RU2777068C1 - Strain of akkermansia muciniphila and application thereof - Google Patents

Abstract

FIELD: pharmaceuticals.

SUBSTANCE: invention relates to an application of protein B2UM07 containing an amino acid sequence with SEQ ID NO: 2 for suppressing appetite in a subject with reduced hormone GLP-1 and for preventing or treating metabolic disorders associated with hormone GLP-1. Also proposed are pharmaceutical compositions and healthy nutrition products containing said protein B2UM07 as an active substance.

EFFECT: protein B2UM07 provides a high capacity for GLP-1 induction, a capacity for maintaining the glucose homeostasis in the body, and an impact on weight loss.

20 cl, 12 dwg, 11 ex

Description

[TECHNICAL FIELD]

Cross-reference to related application(s)

This application claims benefit on the basis of Korean Patent Application No. 10-2018-0121137, filed on October 11, 2018, and Korean Patent Application No. 10-2019-0125670, filed on October 10, 2019, with the Intellectual Property Office of the Republic of Korea, each of which is incorporated herein by reference.

The present invention relates to a strain of Akkermansia muciniphila SNUG-61027 (accession number KCTC 13530BP) effective in appetite suppression and prevention, treatment, relief, improvement and treatment of metabolic disorders, as well as to the use of this strain.

[PRIOR ART]

Obesity is a condition in which an excessive amount of fat accumulates in the human body as a result of changes in eating habits, such as a high-calorie diet, lack of exercise, etc.; Obesity is associated with the development of type 2 diabetes, cardiovascular disease, liver disease, and various cancers, and therefore is of great clinical importance. On the other hand, intestinal microorganisms are known to be closely associated with metabolic disorders such as obesity and diabetes, in particular, an increase in the Akkermansia muciniphila strain in the intestines of mice treated with the antidiabetic drug metformin was observed, as well as an improvement in glucose homeostasis when this strain was administered to mice treated with high-fat diet, this strain is attracting attention as a potential anti-obesity agent and represents a new paradigm in anti-obesity drug research.

Various studies have been conducted to elucidate the mechanism of action of the Akkermansia muciniphila strain against obesity, the effectiveness of which against obesity has been tested in intestinal microorganisms, close to 10 times the total number of human cells, however, in the course of conventional studies, the focus has been on obesity-related indicators, such as weight loss, chronic metabolic inflammation, repairing damaged barriers, or improving blood lipid levels.

However, the anti-obesity action has different mechanisms in addition to the aforementioned indicator, and in particular, it has recently been reported that brown fat interacts with intestinal microorganisms in connection with a homeostasis mechanism that maintains body temperature. Adipose tissue divides into white adipose tissue, which stores energy in the form of triglycerides, and brown adipose tissue, which releases energy in the form of heat; brown adipose tissue leads to energy uptake through tissue-specific factors UCP-1 and thus acts to regulate glucose homeostasis and increase insulin sensitivity.

Meanwhile, glucagon-like peptide (GLP-1), an appetite-regulating hormone, is secreted in the ileum during meals, which increases satiety, regulates appetite, and induces the release of insulin from the pancreas, thereby regulating blood glucose levels.

GLP-1 is secreted from L cells, which are a type of intestinal endocrine cells found in the ileum and colon.

GLP-1 is known to have a therapeutic effect in diabetes, obesity, cardiovascular and cerebrovascular diseases, and nerve cell inflammation (Salcedo I et al., Neuroprotective and neurotrophic actions on glucagon-like peptide-1 (GLP-1) : an emerging opportunity to treat neurodegenerative and cerebrovascular disorders British Journal of Pharmacology (2012) 166, 1586-1599), therapeutic effect in atherosclerosis (Burgmaier M et al., Glucagon-like peptide-1 (GLP-1) and its split products GLP-1(9-37) and GLP-1(28-37) stabilize atherosclerotic lesions in apoe-/- mice Atherosclerosis (2013) 231, 427-435), and so on.

In addition, GLP-1 is involved in demonstrating a therapeutic effect in diabetes by stimulating glucose-dependent pancreatic insulin secretion, enhancing insulin gene expression, showing effects on pancreatic beta cell proliferation, effects on pancreatic beta cell survival, effects on glucagon secretion inhibition , reducing blood glucose levels, etc., and is involved in demonstrating a therapeutic effect in obesity by slowing down the rate of gastric emptying, suppressing appetite, increasing satiety, and reducing food intake. In addition, GLP-1 has a therapeutic effect in cardiovascular diseases by protecting cardiomyocytes from local ischemia and enhancing cardiac function in patients at risk of heart attack (Sokos, G.G. et al., Glucagon-like peptide-1 infusion improves left ventricular ejection fraction and functional status in patients with chronic heart failure J. Card Fail (2006) 12: 694-699., Ban, K., et al., Cardioprotective and vasodilatory actions of glucagon-like peptide-1 receptor are mediated through both glucagon-like peptide-1 receptor-dependent and -independent pathways Circulation (2008) 117: 2340-2350.

GLP-1 secretion is known to be stimulated by the activation of TGR5 and GPR119, which are a type of G-protein coupled receptor (GPCR) (Reimann, F., et al., Glucose sensing in L cells: a primary cell study. Cell Metab. ( 2008) 8: 532-539; Lauffer, L. M., et al., GPR119 is essential for oleoylethanolamide-induced glucagon-like peptide-1 secretion from the intestinal enteroendocrine L-cell. Diabetes (2009) 58:1058-1066), or activation of α-gustducin (Jang, H.J., et al., 2007. Gut expressed gustducin and taste receptors regulate secretion of glucagon-like peptide-1. Proceeding of the National Academy of Science 104, 1506915074.). In particular, it is known that activation of the G-protein-coupled receptor (GPCR) TGR5 (GPR131), expressed in brown adipose tissue and muscle, increases energy expenditure and thus exhibits a therapeutic effect in obesity, which is associated with positive dynamics in the disease. liver (Lieu T et al., GPBA: A G protein-coupled receptor for bile acids and an emerging therapeutic target for disorders of digestion and sensation. British Journal of Pharmacology (2013), in press), suppression of arteriosclerosis has also been reported (Pols TWH et al., TGR5 activation inhibits atherosclerosis by reducing macrophage inflammation Cell Metabolism (2011) 14, 747).

