Composition for preventing or treating metabolic diseases using strain expressing tarbonate dehydrate or combination therapy

Abstract

The present invention relates to a composition for preventing or treating a metabolic disease using a strain expressing a tartrate dehydratase or a combination therapy comprising the same, according to one aspect, when a fermentative Lactobacillus mucus strain expressing a tartrate dehydratase is administered in combination with tartaric acid, citric acid or a substance each comprising the same, due to the synergistic effect of anti-obesity activity, the composition is very effective for preventing or treating metabolic diseases.

Description

Technical Field

[0001] This relates to a composition for the prevention or treatment of metabolic diseases that utilizes a strain expressing tartrate dehydratase or a combination therapy containing it. Background Technology

[0002] Obesity is a state of excessive accumulation of adipose tissue in the body due to an increase in the size or number of fat cells, meaning the amount of fat exceeds the body's needs. Obesity is considered a leading modifiable cause of death in modern society and is a chronic disease that must be managed to ensure a healthy and active life. It is well known that obesity is associated with a variety of diseases, including hypertension, diabetes, insulin resistance, glucose intolerance, hyperlipidemia, angina pectoris, fatty liver disease, cancer, and osteoarthritis. Therefore, as the metabolic diseases caused by obesity become increasingly diverse and severe, there is an urgent need for prevention and treatment.

[0003] The development of obesity treatments primarily aims to reduce weight, inhibit fat accumulation, and improve metabolism. Many obesity treatments currently under development and commercialized utilize mechanisms that reduce weight by suppressing appetite. While these appetite suppressants offer excellent weight-loss efficacy, their mechanism of action—decreases food intake by acting on the central nervous system to reduce appetite—presents a significant challenge: the efficacy may not be sustained long-term, or serious side effects may occur with prolonged use. Therefore, there is an urgent need to develop obesity treatments that do not cause serious side effects.

[0004] Furthermore, recent research indicates that certain compounds, such as succinic acid, may play an important role in regulating metabolism and maintaining energy balance. Succinic acid is an important intermediate in the tricarboxylic acid cycle and can inhibit fat accumulation in the body by promoting efficient energy production and lipolysis (Physiological Reports. 2020; 8: e14630).

[0005] Furthermore, succinic acid has been reported to activate energy expenditure in brown adipose tissue (Nature. 2018 Aug; 560(7716): 102-106). Unlike white adipose tissue, brown adipose tissue expends energy through thermogenesis in an individual's adipocytes, and its activity is negatively correlated with age or metabolic state, particularly with degree of obesity. Accumulation of succinic acid induces uncoupling protein 1 (UCP1)-dependent thermogenesis in brown adipose tissue (BAT), thereby preventing diet-induced obesity and improving glucose tolerance.

[0006] Succinic acid is an intermediate metabolite produced and utilized by many organisms except viruses. Cases of side effects caused by succinic acid intake are relatively rare. The mechanisms by which it inhibits fat accumulation and activates energy consumption in brown adipose tissue are the basis for the attention given to succinic acid as a promising candidate for the treatment of metabolic diseases such as obesity.

[0007] However, it has been reported that succinic acid exists in extremely low concentrations in germ-free mice, but its concentration increases in the gut where microbes reside (Sci Transl Med. 2014 Jan 22; 6(220): 220ra11.), which suggests that the accumulation of succinic acid in the gut is caused by gut microbes rather than by succinic acid intake. This means that in order to develop therapeutic agents for metabolic diseases such as obesity, it is necessary to discover strains that can accumulate succinic acid in the gut.

[0008] Therefore, the inventors discovered strains capable of accumulating succinic acid by expressing tartrate dehydratase and / or citrate lyase, and developed compositions utilizing these strains for the prevention or treatment of metabolic diseases. Summary of the Invention

[0009] Technical problem In one approach, a strain expressing tartrate dehydratase is provided.

[0010] In another approach, a strain expressing citrate lyase is provided.

[0011] Another option is to provide a strain expressing tartaric acid dehydratase; and a complex of tartaric acid or a substance containing tartaric acid, or citric acid or a substance containing citric acid.

[0012] Another approach provides a pharmaceutical composition for the prevention or treatment of metabolic diseases, comprising a first active substance and a second active substance; the first active substance comprises a strain expressing tartrate dehydratase, and the second active substance comprises tartrate or contains tartrate, or citric acid or contains citric acid.

[0013] Another approach provides a health food for preventing or improving metabolic diseases, comprising a first active substance and a second active substance; the first active substance comprises a strain expressing tartaric acid dehydrating enzyme, and the second active substance comprises tartaric acid or a substance containing tartaric acid, or citric acid or a substance containing citric acid.

[0014] Another embodiment provides the use of a composition in the prevention or treatment of metabolic diseases, the composition comprising a first active substance and a second active substance as active ingredients, the first active substance comprising a strain expressing tartrate dehydratase, a culture of the strain, a lysate of the strain, or a mixture thereof, the second active substance comprising tartaric acid or a substance containing tartaric acid, or citric acid or a substance containing citric acid; or, the composition comprising the first active substance as an active ingredient, the second active substance being administered in combination with it.

[0015] Another approach provides a method for improving, preventing, or treating metabolic diseases, the method comprising the steps of: administering a composition to a desired individual, the composition comprising a first active substance and a second active substance as active ingredients, the first active substance comprising a strain expressing tartrate dehydratase or citrate lyase, a culture of the strain, a lysate of the strain, or a mixture thereof, the second active substance comprising tartaric acid or a substance containing tartaric acid, or citric acid or a substance containing citric acid.

[0016] Another approach provides a method for improving, preventing, or treating metabolic diseases, the method comprising the steps of: administering to a desired individual a first active substance comprising a strain expressing tartaric acid dehydratase or citrate lyase, a culture of the strain, a lysate of the strain, or a mixture thereof; and administering a second active substance comprising tartaric acid or a substance containing tartaric acid, or citric acid or a substance containing citric acid.

[0017] Another embodiment provides the use of a composition in the preparation of a medicament for improving, preventing or treating metabolic diseases, the composition comprising a first active substance and a second active substance as active ingredients, the first active substance comprising a strain expressing tartrate dehydratase, a culture of the strain, a lysate of the strain, or a mixture thereof, the second active substance comprising tartaric acid or a substance containing tartaric acid, or citric acid or a substance containing citric acid; or, the composition comprising the first active substance as an active ingredient, the second active substance being administered in combination therewith.

[0018] Technical solution In one approach, a strain expressing tartrate dehydratase is provided.

[0019] In one specific example, the strain may also express citrate lyase or ATP-citrate lyase. In the strain, tartrate dehydratase and citrate lyase are expressed independently. The tartrate dehydratase is an enzyme that dehydrates tartrate and converts it to succinate, and the citrate lyase is an enzyme involved in the breakdown of citric acid to produce succinate.

[0020] In one specific instance, strains expressing tartrate dehydratase and / or citrate lyase are characterized by succinic acid accumulation.

[0021] In one specific instance, the strain may be a Lactobacillus strain, specifically Lactobacillus fermentum (… Limosilactobacillus fermentum ).

[0022] The genus *Lactobacillus* is a group of aerobic or facultative anaerobic Gram-positive bacilli widely distributed in nature. Microorganisms belonging to the genus *Lactobacillus* include *Lactobacillus fermentum* (…). Limosilactobacillus leaven Lactobacillus plantarum ( Lactiplantibacillus plantarum Lactobacillus sakei () Lactobacillus sakei )wait.

