Composition for preventing or treating metabolic diseases by using strain, that expresses tartaric acid dehydratase, or combination therapy comprising same

Abstract

The present invention relates to a composition for preventing or treating metabolic diseases by using a strain, that expressing tartaric acid dehydratase, or combination therapy comprising same. If a Limosilactobacillus fermentum strain expressing tartaric acid dehydratase is co-administered with tartaric acid, citric acid, or materials containing each of tartaric acid and citric acid, according to one aspect, the present invention is very effective in preventing or treating metabolic diseases through a synergistic effect of anti-obesity activity.

Description

Composition for preventing or treating metabolic diseases using a strain expressing tartaric acid dehydratase or a combination therapy comprising the same

[0001] The present invention relates to a composition for preventing or treating metabolic diseases using a strain expressing tartaric acid dehydratase or a combination therapy comprising the same.

[0002] Obesity is a condition in which the body accumulates excessive fat tissue due to an increase in the size or number of fat cells, i.e., the body has more fat than it needs. Obesity is considered the leading correctable cause of death among modern people and is a chronic disease that must be managed to lead a healthy and vibrant life. Obesity is known to be associated with various diseases, including hypertension, diabetes, insulin resistance, glucose intolerance, hyperlipidemia, angina, fatty liver disease, cancer, and degenerative arthritis. As the metabolic diseases caused by obesity are becoming more diverse and more severe, prevention and treatment are urgently needed.

[0003] The development of obesity treatments primarily aims to reduce weight, suppress fat accumulation, and improve metabolism. Many obesity treatments currently in development and commercialization utilize mechanisms that reduce appetite to promote weight loss. While these appetite suppressants offer significant weight loss benefits, their mechanism of action, acting on the central nervous system to suppress appetite and thus food intake, poses a significant challenge: their effects may not last long or cause serious side effects with long-term use. Therefore, there is an urgent need to develop obesity treatments that do not cause serious side effects.

[0004] Meanwhile, recent research has revealed that certain compounds, such as succinic acid, may play a crucial role in regulating metabolism and maintaining energy balance. Succinic acid, a key intermediate in the Krebs cycle, promotes efficient energy production and lipolysis, thereby inhibiting fat accumulation in the body (Physiological Reports. 2020;8:e14630).

[0005] Furthermore, succinic acid has been reported to activate energy expenditure in brown fat (Nature. Aug. 2018; 560(7716): 102-106.). Unlike white fat, brown fat consumes energy through thermogenesis among adipocytes in an individual, and the activity of brown fat is inversely correlated with age and body metabolic status, especially obesity. Accumulation of succinic acid induces uncoupling protein 1 (UCP1)-dependent thermogenesis in brown adipose tissue (BAT), which prevents diet-induced obesity and improves glucose tolerance.

[0006] Succinic acid is an intermediate metabolite produced and utilized by many living organisms, excluding viruses. There are few reported cases of adverse effects due to the ingestion of succinic acid. The above-mentioned mechanisms of suppressing fat accumulation and activating energy consumption of brown fat are the basis for succinic acid being considered a promising candidate for the treatment of metabolic diseases such as obesity.

[0007] However, intestinal succinic acid exists in very low concentrations in germ-free mice, and its concentration has been reported to increase in the intestines where microorganisms reside (Sci Transl Med. 2014 January 22; 6(220): 220ra11.), which suggests that the accumulation of succinic acid in the intestines is due to intestinal microorganisms rather than succinic acid intake, and it means that the discovery of strains capable of accumulating succinic acid in the intestines is necessary for the development of treatments for metabolic diseases such as obesity.

[0008] Accordingly, the present inventors discovered a strain capable of accumulating succinic acid by expressing tartrate dehydratase and / or citrate lyase, and developed a composition for preventing or treating metabolic diseases using the strain.

[0009] One aspect is to provide a strain expressing tartrate dehydratase.

[0010] Another aspect is to provide a strain expressing citrate lyase.

[0011] Another aspect is to provide a strain expressing tartrate dehydratase; and a complex of tartrate or a substance comprising tartrate, or citrate or a substance comprising citrate.

[0012] Another aspect provides a pharmaceutical composition for preventing or treating a metabolic disease, comprising a first active substance comprising a strain expressing tartaric acid dehydratase; and a second active substance containing tartaric acid or tartaric acid, or citric acid or citric acid.

[0013] Another aspect provides a health functional food for preventing or improving metabolic diseases, comprising a first active substance including a strain expressing tartaric acid dehydratase; and a second substance containing tartaric acid or tartaric acid, or citric acid or citric acid.

[0014] Another aspect provides a use for preventing or treating a metabolic disease, comprising a first active substance comprising a strain expressing tartrate dehydratase, a culture of the strain, a lysate of the strain, or a mixture thereof; and a second active substance comprising tartaric acid or a substance comprising tartaric acid, or citric acid or a substance comprising citric acid, as active ingredients; or a composition comprising the first active substance as an active ingredient and co-administering the second active substance.

[0015] Another aspect provides a method for improving, preventing or treating a metabolic disease, comprising administering to a subject in need thereof a composition comprising as active ingredients a 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 a second active substance comprising tartaric acid or a material comprising tartaric acid, or citric acid or a material comprising citric acid.