In addition, triglycerides, which are excessively accumulated in obese patients, are found not only in adipose tissues, but also in the liver or muscles, which leads to insulin resistance. Therefore, the expenditure of excessively accumulated triglycerides can be the prevention and treatment of obesity as a major disease and associated metabolic disorders. Adipocytes are broadly divided into white adipocytes, brown adipocytes, and beige adipocytes. White adipocytes are found in large triglyceride fat globules, mostly found in the abdominal cavity, and are known to play a negative role in health. It has been reported that brown adipocytes contain more mitochondria and small fat globules compared to white adipocytes and can be produced while maintaining body temperature through heat generation and appropriate exercise. Increasing brown adipocytes in mice has been shown to be effective in treating obesity and metabolic disorders, resulting in weight loss and increased energy intake in high-fat diet-induced obesity. In addition, brown adipocytes express a large amount of the protein UCP-1 (uncoupling protein-1), which is known to play a critical role in the production of heat with calorie intake instead of the accumulation of calories in adipocytes. In addition to brown adipocytes, the role of beige adipocytes is important. Beige adipocytes are derived from unhealthy white adipocytes upon stimulation such as exercise or exposure to cold, white adipocyte manifestations are reduced, but they turn into brown adipocyte manifestations, resulting in an increase in UCP-1 expression. Such beige adipocytes are also known to be useful in obesity and metabolic disorders, similar to brown adipocytes in mice.

[Prior Art Literature]

1. Korea Patent No. 10-1809172

2. Korean Published Patent Application No. 10-2015-0133646

[DETAIL DISCLOSURE]

[Technical issue]

Under these circumstances, with the aim of effectively preventing and treating metabolic disorders, the present inventors have found that Akkermansia muciniphila enhances the UCP-1 factor affecting brown fat activity and induces the secretion of GLP-1 as an appetite-regulating hormone in the small intestine when using a standard strain Akkermansia muciniphila (Akk; American Type Culture Collection, accession number ATCC BAA-835), currently used in anti-obesity research, and an isolated strain of Akkermansia muciniphila SNUG-61027 (accession number: KCTC13530), isolated from the faeces of healthy Koreans.

In addition, the present inventors found that this pathway is induced depending on the host cytokine IL-6, and finally isolated a strain of Akkermansia muciniphila that promotes the induction of GLP-1 secretion in the anti-obesity mechanism, culture solution, bacterial cell, supernatant, an extract or fraction of a strain, or a target protein isolated from a strain, thereby completing the invention.

[Technical solution]

As one of the aspects, the task was solved by Akkermansia muciniphila (Akk) strain SNUG-61027 with accession number KCTC 13530BP. Below is information about the strain.

Name of the depositing organization: Korea Research Institute of Biological Sciences and Biotechnology

Access number: KCTC 13530BP

Accessed: May 25, 2018

The strain according to the present invention contains 16S rDNA, consisting of the nucleotide sequence with SEQ ID NO (identification number): 1.

According to another embodiment, the present invention provides a pharmaceutical composition for appetite suppression or the prevention, relief or treatment of metabolic disorders, containing Akkermansia muciniphila SNUG-61027 (accession number KCTC 13530BP), or its culture solution as an active substance.

By "culture solution" in the sense of this document is meant a solution of the entire medium containing the strain, its metabolite, additional nutrients, etc., obtained by culturing the strain for a certain period of time in an environment capable of providing nutrients in such a way that Akkermansia muciniphila strain SNUG-61027 (accession number KCTC 13530BP) could grow and survive in vitro, which includes all cell-free supernatants, extracts and culture fractions. The liquid obtained by removing cells from the culture solution is also called the "supernatant" (supernatant); the supernatant can be obtained by settling the culture solution for some time and separating only the upper liquid layer without the precipitated part in the lower layer, by removing bacterial cells by filtration or centrifugation of the culture solution to remove the sediment in the lower part and collecting only the liquid in the upper part.

By "bacterial cell" is meant the actual strain according to the invention, in particular the strain itself isolated and isolated from the fermented product, or the strain isolated from the culture solution obtained by culturing the isolated strain. Bacterial cells can be obtained by centrifuging the culture solution to obtain the sedimented portion in the lower layer, or keeping the culture solution for a certain period of time and then removing the liquid in the upper part, since the cells are deposited in the lower layer of the culture solution by gravity.

Further, the extract of the culture solution of Akkermansia muciniphila SNUG-61027 strain, bacterial cells or supernatant (accession number KCTC 13530BP) according to the present invention may be, but not limited to, an extract extracted with ethyl acetate (EtOAc) or ethanol (ethyl alcohol; EtOH). In addition, the fraction of the culture solution, bacterial cells or supernatant of Akkermansia muciniphila SNUG-61027 strain according to the present invention may be a fraction obtained by fractionating an ethyl acetate extract with methanol, but not limited to this. The culture solution, supernatant or extract fraction of the Akkermansia muciniphila strain (accession number KCTC 13530BP) according to the present invention can be obtained by a conventional fractionation method well known in the art, such as a chromatographic method using an anion exchange column, a size exclusion column, and the like.

By "metabolic disorder" in the sense of this document is meant one, two or more disorders in various diseases, such as impaired glucose tolerance, diabetes, fatty liver, hypertension, dyslipidemia, obesity, cardiovascular atherosclerosis, etc., caused by chronic metabolic disorders in the same person. For example, the metabolic disorder can be any of the following: glucose intolerance, diabetes, atherosclerosis, hyperlipidemia, hypercholesterolemia, fatty liver disease, cardiovascular disease, or obesity.

According to the present invention, the induction of elevated IL-6 levels, increased secretion of GLP-1, and increased activity of brown fat can have beneficial effects on metabolic disorders and prevent, alleviate, or treat metabolic disorders.

In another embodiment, the present invention provides a pharmaceutical composition for appetite suppression or prevention, normalization or treatment of metabolic disorders, containing the protein B2UM07, consisting of the amino acid sequence of SEQ ID NO: 2 as an active substance.

In the identification of the protein by LC/MS-MS analysis in the potency part of the invention, by searching for a match with a common strain in the NCBI database, the B2UM07 protein was identified and the following information was obtained.

Gene: Amuc_1631

UniProtKB-B2UM07

The name of the protein is C-terminal protease

Organism: Akkermansia muciniphila

The B2UM07 protein can be obtained from a strain of Akkermansia muciniphila, in particular the strain of Akkermansia muciniphila can be SNUG-61027 (accession number KCTC 13530BP).

In addition to the protein consisting of the amino acid sequence of SEQ ID NO: 2, other sequence variants are also considered to be within the protected scope of the present invention. Such variants include a protein consisting of an amino acid sequence or an amino acid sequence encoded by a nucleotide sequence, the functional characteristics of which are similar to the amino acid sequence of SEQ ID NO: 2 despite a change in the nucleotide or amino acid sequence. In particular, the protein of the present invention may comprise an amino acid sequence having an affinity for the amino acid sequence of SEQ ID NO: 2 of at least 70%, preferably at least 80%, more preferably at least 90%, and most preferably at least 95%.

In addition, the present invention provides a gene encoding the B2UM07 protein. The gene according to the present invention includes, respectively, both genomic DNA and cDNA encoding the B2UM07 protein. Preferably, the gene may contain a protein-encoding nucleotide sequence of SEQ ID NO: 2.

In addition, various variants of the nucleotide sequence are within the scope of the present invention. In particular, gene variants may comprise a nucleotide sequence having an affinity for the nucleotide sequence of SEQ ID NO: 2 of at least 60%, preferably at least 70%, more preferably at least 80%, and most preferably at least 90%.