[0023] Lactobacillus ( Lactobacillus It has been reclassified as a member of the genus *Lactobacillus*. Limosilactobacillus ), Lactobacillus plantarum ( Lactiplantibacillus ) or Lactobacillus spp. ( Lactobacillus There are 23 taxa, including *Lactobacillus fermentum*, etc. In this specification, the original strain names and the revised strain names are interchangeable. For example, *Lactobacillus fermentum* (… Limosilactobacillus fermentum ) can be combined with Lactobacillus fermentum ( Lactobacillus fermentum ), fermented Lactobacillus plantarum ( Lactiplantibacillus fermentum ) or Lactobacillus fermentation ( Lactobacillus fermentum They can be used interchangeably.

[0024] The inventors conducted research to develop new bacterial strains and screened *Lactobacillus fermentum* GB102 as a candidate strain with anti-obesity activity. This strain was deposited at the Korea Institute of Biomedical Research and Development (KICRD) on September 6, 2019, with accession number SD1335. The same strain was also deposited at KICRD on January 14, 2020, with accession number KCTC14105BP. This strain is a probiotic strain, harmless to humans, and has no side effects when used.

[0025] In this specification, the term "Lactobacillus fermentum GB102" may be used in conjunction with... L. fermentum GB102 strain, Lactobacillus fermentum ( Lactobacillus fermentum GB102 strain, Lactobacillus fermentum ( Limosilactobacillus leaven GB102 strain, GB102 interchangeable description.

[0026] In one specific example, the strain may be a strain having a 16S rRNA consisting of the nucleotide sequence of SEQ ID NO: 1, or a strain having a 16S rRNA containing a nucleotide sequence with at least 98.5% identity to its nucleotide sequence. Specifically, it has at least 93%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.8%, 99.9%, or 100% identity to the nucleotide sequence consisting of SEQ ID NO: 1 in this specification.

[0027] In one specific instance, the strain may be a strain containing a 16S rRNA gene consisting of the nucleotide sequence of SEQ ID NO: 1.

[0028] In one specific example, the strain may be the strain with accession number KCTC14105BP.

[0029] In one specific instance, the strain may be a mutant of a naturally occurring strain.

[0030] In one specific instance, the strain may be a live bacterium, a dead bacterium, or a cytoplasmic fraction obtained by rupturing the strain; preferably, it may be a live bacterium.

[0031] In one specific instance, the strain may possess anti-obesity activity. Specifically, the anti-obesity activity may include one or more activities selected from the group consisting of: inhibiting weight gain, increasing energy metabolism rate or basal metabolic rate, accumulating brown adipose tissue, activating brown adipose tissue, reducing the ratio of fat mass to lean body mass, inhibiting hepatic steatosis (fatty liver), reducing blood AST (alanine aminotransferase), ALT (alanine aminotransferase, GPT), triglycerides, low-density cholesterol, high-density cholesterol, or total cholesterol, and increasing the expression of UCP-1 (uncoupling protein 1). Therefore, the strain may inhibit weight gain.

[0032] In one specific example, the strain contributes to gut health, thereby effectively promoting overall health in the treatment of metabolic diseases such as obesity. Specifically, the strain can control the gut microbiota by producing large amounts of organic acids. When the strain is ingested in combination with prebiotics (e.g., foods containing tartaric acid, citric acid, etc.), it can promote succinic acid production while maintaining gut microbiota balance and alleviate bowel obstruction during metabolic disease diet therapy, thereby enhancing the treatment efficacy of metabolic diseases such as obesity.

[0033] In another embodiment, a pharmaceutical composition for the prevention or treatment of metabolic diseases is provided, the composition comprising a first active substance and a second active substance as active ingredients, the first active substance comprising a strain expressing tartrate dehydratase, a culture of the strain, a lysate of the strain, or a mixture thereof, and the second active substance comprising tartaric acid or a substance containing tartaric acid, or citric acid or a substance containing citric acid; or, the composition comprising the first active substance as an active ingredient, and the second active substance is administered in combination therewith.

[0034] In one specific example, the pharmaceutical composition for the prevention or treatment of metabolic diseases may be a composition or complex comprising: a first active substance and a second active substance as active ingredients; the first active substance comprising a strain expressing *Lactobacillus fermentum*, a culture of the strain, a lysate of the strain, or a mixture thereof; and the second active substance comprising tartaric acid or a substance containing tartaric acid, or citric acid or a substance containing citric acid; or, the composition or complex comprising the first active substance as an active ingredient, and the second active substance being administered in combination with it.

[0035] The “strain” is as described above.

[0036] In this specification, the term "culture" may be used interchangeably with "culture medium," "culture supernatant," "culture upper layer," "conditioned medium," or "adjustment medium," and may refer to the entire culture medium containing the strain, its metabolites, additional nutrients, etc., obtained after culturing the strain in a medium capable of providing nutrients for the growth and survival of the *Lactobacillus* strain in vitro for a certain period of time. The culture refers to the product obtained by culturing probiotic strains in a known culture medium, which may or may not contain the strain itself. The culture medium may be selected from known liquid or solid culture media, such as, but not limited to, MRS liquid medium, GAM liquid medium, MRS agar medium, GAM agar medium, and BL agar medium.

[0037] In this specification, the term "lysate" is used interchangeably with "solution," referring to a solution or suspension of microbial cells, such as ruptured *Lactobacillus fermentum* cells, in an aqueous culture medium. Cell lysates include, for example, macromolecules such as DNA, RNA, proteins, peptides, carbohydrates, lipids, etc., and / or micromolecules such as amino acids, sugars, fatty acids, etc., or components thereof. Furthermore, the lysate also includes cell fragments, which may be smooth or granular in structure.

[0038] The culture medium may include the culture medium itself obtained by culturing the strain, its concentrate, or lyophilized form, or the culture supernatant obtained by removing the strain from the culture medium, its concentrate, or lyophilized form.

[0039] The culture medium can be obtained by culturing *Lactobacillus fermentans* in a suitable culture medium (e.g., MRS plate medium) at any temperature above 10°C or below 40°C for a certain period of time, for example, 4 to 50 hours. The culture medium and culture conditions used to culture the *Lactobacillus fermentans* can be appropriately selected or modified and used by those skilled in the art.

[0040] In one specific instance, the first active substance may contain only a strain of *Lactobacillus fermentans* as the active ingredient, or it may contain one or more pharmaceutically acceptable carriers, excipients, or diluents.

[0041] In one specific instance, the second active substance may contain tartaric acid or a substance containing it.

[0042] Tartaric acid, also known as tartaric acid, is an organic acid with the chemical name 2,3-dihydroxybutanedioic acid, which can be represented by the following chemical formula 1: [Chemical Formula 1]

[0043] The tartaric acid comprises L-tartaric acid, D-tartaric acid, meso tartaric acid, or any combination thereof, preferably L-tartaric acid.

[0044] The tartaric acid may be contained in the form of a salt.