[0016] Another aspect provides a method for improving, preventing or treating a metabolic disease, comprising the steps of: administering to a subject in need thereof a 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 administering a second active substance comprising tartaric acid or a substance comprising tartaric acid, or citric acid or a substance comprising citric acid.

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

[0018] One aspect provides a strain expressing tartrate dehydratase.

[0019] In one specific example, the strain may further express citrate lyase or ATP-citrate lyase. In the strain, tartrate dehydratase and citrate lyase are each independently expressed. The tartrate dehydratase is an enzyme that dehydrates tartrate to convert it into succinate, and the citrate lyase is an enzyme involved in decomposing citrate to produce succinate.

[0020] In one specific example, a strain expressing tartrate dehydratase and / or citrate lyase is characterized by accumulating succinate.

[0021] In one specific example, the strain may be a Lactobacillus group strain, specifically Limosilactobacillus fermentum.

[0022] The Lactobacillus group is a group of microorganisms, including aerobic or facultatively anaerobic Gram-positive rod-shaped bacilli, widely distributed in nature. Microorganisms belonging to this group include Limosilactobacillus fermentum, Lactiplantibacillus plantarum, and Latilactobacillus sakei.

[0023] Lactobacillus has been reclassified into 23 taxa, including Limosilactobacillus, Lactiplantibacillus, and Latilactobacillus, and the existing strain names and the changed strain names can be used interchangeably in this specification. For example, Limosilactobacillus fermentum can be used interchangeably with Lactobacillus fermentum, Lactiplantibacillus fermentum, or Latilactobacillus fermentum.

[0024] The present inventors conducted research to develop a new strain and selected Limosilactobacillus fermentum GB102 as a candidate strain with anti-obesity activity. This strain was deposited with the Biological Resource Center of the Korea Research Institute of Bioscience and Biotechnology on September 6, 2019, under accession number SD1335. The same strain was deposited with the Biological Resource Center of the Korea Research Institute of Bioscience and Biotechnology on January 14, 2020, under accession number KCTC14105BP. This strain is a probiotic strain, is harmless to the human body, and can be used without side effects.

[0025] In this specification, the term "Limosilactobacillus fermentumGB102" may be described interchangeably with L. fermentumGB102 strain, Lactobacillus fermentum GB102 strain, Lactobacillus fermentum GB102 strain, or GB102.

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

[0027] In one specific example, the strain may be a strain comprising a 16S rRNA gene consisting of a nucleotide sequence of sequence number 1.

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

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

[0030] In one specific example, the strain may be a live cell, a dead cell, or a cytoplasmic fraction obtained by disrupting a strain, and preferably a live cell.

[0031] In one specific example, the strain may have anti-obesity activity. Specifically, the anti-obesity activity may include one or more activities selected from the group consisting of suppressing weight gain, increasing energy metabolism or basal metabolic rate, accumulating brown fat, activating brown fat, reducing body fat mass relative to lean body mass, suppressing hepatic steatosis, reducing blood alanine aminotransferase (AST), alanine transaminase (ALT), triglycerides, low-density cholesterol, high-density cholesterol, or total cholesterol, and increasing the expression of Uncoupling protein 1 (UCP-1). Therefore, the strain may suppress weight gain.

[0032] In one specific example, the strain may be effective in promoting complex health when treating metabolic diseases such as obesity by helping intestinal health. Specifically, the strain may control intestinal microflora through the production of large amounts of organic acids. When the strain is taken in combination with prebiotics (e.g., foods containing tartaric acid, citric acid, etc.), the strain may promote succinic acid production while maintaining intestinal microflora balance, and may alleviate bowel movement disorders felt during dietary therapy for metabolic disease treatment, thereby achieving an enhanced effect in the treatment of metabolic diseases such as obesity.

[0033]

[0034] Another aspect provides a pharmaceutical composition for preventing or treating a metabolic disease, comprising a first active substance comprising a strain expressing tartrate dehydratase, a culture of the strain, a lysate of the strain, or a mixture thereof; and a second active substance comprising tartrate or a substance comprising tartrate, or citrate or a substance comprising citric acid, as active ingredients; or comprising the first active substance as an active ingredient and the second active substance being administered in combination.

[0035] In one specific example, the pharmaceutical composition for preventing or treating a metabolic disease comprises a first active substance comprising a strain of Limosilactobacillus fermentum, a culture of the strain, a lysate of the strain, or a mixture thereof, and a second active substance comprising tartaric acid or a substance comprising tartaric acid, or citric acid or a substance comprising citric acid as active ingredients; or the first active substance may be an active ingredient, and the second active substance may be a composition or complex for co-administration.

[0036] The above “strain” is as described above.

[0037] The term "culture" in this specification may be used interchangeably with "culture solution", "culture supernatant", "culture supernatant", "conditioned culture solution" or "conditioned medium", and may mean the entire medium including the strain, its metabolites, extra nutrients, etc. obtained by culturing the strain for a certain period of time in a medium capable of supplying nutrients so that the Lactobacillus group strain can grow and survive in vitro. The culture means a product obtained by culturing a probiotic strain in a known medium, and the product may or may not include the strain itself. The medium may be selected from known liquid media or solid media, and may be, for example, but is not limited to, MRS liquid medium, GAM liquid medium, MRS agar medium, GAM agar medium, BL agar medium.