Another aspect of the present invention provides a recombinant vector containing a gene encoding the B2UM07 protein according to the present invention. The term "recombinant" as used herein refers to a cell in which the cell replicates a heterologous nucleic acid, expresses a nucleic acid, or expresses a peptide, a heterologous peptide, or a protein encoded by a heterologous nucleic acid. The recombinant cell may express genes or segments of the cell's genes that are not naturally present in the cells, in either sense or antisense form. Further, the recombinant cell may express genes naturally present in cells, but the gene is modified and reintroduced into the cell by artificial means.

By "vector" is meant fragment(s) of DNA or nucleic acid molecules delivered into the cell. The vector can replicate DNA and reproduce independently in host cells. Further, the present invention provides a transformant transformed with a recombinant vector. As a method for transforming a vector into Escherichia coli (E. coli), a method widely known in the art, such as using a competent cell using CaCl ₂ buffer, electroporation, and heat shock, can be used. As a culture method for the transformed E. coli, a commonly used E. coli culture method can be used.

The pharmaceutical composition according to this invention can be administered to mammals, including humans, in various ways. By "administration" in the sense of this document is meant the introduction of a predetermined substance into the body of an individual using any suitable method, and the method of administration can be any of the methods commonly used in the prior art, for example, the substance can be administered orally, externally, intravenously, intramuscularly, subcutaneously, etc., with oral administration being preferred. The pharmaceutical composition according to the invention, once prepared, can be used in preparations for oral administration, for example, in powders, granules, tablets, capsules, suspensions, emulsions or syrups, or in preparations involving a different route of administration, for example, ointments, aerosols, transdermal preparations, suppositories or sterile solutions for injection, in accordance with the usual method. The pharmaceutical composition according to this invention may further contain pharmaceutically acceptable and physiologically acceptable adjuvants such as carriers, excipients, diluents, etc.

Carriers, excipients and diluents that may be contained in the pharmaceutical composition according to this invention may be lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum arabic, alginate, gelatin, calcium phosphate, silicate calcium, cellulose, methylcellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil. A diluting agent or excipient may be used in the formulation, such as commonly used fillers, emulsifiers, binders, wetting agents, disintegrators and surfactants.

In one of the specific embodiments of this invention, involving the use of a pharmaceutical composition for humans, the pharmaceutical composition can be administered separately, but taking into account the method of administration and standard pharmaceutical practice, in general, it can be administered in admixture with the selected pharmaceutical carrier. For example, a composition containing the Akkermansia muciniphila strain of the present invention may be administered orally, buccally or sublingually in the form of a tablet containing starch or lactose, in the form of a capsule containing only the active ingredient of the present invention or an excipient in addition to the active ingredient, or in the form of an elixir or suspension containing a chemical agent to impart flavor or color.

The dosage of the pharmaceutical composition according to the present invention may depend on the age, weight and sex of the patient, the dosage form, the condition of the patient and the severity of the disease, and may be administered one or more times a day, in separate doses at certain intervals, as directed by the physician or pharmacist. . For example, the daily dose may be from 0.1 to 500 mg/kg, preferably from 0.5 to 300 mg/kg, based on the content of the active substance. The above doses are averages and may be increased or decreased depending on the individual.

In another embodiment, the present invention provides a functional health food for appetite suppression or normalization or relief of metabolic disorders, containing Akkermansia muciniphila strain SNUG-61027 (accession number KCTC 13530BP) or a culture solution, supernatant, extract or fraction of this strain as an active substance .

The metabolic disorder may be glucose intolerance, diabetes, atherosclerosis, hyperlipidemia, hypercholesterolemia, fatty liver, cardiovascular disease, or obesity.

According to the present invention, an increased level of IL-6, an increased secretion of GLP-1, and an increased activity of brown fat can be induced, which can have a beneficial effect on metabolic disorders, as well as provide relief or treatment of metabolic disorders.

In addition, the present invention proposes a functional healthy food for appetite suppression, normalization or alleviation of a condition with metabolic disorders, containing as an active substance the B2UM07 protein, consisting of the amino acid sequence with SEQ ID NO: 2.

The metabolic disorder may be glucose intolerance, diabetes, atherosclerosis, hyperlipidemia, hypercholesterolemia, fatty liver, cardiovascular disease, or obesity.

The B2UM07 protein can be obtained from a strain of Akkermansia muciniphila, in particular, the strain of Akkermansia muciniphila can be SNUG-61027 (accession number KCTC 13530BP), details of which are given above.

Functional healthy food can be a variety of drinks, fermented milk, nutritional supplements, etc.

The content of the Akkermansia muciniphila strain as an active ingredient in a functional healthy diet is not specifically limited, and may be appropriately changed depending on the form of the food, the intended use, etc., for example, it can be added to food in an amount of 0.01% up to 15% of the total weight, and the health drink composition may be added in an amount of 0.02 g to 10 g, preferably 0.3 g to 1 g per 100 ml.

For health food beverages according to this invention, there are no special restrictions on the composition of the liquid, except that the composition must include a strain of Akkermansia muciniphila as a mandatory ingredient in the indicated ratio, and various flavors or natural carbohydrates may be present as additional ingredients. like regular drinks.

Examples of the aforementioned natural carbohydrates can be conventional saccharides such as monosaccharides such as glucose, fructose and the like, disaccharides such as maltose, sucrose and the like, and polysaccharides such as dextrin, cyclodextrin and the like, and sugar alcohols such as xylitol, sorbitol, erythritol, etc. As flavors, in addition to those mentioned above, you can use natural flavors (thaumatin, stevia extract (for example, rebaudoside A, glycyrrhizin, etc.) and synthetic flavors (saccharin , aspartame, etc.) The content of natural carbohydrate in 100 ml of the composition according to this invention is usually about 1 to 20 g, preferably about 5 to 12 g.

In addition to the above, the functional health food according to the present invention may contain various nutrients, vitamins, minerals (electrolyte), flavors such as synthetic flavors and natural flavors, colorants and flavor enhancers (cheese, chocolate, etc.), pectin acid and its salts. , alginic acid and its salts, organic acids, protective colloidal thickeners, pH adjusting agents, stabilizers, preservatives, glycerol, alcohol, carbonizing agents used in carbonated beverages, etc.

In addition, the functional health food according to the present invention may include fruits, as used in the preparation of natural fruit juices and fruit juice drinks, as well as herbal drinks. These components may be used singly or in combination. Although the proportion of these additives is not very important, it is usually selected from the range from 0 to about 20 parts by weight per 100 parts by weight of the functional health food according to this invention.

In another embodiment, the use of Akkermansia muciniphila strain SNUG-61027 (accession number KCTC 13530BP) or a culture solution, supernatant, extract or fraction of this strain is provided for the suppression of appetite or the prevention, treatment, normalization or improvement of metabolic disorders.