[0045] Substances containing tartaric acid may include berry extracts, specifically tamarind ( Tamarind ),Grape( Wine grapevine Star fruit () Averrhoa carambola Tree tomato ( Solanum beet ), Roselle ( Hibiscus sabdariffa ), Citrus ( Citrus spp. ),apple( Domestic apple ),plum( Prunus mother ), dates ( Phoenix dactylifera European sweet cherry ( Prunus avium, Prunus cerasus ),raspberry( Rubus idaeus ),blackberries( Rubus fruticosus ), black tea curd ( Red currant ),cranberry( Vaccinium macrocarpon ),pineapple( Pineapple is delicious. ), Hovenia dulcis ( Sweet Hovenia),banana( Muse spp. ),Pomegranate( Pomegranate ), wild chokeberry ( Aronia melanocarpa ), highbush blueberry ( Blueberry Corymbosum ), planting paddy fields ( Korean blackberry ) and mulberry ( White mulberry Extracts of tartaric acid, etc., or wines made using the berries, but not limited thereto. Specifically, the form of the substance containing tartaric acid is not limited, such as liquid, powder, etc.

[0046] In one specific instance, the second active substance may contain citric acid or a substance containing citric acid.

[0047] Citric acid, also known as citric acid, is an organic acid with the chemical name 2-hydroxypropane-1,2,3-tricarboxylic acid, which can be represented by the following chemical formula 2: [Chemical Formula 2]

[0048] The citric acid may be contained in the form of a salt.

[0049] The substances containing citric acid may include those from the Citrus genus (Citrus). Citrus spp. ) extract, specifically orange ( Citrus sinensis ),lemon( Citrus limon ), lime ( Citrus aurantiifolia ), grapefruit Citrus paradise ) and oranges ( Citrus reticulata Extracts of substances containing citric acid, etc., but not limited to these. Specifically, the form of the substance containing citric acid is not limited, such as liquid, powder, etc.

[0050] In one specific example, the second active substance can induce a strain expressing tartrate dehydratase and / or citrate lyase to produce succinic acid. That is, the second active substance can be provided as a substrate for the first active substance, which comprises a strain expressing tartrate dehydratase and / or citrate lyase.

[0051] In this specification, the term "succinic acid" is used interchangeably with succinic acid and can be represented by the following chemical formula 3: [Chemical Formula 3]

[0052] Succinic acid, when administered to an individual, induces the accumulation of brown adipose tissue in adipocytes and activates energy expenditure in brown adipose tissue. In mammals, two main types of adipose tissue exist: white adipose tissue and brown adipose tissue (Gesta S et al., Cell 2007; 131: 242-256). Furthermore, beige adipose tissue is known to contribute to the browning of white adipose tissue. White adipose tissue is used for energy storage in individuals, and therefore, excess of it is associated with obesity. In contrast, brown adipose tissue has a higher concentration of mitochondria and can specifically express uncoupling protein 1 (UCP1). Therefore, brown adipose tissue can mitigate obesity by inducing energy expenditure and thermogenesis. Thus, succinic acid induced by the second active substance exhibits anti-obesity activity by increasing energy metabolism or basal metabolic rate through the accumulation of brown adipose tissue and activation of energy expenditure in brown adipose tissue.

[0053] In one specific instance, the first and second active substances can be administered simultaneously, sequentially, or in reverse order. For example, when administered in combination with the second active substance, the anti-obesity activity of the first active substance can further induce succinic acid production, thereby exhibiting a synergistic effect.

[0054] In this specification, the terms "combined therapy," "combined treatment," "combined administration," or "in combination" refer to any form of simultaneous or concurrent treatment using at least two individual substances that are useful for the treatment of a disease. The components of a combined administration can be administered simultaneously, sequentially, in reverse order, or in any other order. These components can be administered at different doses, at different frequencies, or via different routes, depending on appropriate methods.

[0055] In this specification, the term "administration" means the introduction of a prescribed substance into an individual in an appropriate manner. "Individual" refers to any organism, including humans, that may suffer from a metabolic disease, such as rats, mice, and livestock. As a specific example, it can include mammals, including humans.

[0056] In this specification, the term "simultaneous administration" is not particularly limited and means that the components of a combination therapy are administered substantially simultaneously, such as as a mixture or in a sequential order.

[0057] In this specification, the term "sequential administration" is not specifically limited and means that the components of a combination therapy are not administered simultaneously, but rather sequentially or in batches, with specific time intervals between administrations. The time intervals between administrations of the components in a combination therapy can be the same or different, and can be selected from, for example, ranges from 2 minutes to 96 hours, 1 day to 7 days, or 1 week, 2 weeks, or 3 weeks. Typically, the time intervals between administrations can be from minutes to hours, such as 2 minutes to 72 hours, 30 minutes to 24 hours, or 1 hour to 12 hours. Further examples include time intervals ranging from 24 to 96 hours, 12 to 36 hours, 8 to 24 hours, and 6 to 12 hours.

[0058] The combined administration can refer to the simultaneous, sequential, or individual application of *Lactobacillus fermentum* strains and tartaric acid or a substance containing tartaric acid, or citric acid or a substance containing citric acid, in any order. Specifically, the combined administration can be the simultaneous application of a first active substance containing *Lactobacillus fermentum* strains and a second active substance containing tartaric acid or a substance containing tartaric acid, or citric acid or a substance containing citric acid, or the application of the second active substance after the application of *Lactobacillus fermentum* strains.

[0059] The combined treatment method according to the invention can be defined as providing a synergistic effect, for example, if the efficacy measured by the degree of response, rate of response, disease progression, or survival is superior to the efficacy obtained by administering one or more components of the combined treatment method at conventional doses. For example, the efficacy of the combined treatment method is synergistic if its therapeutic efficacy is superior to that obtained by using each of the components alone. In particular, a synergistic effect is considered to exist if, without impairing one or more of the degree of response, rate of response, disease progression, and survival data, especially without impairing the duration of response, and if problematic side effects are reduced and / or lessened compared to using each component at conventional doses, while the conventional doses of *Lactobacillus fermentum* strain and tartaric acid or substances containing tartaric acid, or citric acid or substances containing citric acid, are reduced.

[0060] In this specification, the term "pharmaceutical composition" means a mixture containing one or more active ingredients and various components that deliver or assist in its delivery for a specific therapeutic, preventive or diagnostic purpose.

[0061] The pharmaceutical composition may further comprise a pharmaceutically acceptable diluent or carrier. The diluent may be lactose, corn starch, soybean oil, microcrystalline cellulose, or mannitol, or a combination thereof. The carrier may be an excipient, disintegrant, binder, lubricant, or a combination thereof. The excipient may be amorphous cellulose, lactose, low-substituted hydroxycellulose, or a combination thereof. The disintegrant may be calcium carboxymethyl cellulose, sodium carboxymethyl starch, anhydrous calcium hydrogen phosphate, or a combination thereof. The binder may be polyvinylpyrrolidone, low-substituted hydroxypropyl cellulose, hydroxypropyl cellulose, or a combination thereof. The lubricant may be magnesium stearate, silica, talc, or a combination thereof.

