit number of which is DSM 22959, a strain of Akkermansia muciniphila cultivated in a mucin-free medium, the strain derived from the strain, a culture of the strain, a concentrate of the culture, a dried product of the culture, Wherein the active ingredient is at least one selected from the group consisting of an extract of the above-mentioned culture. delete The method according to claim 4, further comprising, in addition to the active ingredient, a nutrient, a vitamin, an electrolyte, a flavoring agent, a coloring agent, a enhancer, a pectic acid and a salt thereof, alginic acid and a salt thereof, an organic acid, a protective colloid thickener, Wherein the composition further contains at least one selected from the group consisting of preservatives, glycerin, alcohols and carbonating agents used in carbonated beverages. The deposit number of which is DSM 22959, a strain of Akkermansia muciniphila cultivated in a mucin-free medium, the strain derived from the strain, a culture of the strain, a concentrate of the culture, a dried product of the culture, Wherein the active ingredient is at least one selected from the group consisting of an extract of the above-mentioned culture, an active ingredient,

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