The Akkermansia muciniphila strain SNUG-61027 (accession number KCTC 13530BP) used to practice this invention may contain a 16S rDNA consisting of the nucleotide sequence of SEQ ID NO: 1.

The metabolic disorder to which the present invention applies may be glucose intolerance, diabetes, atherosclerosis, hyperlipidemia, hypercholesterolemia, fatty liver, cardiovascular disease, or obesity.

In addition, in another embodiment of the present invention, the use of the B2UM07 protein, consisting of the amino acid sequence of SEQ ID NO: 2, is provided for the suppression of appetite or the prevention, treatment, improvement or mitigation of metabolic diseases.

The B2UM07 protein used to practice this invention can be obtained from a strain of Akkermansia muciniphila.

The strain of Akkermansia muciniphila used to practice this invention may be strain SNUG-61027 (accession number KCTC 13530BP).

The present invention provides a method for suppressing appetite or preventing, treating, normalizing or alleviating a metabolic disorder, comprising the step of treatment with Akkermansia muciniphila SNUG-61027 (accession number KCTC 13530BP) or a culture solution, supernatant, extract or fraction of this strain.

Akkermansia muciniphila strain SNUG-61027 (accession number KCTC 13530BP) used in a method for suppressing appetite or preventing, treating, normalizing or alleviating metabolic disorders may contain 16S rDNA consisting of the nucleotide sequence of SEQ ID NO: 1.

The metabolic disease to which the method of this invention is applied may be glucose intolerance, diabetes, atherosclerosis, hyperlipidemia, hypercholesterolemia, fatty liver, cardiovascular disease, or obesity.

The present invention provides a method for suppressing appetite or preventing, treating, normalizing or alleviating metabolic disorders, comprising the step of treating with the B2UM07 protein consisting of the amino acid sequence of SEQ ID NO: 2.

The B2UM07 protein used in the method of this invention can be obtained from a strain of Akkermansia muciniphila.

The strain of Akkermansia muciniphila used in the method of this invention may be strain SNUG-61027 (Accession number KCTC 13530BP)

[BENEFICIAL EFFECTS OF THE INVENTION]

The present invention confirmed the effect of brown fat activation and the ability to secrete the appetite regulating hormone GLP-1, in addition to weight loss and regulation of glucose homeostasis among the anti-obesity effects of Akkermansia muciniphila, and confirmed that these effects depend on the specific cytokine IL-6 in the host body . In addition, the present invention identified a new strain of Akkermansia muciniphila SNUG-61027 (accession number KCTC 13530BP) with a significantly increased ability to induce GLP-1, and confirmed that the B2UM07(P9) protein isolated from the culture solution of Akkermansia muciniphila strain showed a high ability to induction of GLP-1, the ability to maintain glucose homeostasis in the body and the effect on weight loss. Thus, the new strain of Akkermansia muciniphila and the B2UM07 protein may be useful for appetite suppression, treatment or prevention of metabolic disorders.

[BRIEF DESCRIPTION OF THE DRAWINGS]

FIG. 1 shows the results of experiments to improve the effects on weight of liver and brown fat after administration of the Akkermansia muciniphila (Akk) strain in a mouse model fed a high fat diet.

FIG. 2 shows the results of qPCR experiments confirming an increase in the expression of UCP-1 and markers associated with brown fat using the Akkermansia muciniphila strain.

FIG. 3 shows the results of qPCR experiments confirming an increase in IL-6 and GLP-1 cytokine levels in the small intestine with Akkermansia muciniphila strain.

FIG. 4 shows experimental results confirming that Akkermansia muciniphila strain-mediated brown fat expression and thermogenesis are dependent on the cytokine IL-6.

FIG. 5 shows the results of in vitro experiments (ELISA) confirming that Akkermansia muciniphila GLP-1 secretion is due to a bacterial secretion.

FIG. 6 shows the results of in vitro experiments confirming that GLP-1 secretion by Akkermansia muciniphila is caused by elements other than short chain fatty acid (SCFA).

FIG. 7A shows the results of in vitro experiments to monitor GLP-1 inducibility for exclusion fractions of Akkermansia muciniphila, and FIG. 7B shows the results of experiments monitoring GLP-1 secretion by GLP-1 inducing fractions (100K, 300K) after proteinase K (PK) treatment.

FIG. 8 shows the results of fractionation experiments on an anion exchange column and size exclusion column inducing GLP-1 fractions (100K) for GLP-1 inducing fractions.

FIG. 9 shows the result of a qualitative protein analysis of GLP-1 inducing fractions (100K, m2-m4, G17-G20) of Akkermansia muciniphila using LC/MS-MS.

FIG. 10 shows the results of GLP-1 inducibility monitoring experiments with purified candidate proteins (SDS-PAGE gel).

FIG. 11 shows the result of the experiment, confirming the ability to maintain glucose homeostasis in the body with intraperitoneal administration of the target protein.

FIG. 12 shows the result of an experiment confirming the ability to maintain glucose homeostasis in the body when the target protein is orally administered.

[DETAIL DISCLOSURE OF EMBODIMENTS]

The present invention is described below with reference to examples. However, these examples are for illustrative purposes only and do not limit the scope of the present invention.

Example 1 Analysis of Liver Weight Loss and Brown Fat Weight Loss Effect After Administration of Akkermansia muciniphila (Akk) Strain in a High Fat Feeding Mouse Model

Akkermansia muciniphila strain (ATCC BAA-835, Akk) was obtained by anaerobic cultivation for 72 hours in brain heart extract (BHI) solid medium supplemented with 0.5% mucin, and a stock was created. The strain was administered daily orally to 6-week-old male C57BL/6 mice at a concentration of 4 x 10 ⁸ CFU/200 μl/mouse along with a high fat (60% fat) diet (HV+Akk, n=8 per group). The control groups were a low-fat (10% fat) (LF) diet and a high-fat (HF) only diet. Comparison between the control groups and the group in which the test strain was administered was made after 14 weeks. After 16 hours without food, adipose tissue and liver tissue were sampled and tissue weight was measured (FIG. 1A).

As a result, it was confirmed that there was no significant change in the weight of inguinal white adipose tissue (igWAT) and epididymal white adipose tissue (EpiWAT) in the HP+Akk group compared to the HP group, but the weight of interscapular brown adipose tissue (iBAT) was significantly reduced. In addition, when conducting a correlation analysis of brown adipose tissue (iBAT) and hepatic tissue mass with body weight FIG. 1B) it was confirmed that the weight of brown adipose tissue and liver tissue were significantly proportional to body weight, thus, the reduction in the weight of brown fat and liver showed possible target tissues for Akkermansia muciniphila.

In addition, when comparing fat size in brown adipose tissue and liver tissue by group by hematoxylin-eosin (HE) staining, it was noted that the size of adipocytes in brown adipose tissue and liver tissue were significantly reduced in the Akkermansia muciniphila group compared to the control group ( FIG 1C and D).