[0062] When the pharmaceutical composition is formulated, it can be prepared using commonly used lubricants, sweeteners, flavorings, emulsifiers, suspending agents, preservatives, fillers, extenders, binders, wetting agents, disintegrants, surfactants, and other diluents or excipients. Solid dosage forms for oral administration can include tablets, pills, powders, granules, capsules, etc., which can be prepared by mixing at least one or more excipients such as starch, calcium carbonate, sucrose or lactose, gelatin, etc., with the composition. In addition to simple excipients, lubricants such as magnesium stearate and talc can also be used. Liquid dosage forms for oral administration include suspensions, oral solutions, emulsions, syrups, etc., and can include various excipients such as wetting agents, sweeteners, flavorings, preservatives, etc., in addition to commonly used simple diluents such as water and liquid paraffin. Dosage forms for non-oral administration can include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized formulations, and suppositories. As non-aqueous solvents and suspending agents, propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate can be used. As a base for suppositories, witepsol, polyethylene glycol, Tween 61, cocoa butter, glyceryl laurate, glycerin gelatin, etc. can be used, and when formulated into eye drops, known diluents or excipients can be used.

[0063] To improve stability or absorption, the pharmaceutical composition may use carbohydrates such as glucose, sucrose, or dextran, antioxidants such as ascorbic acid or glutathione, as well as chelating agents, low molecular weight proteins, or other stabilizers as pharmaceutical agents.

[0064] In one specific example, the pharmaceutical composition can be formulated into an oral or non-oral dosage form. Oral dosage forms may be granules, powders, liquids, tablets, capsules, dry syrups, or combinations thereof. For non-oral administration, injection methods may include topical application, intraperitoneal injection, rectal injection, subcutaneous injection, intravenous injection, intramuscular injection, or intrapleural injection.

[0065] In one specific example, the routes of administration of the pharmaceutical composition include, but are not limited to, oral, intravenous, intramuscular, intraarterial, intramedullary, intradural, intracardiac, percutaneous, subcutaneous, intraperitoneal, intranasal, intestinal, local, sublingual, or rectal administration. Specifically, a first active substance comprising a strain of *Lactobacillus fermentum* and a second active substance comprising tartaric acid or containing tartaric acid, or citric acid or containing citric acid, may be administered orally in granules, powders, liquids, tablets, capsules, dry syrups, or combinations thereof, or non-oral via injection methods such as topical application, intraperitoneal injection, rectal injection, subcutaneous injection, intravenous injection, intramuscular injection, or intrapleural injection.

[0066] In one specific example, the pharmaceutical composition includes a first oral formulation containing the first active substance and a second oral formulation containing a second active substance, the first and second oral formulations being orally administered.

[0067] In one specific example, the pharmaceutical composition may contain the first active substance and the second active substance in a single oral formulation for oral administration.

[0068] The pharmaceutical composition is administered in a pharmaceutically effective amount. The term "pharmaceuticalally effective amount" means an amount sufficient to treat a disease with a reasonable benefit / risk ratio suitable for medical treatment. The effective dose level may depend on factors including the type and severity of the patient's disease, the activity of the drug, the sensitivity to the drug, the timing of administration, the route of administration and excretion rate, the duration of treatment, factors of concomitant medications, and other factors well known in the medical field.

[0069] According to a specific example, the composition may contain 0.001% to 80% by weight of *Lactobacillus fermentum* strain based on the total weight of the composition. Furthermore, the dosage of the *Lactobacillus fermentum* strain may be 0.01 mg to 10,000 mg, 0.1 mg to 1,000 mg, 1 mg to 100 mg, 0.01 mg to 1,000 mg, 0.01 mg to 100 mg, 0.01 mg to 10 mg, or 0.01 mg to 1 mg. The strain is contained in the composition at a therapeutically effective amount or a nutritionally effective concentration; for example, the strain may be contained in a 10... 3 Up to 10 16 CFU / g, 10 3 Up to 10 15 CFU / g, 103 Up to 10 14 CFU / g, 10 3 Up to 10 13 CFU / g, 10 3 Up to 10 12 CFU / g, 10 4 Up to 10 16 CFU / g, 10 4 Up to 10 15 CFU / g, 10 4 Up to 10 14 CFU / g, 10 4 Up to 10 13 CFU / g, 10 4 Up to 10 12 CFU / g, 10 5 Up to 10 16 CFU / g, 10 5 Up to 10 15 CFU / g, 10 5 Up to 10 14 CFU / g, 10 5 Up to 10 13 CFU / g, 10 5 Up to 10 12 CFU / g, 10 6 Up to 10 13 CFU / g, 10 6 Up to 10 12 CFU / g, 10 7 Up to 10 13 CFU / g, 10 7 Up to 10 12 CFU / g, 10 8 Up to 10 13 CFU / g or 10 8 Up to 10 12 The CFU / g content is included in the composition, or in an equal volume of live or dead bacterial cultures. Specifically, for adult patients, 1×10⁻⁶ can be administered in single or multiple doses. 3 Up to 1×10 16 The composition contains live or dead bacteria at CFU / g. However, the dosage can be prescribed in various ways depending on factors such as the formulation method, route of administration, patient's age, weight, sex, pathological state, food intake, administration time, route of administration, excretion rate, and response sensitivity. Those skilled in the art can adjust the dosage appropriately based on these factors. According to one embodiment, the composition comprises inactivated dried bacterial strains and can be administered in doses of 1g to 10g, 0.5g to 1.5g, 2.5g to 3.5g, or 4.5g to 5.5g.

[0070] On the one hand, the drug composition can be administered once daily or in multiple divided doses. Specifically, based on a 7-day period, the administration can be carried out in cycles of 6 days followed by 1 day of rest, 5 days followed by 2 days of rest, or 4 days followed by 3 days of rest. More specifically, the administration can be carried out in cycles of 5 days followed by 2 days of rest.

[0071] Furthermore, the pharmaceutically effective amount and effective dosage of the pharmaceutical composition can vary depending on the formulation method, administration method, administration time, and / or route of administration. Additionally, it can vary depending on various factors such as the type and extent of the desired response achieved through the administration of the pharmaceutical composition, the type of individual as a test subject, age, weight, general health status, disease symptoms or severity, sex, diet, excretion, and the composition of other pharmaceutical compositions administered simultaneously or concurrently to the individual, as well as similar factors known in the pharmaceutical field. Those skilled in the art can easily determine and prescribe an effective dosage for the target treatment. The pharmaceutical composition according to the invention can be administered once daily or in multiple divided doses. Therefore, the dosage does not limit the scope of the invention in any way. The dosage of the pharmaceutical composition can range from 1 μg / kg / day to 1,000 μg / kg / day.

[0072] The individual can be a mammal, such as a human, cow, horse, pig, dog, sheep, goat, or cat. The individual can be someone who needs treatment for side effects caused by taking medication for a metabolic disease.

[0073] In one specific example, the pharmaceutical composition for the prevention or treatment of metabolic diseases may comprise a first active substance and a second active substance as active ingredients. The first active substance comprises a strain expressing citrate lyase, a culture of the strain, a lysate of the strain, or a mixture thereof. The second active substance comprises tartaric acid or a substance containing tartaric acid, or citric acid or a substance containing citric acid. Alternatively, the first active substance may be included as an active ingredient, and the second active substance may be administered in combination with it.

[0074] In this specification, the term "treatment" can refer to the cure of metabolic diseases, etc., in a shorter time compared to natural healing. The treatment may include improvement and / or relief of metabolic diseases. Furthermore, the treatment may refer to the cure and / or recovery of symptoms caused by metabolic diseases.