Thus, as a result of the experiment, it was confirmed that Akkermansia muciniphila contributes to the reduction of brown adipose tissue weight, adipocyte size, and hepatic tissue weight (FIG. 1A~1D).

Example 2 Increased Expression of UCP-1 and Brown Fat Markers with Akkermansia muciniphila

Brown adipose tissue (iBAT) was subjected to immunohistochemical staining (IHC), after which the levels of uncoupling protein (UCP-1), which is a marker of brown fat activation, were compared between groups (FIG. 2A). After tissue RNA isolation and cDNA synthesis, expression of the UCP-1 gene was confirmed by qPCR, as well as brown fat differentiation markers (CIDEA, PRDM16, PPARGC1α, Apelin) (FIG. 2B).

As a result of the experiment, it was confirmed that brown fat markers in brown adipose tissue were significantly higher in mice fed a high-fat diet and Akkermansia muciniphila, compared with the group not fed Akkermansia muciniphila, and the increase was also confirmed by staining tissues with UCP factor -1 associated with the activity of brown fat. Thus, the mechanism of brown adipose tissue induction by Akkermansia muciniphila was confirmed.

Example 3 Elevation of Cytokine IL-6 and GLP-1 in the Ileum and Colon by Akkermansia muciniphila

After RNA extraction from ileum and colon tissues and cDNA synthesis, expression levels of immune cytokine markers (TNF-α, IL-1β, IL-18, IL-6, IL-10) were compared between groups (FIG. 3A and B).

When a mouse intestinal cell line (CT26 cell) was treated with three types of Lactobacillus (KCTC2180, KCTC3112, KCTC1048), three types of Bifidobacterium (KCTC3127, KCTC3128, KCTC3352), or Akkermansia muciniphila (Akk), the ability to express the cytokine IL-6 was compared. Lipopolysaccharide (LPS) from E. coli was used as a positive control (FIG. 3C).

Related genes (gcg, pcsk1, pcsk2) were identified by qPCR to induce secretion of intestinal secreted appetite-regulating hormone, glucagon-like peptide-1 (GLP-1) in ileal tissue (FIG. 3D).

As a result of the experiment, a significant increase in the level of the cytokine IL-6 in the cells of the ileum and colon of mice with the introduction of Akkermansia muciniphila was confirmed, as well as a significant increase in the secretion of the regulatory hormone, glucagon-like peptide-1 (GLP-1) in serum (FIG. 3A - 3D) . In particular, mouse ileum cell lines showed significantly elevated levels of IL-6 with Akkermansia muciniphila compared to other strains of Lactobacillus and Bifidobacterium.

Example 4. Dependence of the manifestation of brown fat and exothermic reaction when using the Akkermansia muciniphila strain on the cytokine IL-6

An observation was made regarding the dependence of the efficiency of activation of brown fats under the action of Akkermansia muciniphila on the cytokine IL-6.

For this purpose, 6-week-old male C57BL/6 wild-type (WT) and IL-6 gene-deficient (IL-6KO) mice were fed a high-fat diet (60% fat; FF), respectively, the strain was administered daily orally into concentration 4×10 ⁸ cfu/200 μl/mouse (N=6 in the IL-6KO group, n=8 in each of the other groups). Groups fed a low-fat diet (10%; HF) or a high-fat diet (no strain) were used as controls. After 14 weeks, groups of WT mice and IL-6KO mice fed only a high fat diet and treated with strain were compared.

After 16 hours without food, brown adipose tissue was collected, RNA extraction, cDNA synthesis, and then UCP-1 expression was confirmed by qPCR (FIG. 4A). Rectal temperature was measured with a digital thermometer (TESTO925) (FIG. 4B). Brown adipose tissue skin temperature was measured with a thermal imager (FLIR) (FIGS. 4C and D).

To measure the concentration of GLP-1 in blood serum, in the morning after not eating for 5 hours, glucose was orally administered at a concentration of 2 g/kg. After 10 minutes, plasma was taken using a blood sample from the retro-orbital sinus and placed in a refrigerated tube with the addition of 1 μg/ml diprotin A (6019; Tocris) to prevent the half-life of GLP-1. After centrifugation (4000 × g, 10 min), the supernatant was frozen at -80°C. GLP-1 secretion was then determined using the GLP-1 ELISA kit (FIG. 4E).

Genes associated with induction of GLP-1 secretion (gcg, pcsk1, pcsk2) in ileum and colon tissues were evaluated by qPCR in WT mice and IL-6KO mice (FIG. 4F~H).

As a result of the experiment, expression of the brown fat-associated UCP-1 gene, increased by administration of Akkermansia muciniphila, was not increased in mice deficient in the IL-6 gene (FIG. 4A). In addition, by observing the temperature of the skin surface over the brown adipose tissue area using an infrared camera or measuring with a rectal thermometer, it was confirmed that IL-6KO mice did not show heat due to brown fat activation (FIGS. 4B~4D). Further, in contrast to WT mice, in IL-6KO mice, the serum concentration of GLP-1 was rather reduced and no change in the level of genes (gcg, pcsk1, pcsk2) inducing GLP-1 secretion was observed, therefore, it was confirmed that increased levels of GLP-1 in appetite-regulating hormone in the ileum is also dependent on IL-6 (FIG. 4E~H).

Example 5: Confirmation of GLP-1 secretion association using Akkermansia muciniphila with a substance secreted by bacteria (in vitro)

To obtain a liquid culture, the Akk strain (Akkermansia muciniphila ATCC BAA-835) or Akkermansia muciniphila SNUG-61027 was cultured in 0.5% mucin medium, then in brain heart extract (BHI) broth medium supplemented with 0.1% or 5 % fetal bovine serum (FBS) within 36 hours.

The cell line NCI-H716 (ATCC CCL-251) secreting GLP-1 was seeded into a 96-well collagen-coated plate at a concentration of 2 × 10 ⁵ cells/mL, after which, to synchronize cell exchange with glucose between cells, the cells were cultured for 2 hours in Hank's balanced salt solution (HBSS) supplemented with 0.2% bovine serum albumin (BSA). Then the bacterial pellet of Akk strain (ATCC BAA-835) or Akkermansia muciniphila SNUG-61027 (bacteria cell ratio: 1:20) or cell-free supernatant (CFS) was treated at a concentration of 10% v/v. After 2 hours, the supernatant was obtained and the level of GLP-1 secretion in the supernatant was determined using an ELISA kit (FIG. 5A). To control the concentration-dependent efficiency of GLP-1 secretion, the supernatant of strain SNUG-61027 was treated at a concentration of 10 to 100% v/v, or the supernatant of Bifidobacterium bifidum (KBL483; isolated strain obtained from Korean faeces) as a control (con), treated at a concentration of 10-100% v/v as above; after 2 hours of treatment, supernatants were obtained and GLP-1 secretion was observed in the supernatant (FIG. 5B).