[0075] In this specification, the term "prevention" refers to a method of partially or completely delaying or preventing the onset or recurrence of a disease, disorder, or its accompanying symptoms, or preventing the acquisition or re-acquisition of a disease or disorder, or reducing the risk of acquiring a disease or disorder. For example, prevention refers to all actions that inhibit or delay the occurrence of metabolic diseases by applying the compositions according to the invention.

[0076] In this specification, the term "comprising as an active ingredient" means adding *Lactobacillus fermentum* strain, vesicles derived from said strain, lysate of said strain, culture medium, or extract of culture medium thereof, or tartaric acid or citric acid as a main component, and also includes adding various components as secondary components for drug delivery and stabilization, etc., to formulate various dosage forms.

[0077] In this specification, the term "obesity" refers to a state of excessive body fat. Clinically, obesity is defined as a Body Mass Index (BMI) of 25 or higher in South Korea and 30 or higher according to the World Health Organization (WHO). Generally, obesity refers to a weight exceeding the normal range, but even if the weight is not very high, a high body fat percentage may constitute obesity. Obesity can occur in both adults and children. Obesity not only leads to weight gain but can also trigger obesity-related metabolic diseases such as binge eating, excessive alcohol consumption and binge eating disorder, hypertension, diabetes, elevated plasma insulin levels, insulin resistance, hyperlipidemia, metabolic syndrome, insulin resistance syndrome, obesity-related gastroesophageal reflux, arteriosclerosis, hypercholesterolemia, hyperuricemia, cardiac hypertrophy and left ventricular hypertrophy, lipodystrophy, non-alcoholic steatohepatitis, cardiovascular disease, or polycystic ovary syndrome. Therefore, the composition can be used not only for obesity but also for the prevention or treatment of obesity-related metabolic diseases. Furthermore, the composition can be used by individuals who are not obese but have a need to lose weight or reduce body fat.

[0078] Obesity can be caused by a variety of factors. These factors may include a high-fat diet, reduced physical activity, genetics, psychological factors, endocrine system abnormalities, metabolic abnormalities, and social and environmental factors. In particular, obesity can be caused by a high-fat diet.

[0079] In this specification, the term "metabolic disease" refers to a general term for various disorders caused by abnormal metabolic processes in the body, also known as metabolic disorders. These are typically caused by imbalances in carbohydrates, lipids, proteins, vitamins, electrolytes, and water. Specifically, metabolic diseases include, but are not limited to, metabolic syndrome, type 1 diabetes, type 2 diabetes, hypertension, hyperlipidemia, obesity, fatty liver, insulin resistance, glucose intolerance syndrome, angina pectoris, coronary artery disease, and arteriosclerosis.

[0080] According to an embodiment of the present invention, the metabolic disease may be selected from the group consisting of: type 1 diabetes, type 2 diabetes, impaired glucose tolerance, abnormal fasting blood glucose, dyslipidemia, lipid metabolism disorder, obesity, fatty liver, insulin resistance syndrome and glucose intolerance syndrome.

[0081] Another approach provides a health food for preventing or improving metabolic diseases, the food comprising a first active substance and a second active substance as active ingredients, the first active substance comprising a strain expressing tartaric acid dehydratase, a culture of the strain, a lysate of the strain, or a mixture thereof, the second active substance comprising tartaric acid or a substance containing tartaric acid, or citric acid or a substance containing citric acid; or, the composition comprises the first active substance as an active ingredient, and the second active substance is administered in combination with it.

[0082] In the aforementioned health functional food, the terms "strain", "first active substance", "tartaric acid", "citric acid", "second active substance", "combined administration", etc., can be within the aforementioned range.

[0083] In this specification, the term "health functional food" refers to food made with specific ingredients as raw materials for the purpose of assisting health, or food made by extracting, concentrating, purifying, or mixing specific ingredients contained in food raw materials. It also refers to food designed and processed so that the ingredients can fully exert biological regulatory functions on organisms, such as biological defense, regulation of biological rhythms, and prevention and recovery from diseases. These health functional foods can play a role in the prevention and improvement of metabolic diseases.

[0084] There are no particular restrictions on the types of food products to which the extract may be added. Examples of food products to which the extract may be added include preparations selected from powders, granules, tablets, capsules, pills, gels, jellies, suspensions, emulsions, syrups, tea bags, infused teas, chewing gum, confectionery, and health beverages, and include all health foods in the conventional sense.

[0085] The health functional food may include food additives that are acceptable in food science, and may include appropriate carriers that are commonly used in the manufacture of the health functional food.

[0086] In this specification, the term "food-grade acceptable" means that the compound is non-toxic to cells or the human body exposed to it.

[0087] The aforementioned health functional foods also include one or more of the following: carriers, diluents, excipients, and additives, and can be formulated into one of the following dosage forms: tablets, pills, powders, granules, capsules, and liquids. Foods to which compounds according to a scheme can be added include various food categories, powders, granules, tablets, capsules, syrups, beverages, chewing gum, tea, vitamin complexes, and other health functional foods.

[0088] In addition to the aforementioned active ingredients, the health functional food may also contain other ingredients as essential components without particular limitation. For example, it may contain various flavoring agents or natural carbohydrates as additional ingredients, similar to ordinary beverages. Examples of natural carbohydrates may be monosaccharides, such as glucose and fructose; disaccharides, such as maltose and sucrose; and polysaccharides, such as conventional sugars like dextrin and cyclodextrin, as well as sugar alcohols such as xylitol, sorbitol, and erythritol. As flavor enhancers other than those mentioned above, natural flavor enhancers (such as sematriene, stevia extract (e.g., rebaudioside A, glycyrrhizic acid, etc.) and synthetic flavor enhancers (such as saccharin and aspartame) may be advantageously used. The proportion of the natural carbohydrates can be appropriately determined by those skilled in the art.

[0089] In addition to the above, the health functional food according to one scheme may contain various nutrients, vitamins, minerals (electrolytes), flavoring agents such as synthetic and natural flavoring agents, coloring agents and flavor enhancers (cheese, chocolate, etc.), pectic acid and its salts, alginic acid and its salts, organic acids, protective colloid thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohol, and carbonating agents used in carbonated beverages. These ingredients may be used alone or in combination, and the proportions of these additives may be appropriately selected by those skilled in the art.

[0090] In the aforementioned health functional foods, the active ingredients can be added directly to the food or used together with other foods or food ingredients, and can be used appropriately according to conventional methods. The amount of active ingredients mixed can be appropriately determined according to its intended use (for prevention or improvement). Generally, when preparing food or beverages, the amount added to the health functional foods can be specifically about 15% by weight or less, more specifically about 10% by weight or less, of the raw materials. However, in cases of long-term intake for health and hygiene or health regulation purposes, the amount can be lower than the aforementioned range.

[0091] The health functional food may be provided in combination with existing known health functional foods for the prevention or improvement of metabolic diseases or other existing health functional foods. The other health functional foods for the prevention or improvement of metabolic diseases may be existing known health functional foods for the prevention or improvement of metabolic diseases, existing health functional foods, or newly developed health functional foods.

[0092] When the health functional food contains other health functional foods that have the effect of preventing or improving metabolic diseases, it is important to mix them in the amount that can achieve the maximum effect with the minimum amount without side effects, which can be easily determined by those skilled in the art.

[0093] Another embodiment provides the use of a composition in improving, preventing or treating metabolic diseases, the composition comprising a first active substance and a second active substance as active ingredients, the first active substance comprising a strain expressing tartrate dehydratase or citrate lyase, a culture of the strain, a lysate of the strain, or a mixture thereof, and the second active substance comprising tartaric acid or a substance containing tartaric acid, or citric acid or a substance containing citric acid.

[0094] Another embodiment provides the use of a composition in improving, preventing or treating metabolic diseases, the composition comprising a first active substance as an active ingredient, tartaric acid or a substance containing tartaric acid, or citric acid or a substance containing citric acid administered thereto, the first active substance comprising a strain expressing tartaric acid dehydratase or citrate lyase, a culture of said strain, a lysate of said strain, or a mixture thereof.

[0095] Another approach provides a method for improving, preventing, or treating metabolic diseases, the method comprising the steps of: administering a composition to a desired individual, the composition comprising a first active substance and a second active substance as active ingredients, the first active substance comprising a strain expressing tartrate dehydratase or citrate lyase, a culture of the strain, a lysate of the strain, or a mixture thereof, the second active substance comprising tartaric acid or a substance containing tartaric acid, or citric acid or a substance containing citric acid.

[0096] Another approach provides a method for improving, preventing, or treating metabolic diseases, the method comprising the steps of: administering to a desired individual a first active substance comprising a strain expressing tartaric acid dehydratase or citrate lyase, a culture of the strain, a lysate of the strain, or a mixture thereof; and administering a second active substance comprising tartaric acid or a substance containing tartaric acid, or citric acid or a substance containing citric acid.

[0097] Another embodiment provides the use of a composition in the preparation of a medicament for improving, preventing or treating metabolic diseases, the composition comprising a first active substance and a second active substance as active ingredients, the first active substance comprising a strain expressing tartrate dehydratase or citrate lyase, a culture of the strain, a lysate of the strain, or a mixture thereof, and the second active substance comprising tartaric acid or a substance containing tartaric acid, or citric acid or a substance containing citric acid.

[0098] Another embodiment provides the use of a composition in the preparation of a medicament for improving, preventing or treating metabolic diseases, the composition comprising a first active substance as an active ingredient, tartaric acid or a substance containing tartaric acid, or citric acid or a substance containing citric acid administered therewith, the first active substance comprising a strain expressing tartaric acid dehydratase or citrate lyase, a culture of said strain, a lysate of said strain, or a mixture thereof.

[0099] The terms and methods described in the invention are equally applicable across the inventions.

[0100] invention effect According to one protocol, when fermentative Lactobacillus strains expressing tartrate dehydratase are administered in combination with tartrate, citric acid, or substances containing either of these, they are highly effective in the prevention or treatment of metabolic diseases due to the synergistic effect of their anti-obesity activity. Attached Figure Description

[0101] Figure 1 This table shows the fold changes in the content of intermediates in the energy metabolism pathway, relative to the overall average, in the culture supernatant of GB102, GB103, and fermentation *Lactobacillus mucinus* strains, as well as the MRS culture supernatant (control group).

[0102] Figure 2 The distribution of dicarboxylic acid metabolism-related genes in the genome of fermenting Lactobacillus strains is shown.

[0103] Figure 3 The figure shows the results confirming the effect of adding tartaric acid or citric acid on the change of succinic acid content in the bacterial culture medium.

[0104] Figure 4 The graph shows the weight gain of mice on a high-fat diet when they were given GB102, tartaric acid or citric acid separately, and when they were given GB102 and tartaric acid or GB102 and citric acid in combination. Detailed Implementation

[0105] Preferred embodiments are given below to aid in understanding the present invention. However, the following embodiments are provided merely to facilitate a better understanding of the present invention, and the present invention is not limited to the following embodiments. Various changes can be made to the embodiments, and the embodiments are not limited to the embodiments disclosed below, but can be implemented in various forms.

[0106] Example 1: Isolation and Identification of Fermenting Lactobacillus strains The method described in Korean Patent Application No. 10-2022-0080568 was used to isolate and identify the *Lactobacillus fermentans* GB102 strain. The entire contents of the aforementioned document are incorporated herein by reference.

[0107] In summary, the *Lactobacillus fermentatus* strain of this invention was isolated from vaginal samples of healthy women undergoing physical examinations at a hospital. Specifically, vaginal samples were collected by swab and streaked onto Rogosa SL (MRS) agar plates, incubated at 37°C in an anaerobic chamber for 48 hours. After bacterial colony growth, single colonies were subcultured on fresh MRS agar plates for pure isolation. After pure isolation, the strain was cultured on MRS medium. To identify the final screened *Lactobacillus fermentatus* strain GB102, the 16S rRNA gene sequence was analyzed. The 16S rRNA gene sequence was obtained by PCR using primers 27F and 1492R targeting the bacterial 16S rRNA gene. After analysis using Sanger sequencing, the 16S rRNA sequence of *Lactobacillus fermentatus* GB102 is listed in Sequence Listing 1. The inventors have named strain GB102 "*Lactobacillus fermentatus*". Lactobacillus fermentum The image was deposited on January 14, 2020, at the Korean Collection for Type Cultures (KCTC), which houses the Korea Institute of Biotechnology.

[0108] At the same time, fermented lactobacillus ( Lactobacillus fermentum The strain name has been changed to Lactobacillus fermentans ( Limosilactobacillus fermentum In the following experimental examples, the existing strain names and the changed strain names are described interchangeably.

[0109] Experimental Example 1: Confirmation of changes in the content of energy metabolism-related intermediates in strain GB102 Energy metabolism-related intermediates were analyzed in the supernatant of MRS culture (control group), GB102, GB103, and fermentation Lactobacillus culture. Specifically, the changes in the contents of lactic acid, pyruvic acid, fumaric acid, succinic acid, and malic acid were measured in the supernatant of each strain.

[0110] The results are shown in Figure 1 .

[0111] Figure 1 This table shows the fold changes in the content of intermediates in the energy metabolism pathway, relative to the overall average, in the culture supernatant of GB102, GB103, and fermentation *Lactobacillus mucinus* strains, as well as the MRS culture supernatant (control group).

[0112] like Figure 1 As shown, succinic acid, which is known to be involved in the energy consumption activity of brown fat, was significantly increased during GB102 culture.

[0113] This means that GB102 is very effective in preventing or treating metabolic diseases such as obesity.

[0114] Example 2: Identification of the encoding genes for tartrate dehydrogenase and citrate lyase in strain GB102 2.1. Culture and genome extraction of *Lactobacillus fermentans* isolates To understand the genomic characteristics of strain GB102, the whole genome sequence of strain Lactobacillus fermentans GB102 was performed using next-generation sequencing technology (NGS) and bioinformatics techniques.

[0115] The strain was cultured in MRS liquid medium in an anaerobic chamber at 37°C for 4 hours, and genomic DNA was extracted from the culture using the MG Genomic DNA Purification Kit (MGMED, Inc., Korea). To obtain long reads with an average length of over 10 kb, sequence analysis was performed using a PacBio RS II instrument; to generate short sequence fragments with high precision and a length less than 500 bp to compensate for the lower precision of the long reads, sequence analysis was performed using a NovaSeq 6000 instrument. The long reads were assembled into a high-quality genome draft using the HGAP2 workflow of the SMRT analysis server. SNPs and InDel errors that might exist in the assembled genome draft sequence were corrected using both long reads and short sequence fragments. From the completed genome sequence, coding DNA sequences (CDS) were predicted using the Prodigal program, and rRNA and tRNA were predicted using the RFAM tool. The predicted CDS is based on the publicly available UniProt database, GenBank nr database, subsystem database, PFAM database, and COG database, and is predicted by performing a homology search (using the BLAST algorithm).