The experiment showed that when a GLP-1 inducing cell line (L-cells) was treated with live bacterial cells and Akkermansia muciniphila supernatant, GLP-1 was not detected when treated with live bacteria, while strong GLP secretion was observed when treated with the supernatant. -1, and the level of secretion was significantly increased when treated with strain SNUG 61027 compared to ATCC BAA-835 (FIG. 5A). Further, upon treatment with the culture supernatant of strain SNUG-61027, the secretion level of GLP-1 increased in a dose dependent manner (FIG. 5B).

Example 6 Confirmation of the association of GLP-1 secretion of Akkermansia muciniphila with factors other than short chain fatty acids (in vitro)

For the analysis of short chain fatty acids (SCFA) secreted by Akkermansia muciniphila, the production of representative short chain fatty acids, acetate, propionate and butyrate, was monitored by GC-MS (FIG. 6A). Two hours after treatment with acetate, propionate (1 mm, 10 mm) and strain culture supernatant (100% v/v), the level of GLP-1 secretion was monitored (FIG. 6B).

As a result of the experiment, it was confirmed that Akkermansia muciniphila secretes acetate and propionate (FIG. 6A). However, the amount of GLP-1 induced by acetate and propionate was significantly lower than the amount of GLP-1 induced by Akkermansia muciniphila culture supernatant (FIG. 6B). Thus, another element other than acetate and propionate is involved in the production of GLP-1 induced by Akkermansia muciniphila.

Example 7 Fractionation and Identification of the GLP-1 Inducing Fraction (100K) Using Size Exclusion Filter, Anion Exchange Column, and Size Exclusion Column

To isolate the active substance in the culture solution, fractions were obtained using size exclusion filters. After concentration, monitoring of GLP-1 induction confirmed the achievement of a high level of secretion of GLP-1 with a fraction of 100-300 kDa. In addition, to remove protein from effective fractions (100K~300K, 30K~100K), proteinase K (PK) at a concentration of 100 µg/ml was treated at 55°C for 1 hour, followed by inactivation at 90°C for 10 minutes. , after which the secretion of GLP-1 was measured. As a result, it was confirmed that GLP-1 secretion was not induced by the protein-deleted fraction. Thus, it was confirmed that the secretion of GLP-1 is induced by the protein in the fraction. To refraction the 100 kD ~ 300 kD (100 K) supernatant fraction of Akkermansia muciniphila, rapid resolution liquid chromatography (FPLC) was performed on proteins using a MonoQ anion exchange column (MonoQ 5/50, GE Healthcare) and an AKTAexplorer system (GE Healthcare). At the same time, 80 μg/ml of the 100K fraction was introduced and the sample was fractionated at a rate of 1 ml/min. Then each fraction was treated with L-cells and the level of secretion of GLP-1 was measured. As a result of the experiment, it was confirmed that GLP-1 was secreted at a high concentration by fractions m2-m4 (FIG. 8A).

Then fractions m2-m4 were concentrated using a 30K filter, the concentrated sample was re-subjected to the FPLC procedure using a GPC size exclusion column (GPC/SEC). For fractionation, the sample was fractionated at a rate of 3 ml/min using an AKTAexplorer HiLoad 16/600 Superdex pg system (GE Healthcare). Similarly, each fraction was treated with L-cells, and the possibility of GLP-1 secretion was confirmed. As a result of the experiment, it was confirmed that GLP-1 is secreted to a high level by fractions G17-G20 (FIG. 8B).

Example 8 Qualitative analysis of the inducible GLP-1 protein fraction (100K, m2-m4, G17-G20) of Akkermansia muciniphila using LC/MS-MS

Sample 1) 100K concentrate, sample 2) MonoQ concentrate, and sample 3) GPC concentrate obtained from Akkermansia muciniphila supernatant were qualitatively analyzed by LC/MS-MS. Proteins of bovine origin that may be present in the basal medium of the supernatant were excluded, and the number of proteins identified in each fraction was tracked.

To do this, each sample obtained through exclusion filters was subjected to a qualitative analysis, 10 types of proteins or peptides were identified in the GPC concentrate, which were considered the final concentrate, they were ranked by intensity and compared with the levels of manifestation in other fractions. LC-MS/MS (nanoflow mass spectrometer Easy-NLC 100/Q Exactive) was used for analysis. Processing was carried out using Maxquant 1.5 software, annotation was performed using the Universal Protein Resource (Uniprot) protein database for protein qualitative analysis. Of the total number of proteins and peptides, only those with a false positive rate of <1% were selected.

As a result of the experiment, 10 proteins of the G17-G20 fraction were found in sample 3, where the highest concentration of the candidate protein was expected (FIG. 9A and B).

Example 9 Confirmation of GLP-1 Induction with a Completely Purified Candidate Protein

In E. coli BL21 cells, 10 proteins were cloned and expressed from a concentrated fraction of Akkermansia muciniphila, after which each protein was purified. One (beta-galactosidase) of 10 proteins was excluded from the following steps because an efficient expression vector could not be cloned. Then the expression and purification of 9 proteins was checked using SDS-PAGE. Amuc1100, a protein derived from Akkermansia muciniphila known to have anti-obesity properties, was used as a positive control (Plovier H. et al., A removed membrane protein from Akkermansia muciniphila or the pasteurized bacterium improves metabolism in obese and diabetic mice. Nat Med. (2017) 23:107-113). Each of the isolated proteins was processed on L cells to confirm GLP-1 secretion.

For this purpose, the synthesized target protein genes were introduced into the pET-21b plasmid (Novagen) with an inducible isopropylthiogalactoside promoter, and the proteins were purified using a histidine label. This was confirmed by SDS-PAGE gel. Large-scale production and purification of proteins were achieved by transformation of Escherichia coli strain BL21 with the synthesized plasmid and culture, and the proteins were processed on the NCI-H716 cell line after concentration quantitation.

As a result of the experiment, it was confirmed that GLP-1 secretion was induced by the B2UKW8 (P1), B2URM2 (P5), and B2UM07 (P9) proteins, in particular, that the B2UM07 protein induces GLP-1 significantly more strongly than the Amuc1100 protein at 10 μg/ml and 100 μg/ml (FIG. 10C).

Example 10. Confirmation of the ability of the target protein to glucose homeostasis in the body (normal diet, intraperitoneal administration)

To confirm that the target protein improves the ability of glucose homeostasis in the body, proteins P1 (B2UKW8), P5 (B2URM2) and P9 (B2UM07) were administered intraperitoneally to mice on a normal diet for a week at a concentration of 100 μg/mouse, after which tests were performed for glucose tolerance.

To this end, 3 effective proteins (P1, P5, P9) were administered intraperitoneally to mice on a normal diet at a concentration of 100 μg/200 μl daily, and on day 7, body weight was compared with the group without protein administration (n=8/group, Fig. 11C), 14 days after administration, glucose was orally administered at a concentration of 2 g/kg, and blood glucose was measured from 15 to 120 minutes as a test for glucose tolerance (FIGS. 11A and 11B).