[0116] 2.2 Ortholog Clustering The whole genome sequencing sequences of 34 Lactobacillus fermentans strains disclosed in GenBank were compared with the genome sequence of strain GB102 extracted according to the method in Example 2.1.

[0117] Specifically, the orthoMCL program was used to perform ortholog clustering of protein-coding genes present in the strain's genome. The clustering results confirmed a difference in gene lists between the GB102 strain, which accumulates succinic acid, and isolated strains that do not. It was confirmed that the GB102 strain, unlike other Lactobacillus fermentum strains, contains genes encoding both tartrate dehydrogenase and citrate lyase, which are genes related to tricarboxylic acid / dicarboxylic acid metabolism.

[0118] Figure 2 The results show the distribution of orthologous genes in the genome of the GB102 strain, which was extracted and assembled according to the method in Example 2.1, by comparing the whole genome sequencing sequences of the Lactobacillus fermentans strain published in GenBank with those of the strain. This analysis reveals the distribution of dicarboxylic acid metabolism-related genes in the Lactobacillus fermentans strain genome.

[0119] like Figure 2As shown, among the publicly available Lactobacillus fermentum genomes in GenBank, only strain HFD1 is identical to strain GB102, possessing genes encoding both citrate lyase and tartrate dehydrogenase. Furthermore, it was confirmed that strains containing tartrate dehydrogenase are limited to strains B44 and HFD1.

[0120] These results suggest that it is uncommon for fermenting *Lactobacillus mucilaginosus* strains to simultaneously contain genes encoding both tartrate dehydrogenase and citrate lyase.

[0121] Example 3: Comparison of succinic acid content in the supernatant of bacterial cultures when tartaric acid or citric acid was added. The effects of adding tartaric acid or citric acid on the changes in succinic acid content in the bacterial culture medium were confirmed.

[0122] Specifically, 200 μL of the supernatant from each strain was placed in a 30 Hz blender (MM400; Retsch®, Haan, Germany) and extracted with cold methanol for 10 minutes, followed by sonication for 10 minutes. Then, after centrifugation at 12,000 rpm and 4°C for 10 minutes (Centrifuge 5427R; Eppendorf, Hamburg, Germany), the supernatant was filtered through a 0.2 μm PTFE (polytetrafluoroethylene) membrane (Chromdisc, Daegu, South Korea) and evaporated using a high-speed vacuum concentrator (Labex, Gimpo, South Korea). For metabolite analysis, each dried sample was reconstituted with 80% methanol, adjusting the final concentration to 20,000 ppm. For GC-MS analysis, 100 μL of the reconstituted sample was dried using a high-speed vacuum concentrator and derivatized as follows.

[0123] First, 50 μL of methoxyamine hydrochloride (pyridine solvent, concentration 20 mg / mL) was added to the dried sample and placed in a hot mixer at 30 °C for 90 minutes. Then, 50 μL of MSTFA was added as a silanizing agent, and the mixture was incubated at 37 °C for 30 minutes. 1 μL of each derivatized sample was injected into a CHroZen GC system (Young In Chromass; Anyang, South Korea) equipped with an L-PAL3 autosampler system and a YL9600 mass scanning spectrometer. Metabolite separation was performed using a DB-5MS column (30 m length × 0.25 mm inner diameter × 0.25 μm particle size; Agilent, Santa Clara, USA) with helium as the carrier gas at a constant flow rate of 1.0 mL / min. The GC column temperature was maintained at 60 °C for 3 minutes, then increased to 320 °C at a rate of 10 °C per minute, and held at this final temperature for another 3 minutes.

[0124] For a scan range of 50-600 amu, the Mass data acquisition rate was set to 2750 amu / s, and then the electron ionization (EI) mode was set to -70 eV. The ion source and transfer line temperatures were set to 230 °C and 250 °C, respectively. The amount of metabolites was expressed as peak area in the chromatogram or as a relative proportion of the average value of all samples.

[0125] Figure 3 The figure shows the results confirming the effect of adding tartaric acid or citric acid on the change of succinic acid content in the bacterial culture medium. Figure 3 In the above, culture supernatant (1) shows the succinic acid content measured after culturing GB102 or fermenting Lactobacillus strain for 6 hours and 16 hours in GIP9 medium with citric acid removed; culture supernatant (2) shows the succinic acid content measured after culturing GB102 or fermenting Lactobacillus strain for 6 hours and 16 hours in GIP9 medium with citric acid added; culture supernatant (3) shows the succinic acid content measured after culturing GB102 or fermenting Lactobacillus strain for 6 hours and 16 hours in GIP9 medium with ta added.

[0126] like Figure 3 As shown, it was confirmed that in the culture supernatant of Lactobacillus fermentation strain, the content of succinic acid in the culture supernatant was almost unchanged regardless of whether tartaric acid or citric acid was added; while in the culture supernatant of GB102 strain, the content of succinic acid increased sharply when tartaric acid or citric acid was added.

[0127] This means that the increased production of succinic acid when tartaric acid or citric acid is added is a specific feature of GB102.

[0128] Experiment Example 4: Changes in body weight gain in mice fed a high-fat diet caused by combined administration of tartaric acid or citric acid. When GB102 was administered in combination with tartaric acid or citric acid, changes in the amount of weight gain in mice on a high-fat diet were confirmed.

[0129] Specifically, after acclimatizing 5-week-old C57 / BL6 mice for 2 weeks, their body weight was measured and they were divided into groups to ensure uniform average body weight. Seven-week-old mice in each group were fed a high-fat diet (HFD), and each group was orally administered the corresponding test substance (200 μL / mouse) daily for 8 weeks. (G1: PBS, G2: GB102 (5 x 10 μL)) 9 CFU / animal + PBS, G3: Tartaric acid (1 mg / animal), G4: GB102 (5 x 10) 9 CFU / animal) + Tartaric acid (1mg / animal), G5: Citric acid (1mg / animal), G6: GB102 (5x10 9CFU / animal + Citric acid (1mg / animal).

[0130] Figure 4 The graph shows the weight gain of mice on a high-fat diet when they were given GB102, tartaric acid or citric acid separately, and when they were given GB102 and tartaric acid or GB102 and citric acid in combination.

[0131] like Figure 4 As shown, comparing the weight results of each group after 8 weeks, the weight gain in the GB102 administration group was significantly greater. p <0.05) was significantly lower in the control group than in the untreated group. In particular, when tartaric acid or citric acid was co-administered with GB102, the weight gain ( p The value was <0.01, which was significantly lower than that of the PBS-treated group.

[0132] This means that GB102 alone is effective in preventing or treating metabolic diseases such as obesity, but when GB102 is administered in combination with tartaric acid or citric acid, it is more effective in preventing or treating metabolic diseases such as obesity.

[0133] The above description of the present invention is for illustrative purposes only. Those skilled in the art should understand that the present invention can be readily modified into other specific forms without altering its technical concept or essential features. Therefore, it should be understood that all aspects of the described embodiments are exemplary and not restrictive.