As a result of the experiment, it was confirmed that the group in which the P9 protein (B2UM07) was administered maintained a significantly lower blood sugar level than the other groups. It has been shown to be more effective than the Akkermansia muciniphila-derived Amuc1100 protein, which is known to promote glucose tolerance. P1 (B2UKW8) and P5 (B2URM2) showed only a trend in glucose tolerance, however, a significant weight loss was also confirmed in the case of the P9 group (FIG. 11C).

Example 11 Confirmation of Target Protein's Ability to Homeostasize Glucose in the Body (High Fat Diet, Oral Administration)

To confirm the improvement in the body's glucose homeostasis ability by the identified target protein, mice fed a high fat diet were injected with P9 protein (B2UM07) at a concentration of 100 μg/mouse for 8 weeks, after which glucose tolerance tests were performed. Blood glucose levels were measured within 15–120 minutes after oral administration of glucose (2 g/kg).

As a result of the experiment, the P9 (B2UM07) administration group showed a significant weight gain inhibition effect compared to the high fat diet group, and the effect was higher than that of the Amuc1100 administration group (FIG. 12A). In addition, it was confirmed that 30 minutes after the administration of glucose, the ability to homeostasis of glucose was significantly changed compared to the group of mice fed a high fat diet (FIGS. 12B and 12C).

--->

<110> COBIOLABS, INC.

<120> Akkermansia muciniphila strain and its use

<130>OPP20201536KR

<150> KR 2018/0121137

<151> 2018-10-11

<150> KR 2019/0125670

<151> 2019-10-10

<160> 2

<170> KoPatentIn 3.0

<210> 1

<211> 918

<212> DNA

<213> Unknown

<220>

<223> Akkermansia muciniphila SNUG-61027

<400> 1

gctaataatt ctctagtggc gcacgggtga gtaacacgtg agtaacctgc ccccgagagc 60

gggatagccc tgggaaactg ggattaatac cgcatagtat cgaaagatta aagcagcaat 120

gcgcttgggg atgggctcgc ggcctattag ttagttggtg aggtaacggc tcaccaaggc 180

gatgacgggt agccggtctg agaggatgtc cggccacact ggaactgaga cacggtccag 240

acacctacgg gtggcagcag tcgagaatca ttcacaatgg gggaaaccct gatggtgcga 300

cgccccgtgg gggaatgaag gtcttcggat tgtaaacccc tgtcatgtgg gagcaaatta 360

aaaagatagt accacaagag gaagagacgg ctaactctgt gccagcagcc gcggtaatac 420

agaggtctca agcgttgttc ggaatcactg ggcgtaaagc gtgcgtaggc tgtttcgtaa 480

gtcgtgtgtg aaaggcgcgg gctcaacccg cggacggcac atgatactgc gagactagag 540

taatggaggg ggaaccggaa ttctcggtgt agcagtgaaa tgcgtagata tcgagaggaa 600

cactcgtggc gaaagcgggt tcctggacat taactgacgc ttaggcacga aggccagggg 660

agcgaaaggg attagatacc cctgtagtcc tggcagtaaa cggtgcacgc ttggtgtgcg 720

gggaatcgac cccctgcgtg ccggaactaa cgcgttaagc gtgccgcccg ggggagtacg 780

gtcgcaagat taaaactcaa agaaattgac ggggacccgc acaagcggtg gaattatgtg 840

gcttaattcg atgcaacgcg aagaacctta cctgggcttg acatgtaatg aacaacatgt 900

gaaagcatgc gactcttc 918

<210> 2

<211> 748

<212> PRT

<213> Unknown

<220>

<223> Akkermansia muciniphila B2UM07 Cytoplasmic__Membrane (Amuc_1631)

<400> 2

Met Glu Lys Asn Ala Pro Phe Ser Val Met Asn Met His Ser Phe Arg

1 5 10 15

Trp Ile Arg Leu Thr Ala Phe Ser Ala Leu Ala Ala Ala Ala Ile Thr

20 25 30

Ser Cys Ala Ser Ala Ala Thr Asp Phe Asn Gln Val Gly Lys Gln Met

35 40 45

Ser Leu Leu Leu Gln Asn Phe His Phe Ser Arg Lys Glu Phe Ser Asp

50 55 60

Glu Leu Ser Thr Lys Phe Leu Glu Thr Tyr Leu Arg Lys Val Asp Pro

65 70 75 80

Asn Lys Ile Phe Phe Thr Gln Gln Asp Val Asp Ala Leu Lys Arg Lys

85 90 95

Tyr Gly Lys Glu Leu Asp Asp Tyr Leu Met Ser Gly Gln Met Met Asp

100 105 110

Ala Ala Gln Ala Met His Ala Leu Tyr Arg Gln Arg Ala Met Gln Arg

115 120 125

Ile Ser Tyr Ala Arg Asp Leu Leu Lys Lys Gly Gly Phe Thr Phe Asp

130 135 140

Lys Asp Lys Ser Ile Glu Arg Ser Arg Arg Lys Thr Ala Ala Trp Pro

145 150 155 160

Lys Asp Glu Ala Glu Met Gln Gln Val Trp Lys Asp Met Val Glu Glu

165 170 175

Gln Leu Leu Ser Glu Ile Leu Arg Arg Glu Thr Val Ala Arg Leu Ala

180 185 190

Lys Glu Gln Asn Lys Pro Asp Pro Leu Ala Asn Glu Lys Pro Ala Glu

195 200 205

Glu Lys Leu Leu Met Arg Tyr Glu Arg Ile Gln Arg Asn Ile Gln Glu

210 215 220

Thr Asp Leu Glu Asp Val Ala Glu Thr Leu Leu Ser Ala Val Ala Leu

225 230 235 240

Thr Tyr Asp Pro His Thr Asp Tyr Met Gly Ala Arg Gln Val Asp Arg

245 250 255

Phe Lys Ile Ser Met Gly Thr Glu Leu Thr Gly Ile Gly Ala Leu Leu

260 265 270

Gly Ser Glu Asp Asp Gly Ser Thr Lys Ile Thr Gly Ile Val Val Gly

275 280 285

Gly Pro Ala Asp Lys Ser Gly Glu Leu Lys Leu Asn Asp Arg Ile Val

290 295 300

Ala Ile Asp Ser Asp Asn Ser Gly Glu Met Val Asp Ile Leu Phe Met

305 310 315 320

Lys Leu Asp Lys Val Val Asp Met Ile Arg Gly Ala Glu Asn Thr Gln

325 330 335

Met Arg Leu Lys Val Glu Pro Ala Asp Ala Pro Gly Gln Ala Lys Ile

340 345 350

Ile Thr Leu Thr Arg Ser Lys Val Pro Leu Lys Asp Glu Leu Ala Lys

355 360 365

Gly Glu Ile Ile Glu Leu Thr Gly Ala Pro Glu Gly Arg Asn Arg Ile

370 375 380

Gly Val Leu Ser Leu Pro Ser Phe Tyr Ala Asp Met Glu Gly Gly Asp

385 390 395 400

Arg Arg Cys Ala Lys Asp Val Lys Lys Ile Leu Glu Arg Met Asn Lys

405 410 415

Glu Asn Val Asp Gly Leu Val Ile Asp Leu Arg Ser Asn Gly Gly Gly

420 425 430

Ser Leu Glu Glu Val Arg Leu Met Thr Gly Phe Phe Thr Gly Asn Gly

435 440 445

Pro Val Val Gln Ile Lys Asp Thr Arg Gly Asn Val Asp Ile Lys Ser

450 455 460

Ala His Asn Arg Gln Lys Leu Phe Asn Gly Pro Ile Val Val Leu Ile

465 470 475 480

Asn Lys Leu Ser Ala Ser Ala Ser Glu Ile Leu Ala Ala Ala Leu Gln

485 490 495

Asp Tyr Gly Arg Ala Val Ile Val Gly Asp Glu Ser Thr Phe Gly Lys

500 505 510

Gly Ser Val Gln Gln Pro Val Asp Ile Gly Gln Tyr Leu Pro Phe Phe

515 520 525

Ala Ala Arg Asp Arg Ala Gly Leu Leu Lys Val Thr Thr Gln Lys Phe

530 535 540

Tyr Arg Val Ala Gly Gly Ser Thr Gln Leu Lys Gly Val Glu Ser Asp

545 550 555 560

Ile Gln Leu Pro Thr Ala Thr Ala Ala Phe Glu Leu Gly Glu Asp Ile

565 570 575

Leu Asp Tyr Ala Met Pro Tyr Asp Gln Ile Thr Pro Cys Thr Asn Tyr

580 585 590

Lys Lys Asp Ser Ser Ile Ala Ala Met Leu Pro Val Leu Lys Asp Ala

595 600 605

Ser Ala Lys Arg Val Glu Lys Asp Arg Asp Leu Gln Ile Ala Arg Glu

610 615 620

Asp Ile Ala Met Met Lys Gln Arg Ile Lys Asp Asn Lys Leu Ser Leu

625 630 635 640

Asn Lys Lys Ile Arg Glu Gln Glu Asn Ser Ala Leu Glu Glu Arg Arg

645 650 655

Lys Ser Ile Asn Lys Glu Arg Lys Ile Arg Phe Ala Glu Met Ala Arg

660 665 670

Glu Asp Ala Thr Lys Tyr Lys Ile Tyr Arg Leu Thr Leu Asp Asp Val

675 680 685

Asn Ala Lys Glu Leu Pro Leu Ala Asp Pro Glu Lys Asp Asn Glu Gln

690 695 700

Phe Met His Leu Ala Glu Asp Pro Thr Ala Glu Leu Asp Asp Ser Pro

705 710 715 720

Glu Tyr Pro Ser Gly Leu Asp Pro Glu Leu Arg Glu Gly Ile Asn Ile

725 730 735

Val Gln Asp Met Leu Lys Leu Glu Ser Ser Gly Lys

740 745

<---

Claims (20)

1. Pharmaceutical composition for the prevention or treatment of metabolic disorders associated with GLP-1 hormone, containing the B2UM07 protein, consisting of the amino acid sequence with SEQ ID NO: 2, as an active substance. 2. A pharmaceutical composition for the prevention or treatment of metabolic disorders according to claim 1, wherein the B2UM07 protein is obtained from a strain of Akkermansia muciniphila . 3. A pharmaceutical composition for the prevention or treatment of metabolic disorders according to claim 2, wherein the Akkermansia muciniphila strain is the SNUG-61027 strain with accession number KCTC 13530BP. 4. A pharmaceutical composition for the prevention or treatment of metabolic disorders according to claim 1, wherein the metabolic disorder is glucose intolerance, diabetes, arteriosclerosis, hyperlipidemia, hypercholesterolemia, fatty liver, cardiovascular disease, or obesity. 5. Pharmaceutical composition for suppressing appetite in a subject with reduced GLP-1 hormone, containing the B2UM07 protein, consisting of the amino acid sequence of SEQ ID NO: 2, as an active substance. 6. An appetite suppressant pharmaceutical composition according to claim 5, wherein the B2UM07 protein is obtained from a strain of Akkermansia muciniphila . 7. An appetite suppressant pharmaceutical composition according to claim 6, wherein the strain of Akkermansia muciniphila is strain SNUG-61027 having accession number KCTC 13530BP. 8. A healthy food product for alleviating the metabolic disorders associated with GLP-1 hormone, containing the B2UM07 protein, consisting of the amino acid sequence of SEQ ID NO: 2, as an active substance. 9. The metabolic health food product according to claim 8, wherein the B2UM07 protein is obtained from a strain of Akkermansia muciniphila . 10. A healthy food product for alleviating metabolic disorders according to claim 9, wherein the strain of Akkermansia muciniphila is a strain of SNUG-61027 with accession number KCTC 13530BP. 11. A healthy food product for alleviating metabolic disorders according to claim 8, wherein the metabolic disorder is glucose intolerance, diabetes, arteriosclerosis, hyperlipidemia, hypercholesterolemia, fatty liver, cardiovascular disease, or obesity. 12. A health food product for suppressing appetite in a subject with reduced GLP-1 hormone, containing the B2UM07 protein, consisting of the amino acid sequence of SEQ ID NO: 2, as an active ingredient. 13. The appetite suppressant health food product of claim 12, wherein the B2UM07 protein is derived from a strain of Akkermansia muciniphila . 14. The appetite suppressant health food product of claim 13, wherein the strain of Akkermansia muciniphila is strain SNUG-61027 with accession number KCTC 13530BP. 15. Use of the B2UM07 protein containing the amino acid sequence of SEQ ID NO: 2 for the prevention or treatment of metabolic disorders associated with GLP-1 hormone. 16. Use for the prevention or treatment of metabolic disorders according to claim 15, wherein the B2UM07 protein is obtained from a strain of Akkermansia muciniphila . 17. Use for the prevention or treatment of metabolic disorders according to claim 16, wherein the strain of Akkermansia muciniphila is the strain SNUG-61027 having accession number KCTC 13530BP. 18. Use of the B2UM07 protein containing the amino acid sequence of SEQ ID NO: 2 to suppress appetite in a subject with reduced GLP-1 hormone. 19. Appetite suppressant use according to claim 18, wherein the B2UM07 protein is obtained from a strain of Akkermansia muciniphila . 20. An appetite suppressant use according to claim 19, wherein the strain of Akkermansia muciniphila is strain SNUG-61027 having accession number KCTC 13530BP.

Images