[0134] [Collection Number] Name of the depository: Korea Institute of Life Sciences Accession number: KCTC14105BP Deposit date: 20200114

Claims

1. A pharmaceutical composition for the prevention or treatment of metabolic diseases, wherein, The composition comprises a first active substance and a second active substance as active ingredients, wherein the first active substance comprises a strain expressing tartrate dehydratase, a culture of the strain, a lysate of the strain, or a mixture thereof. The second active substance contains tartaric acid or a substance containing tartaric acid, or citric acid or a substance containing citric acid; Alternatively, the composition may contain the first active substance as an active ingredient, with the second active substance administered in combination therewith.

2. The pharmaceutical composition for the prevention or treatment of metabolic diseases according to claim 1, wherein, The strain expresses citrate lyase.

3. The pharmaceutical composition for the prevention or treatment of metabolic diseases according to claim 1, wherein, The strain is *Lactobacillus fermentum* (… Limosilactobacillus fermentum ).

4. The pharmaceutical composition for the prevention or treatment of metabolic diseases according to claim 1, wherein, The strain has at least 98.5% sequence identity with the 16S rRNA sequence of SEQ ID NO:

1.

5. The pharmaceutical composition for the prevention or treatment of metabolic diseases according to claim 1, wherein, The strain in question is the strain preserved with the accession number KCTC14105BP.

6. The pharmaceutical composition for the prevention or treatment of metabolic diseases according to claim 1, wherein, The strain contains a mutation of a naturally occurring fermenting *Lactobacillus mucinus* strain.

7. The pharmaceutical composition for the prevention or treatment of metabolic diseases according to claim 1, wherein, The first active substance has anti-obesity activity.

8. The pharmaceutical composition for the prevention or treatment of metabolic diseases according to claim 7, wherein, The anti-obesity activity includes one or more of the following: - Inhibit weight gain; -Increases basal metabolic rate; - Accumulation of brown fat; -Activate the energy consumption of brown fat; -Inhibits hepatic steatosis; - Lowering blood levels of AST (alanine aminotransferase), ALT (alanine aminotransferase, GPT), triglycerides, LDL cholesterol, HDL cholesterol, or total cholesterol; and -Increase the expression of UCP-1 (uncoupling protein 1).

9. The pharmaceutical composition for the prevention or treatment of metabolic diseases according to claim 1, wherein, The tartaric acid is L-tartaric acid, D-tartaric acid, meso tartaric acid, or any combination thereof.

10. The pharmaceutical composition for the prevention or treatment of metabolic diseases according to claim 1, wherein, The tartaric acid and citric acid are contained in the form of salts.

11. The pharmaceutical composition for the prevention or treatment of metabolic diseases according to claim 1, wherein, The second active substance is an extract selected from one or more of the following groups: Tamarind ( Tamarindus indica ),Grape( Vitis vinifera Star fruit () Averrhoa carambola Tree tomato ( Solanum betaceum ), Roselle ( Hibiscus sabdariffa ), Citrus ( Citrus spp. ),apple( Malus domestica ),plum( Prunus mume ), dates ( Phoenix dactylifera European sweet cherry ( Prunus avium, Prunus cerasus ),raspberry( Rubus idaeus ),blackberries( Rubus fruticosus ), black tea curd ( Ribes rubrum ),cranberry( Vaccinium macrocarpon ),pineapple( Ananas comosus ), Hovenia dulcis ( Hovenia dulcis ),banana( Musa spp. ),Pomegranate( Punica granatum ), wild chokeberry ( Aronia melanocarpa ), highbush blueberry ( Vaccinium corymbosum ), planting paddy fields ( Rubus coreanus ) and mulberry ( Morus alba ).

12. The pharmaceutical composition for the prevention or treatment of metabolic diseases according to claim 1, wherein, The metabolic disease is selected from one or more of the following groups: type 1 diabetes, type 2 diabetes, impaired glucose tolerance, abnormal fasting blood glucose, dyslipidemia, lipid metabolism disorder, obesity, fatty liver, insulin resistance syndrome, and glucose intolerance syndrome.

13. The pharmaceutical composition for the prevention or treatment of metabolic diseases according to claim 1, wherein, The first active substance and the second active substance are administered simultaneously, sequentially, or in reverse order.

14. The pharmaceutical composition for the prevention or treatment of metabolic diseases according to claim 1, wherein, The composition comprises a first oral formulation and a second oral formulation, the first oral formulation comprising the first active substance, the second oral formulation comprising the second active substance, and the first oral formulation and the second oral formulation being administered orally.

15. A pharmaceutical composition for the prevention or treatment of metabolic diseases, wherein, The composition comprises a first active substance and a second active substance as active ingredients, wherein the first active substance comprises a strain expressing citrate lyase, a culture of the strain, a lysate of the strain, or a mixture thereof. The second active substance contains tartaric acid or a substance containing tartaric acid, or citric acid or a substance containing citric acid; Alternatively, the composition may contain the first active substance as an active ingredient, with the second active substance administered in combination therewith.

16. A health food product used to prevent or improve metabolic diseases, wherein, The health functional food contains a first active substance and a second active substance as effective ingredients. The first active substance comprises a strain expressing tartrate dehydratase, a culture of the strain, a lysate of the strain, or a mixture thereof. The second active substance contains tartaric acid or a substance containing tartaric acid, or citric acid or a substance containing citric acid; Alternatively, the health food may contain the first active substance as an effective ingredient, and the second active substance may be administered in combination with it.

17. Use of a composition in the prevention or treatment of metabolic diseases, wherein, The composition comprises a first active substance and a second active substance as active ingredients, wherein the first active substance comprises a strain expressing tartrate dehydratase, a culture of the strain, a lysate of the strain, or a mixture thereof. The second active substance contains tartaric acid or a substance containing tartaric acid, or citric acid or a substance containing citric acid; Alternatively, the composition may contain the first active substance as an active ingredient, with the second active substance administered in combination therewith.

18. A method for improving, preventing, or treating metabolic diseases, the method comprising the following steps: The composition is applied to a desired individual and comprises a first active substance and a second active substance as active ingredients. The first active substance comprises a strain expressing tartaric acid dehydratase or citrate lyase, a culture of the strain, a lysate of the strain, or a mixture thereof. The second active substance comprises tartaric acid or a substance containing tartaric acid, or citric acid or a substance containing citric acid.

19. A method for improving, preventing, or treating metabolic diseases, the method comprising the following steps: The first active substance is applied to the desired individual, the first active substance comprising a strain expressing tartrate dehydratase or citrate lyase, a culture of the strain, a lysate of the strain, or a mixture thereof; as well as Apply a second active substance, which comprises tartaric acid or a substance containing tartaric acid, or citric acid or a substance containing citric acid.

20. Use of a composition in the preparation of a medicament for improving, preventing or treating metabolic diseases, wherein, The composition comprises a first active substance and a second active substance as active ingredients, wherein the first active substance comprises a strain expressing tartrate dehydratase, a culture of the strain, a lysate of the strain, or a mixture thereof. The second active substance contains tartaric acid or a substance containing tartaric acid, or citric acid or a substance containing citric acid; Alternatively, the composition may contain the first active substance as an active ingredient, with the second active substance administered in combination therewith.

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