[0038] The term "lysate" herein may be used interchangeably with "lysate" and refers to a solution or suspension of broken cells of a microorganism, such as Rimosylactobacillus fermentum, in an aqueous medium. A cell lysate may contain macromolecules, such as DNA, RNA, proteins, peptides, carbohydrates, lipids, etc., and / or micromolecules, such as amino acids, sugars, fatty acids, etc., or fractions thereof. The lysate may also contain cell debris, which may be smooth or granular in structure.

[0039] The above culture solution may include the culture solution itself, a concentrate thereof, or a lyophilized product obtained by culturing the strain, or a culture supernatant, a concentrate thereof, or a lyophilized product obtained by removing the strain from the culture solution.

[0040] The above culture solution may be obtained by culturing Rimosylactobacillus fermentum in an appropriate medium (e.g., MRS plate medium) at a temperature of more than 10°C or less than 40°C for a certain period of time, for example, 4 to 50 hours. A person of ordinary skill in the art may appropriately select or modify the culture medium and culture conditions for culturing Rimosylactobacillus fermentum.

[0041] In one specific example, the first active ingredient may comprise the strain of Rimosyl Lactobacillus fermentum as an active ingredient alone, or may comprise one or more pharmaceutically acceptable carriers, excipients or diluents.

[0042] In one specific example, the second active material may include tartrate or a material containing it.

[0043] The above-mentioned succinic acid is a type of organic acid also called tartaric acid, and its chemical name is 2,3-dihydroxybutanedioic acid, and may be represented by the following chemical formula 1:

[0044] [Chemical Formula 1]

[0045]

[0046] The above tartaric acid comprises L-tartaric acid, D-tartaric acid, meso-tartaric acid, or any combination thereof, and preferably, L-tartaric acid.

[0047] The above-mentioned citrate may be included in the form of a salt.

[0048] The substance containing the above main acid is a berry extract, specifically, tamarind (Tamarindus indica), grape (Vitis vinifera), carambola (Averrhoa carambola), tamarillo (Solanum betaceum), damasonia (Hibiscus sabdariffa), citrus (Citrus spp.), apple (Malus domestica), plum (Prunus mume), date palm (Phoenix dactylifera), cherry (Prunus avium, Prunus cerasus), raspberry (Rubus idaeus), blackberry (Rubus fruticosus), redcurrant (Ribes rubrum), cranberry (Vaccinium macrocarpon), pineapple (Ananas comosus), hawthorn (Hovenia dulcis), banana (Musa spp.), pomegranate (Punica granatum), chokeberry (Aronia melanocarpa), blueberry (Vaccinium It may include extracts of berries such as corymbosum, black raspberry (Rubus coreanus), and morus alba, or wine made using the berries mentioned above, but is not limited thereto. Specifically, the substance including the tartaric acid is not limited in form, such as liquid or powder.

[0049] In one specific example, the second active material may include citrate or a material containing the same.

[0050] The above citric acid is a type of organic acid also called citric acid, and its chemical formula is 2-hydroxypropane-1,2,3-tricarboxylic acid, and may be represented by the following chemical formula 2:

[0051] [Chemical Formula 2]

[0052]

[0053] The above citric acid may be included in the form of a salt.

[0054] The substance containing the above citric acid may include, but is not limited to, an extract of citrus (Citrus spp.), specifically, extracts of orange (Citrus sinensis), lemon (Citrus limon), lime (Citrus aurantiifolia), grapefruit (Citrus paradisi), tangerine (Citrus reticulata), etc. Specifically, the substance containing the above citric acid is not limited in its form, such as liquid or powder.

[0055] In one specific example, the second active substance may induce succinic acid production in a strain expressing tartrate dehydratase and / or citrate lyase. That is, the second active substance may be provided as a substrate for the first active substance comprising a strain expressing tartrate dehydratase and / or citrate lyase.

[0056] The term succinic acid in this specification may be used interchangeably with succinic acid and may be represented by the following chemical formula 3:

[0057] [Chemical Formula 3]

[0058]

[0059] The above succinic acid, when administered to an individual, induces the accumulation of brown fat among the individual's adipocytes and activates energy consumption of brown fat. Mammals have two main types of adipose tissue: white adipose tissue and brown adipose tissue (Gesta S et al., Cell 2007;131:242-256). In addition, beige adipose tissue is known to contribute to the brownization of white fat. Since white adipose tissue is used for energy storage in an individual, its excess is associated with obesity. In contrast, brown adipose tissue has a high concentration of mitochondria and can uniquely express uncoupling protein 1 (UCP1). Therefore, brown adipose tissue can play a role in improving obesity by inducing energy consumption and thermogenesis. Therefore, succinic acid produced by the second active substance exhibits anti-obesity activity by increasing energy metabolism or basal metabolic rate through the accumulation of brown fat and the activation of energy consumption of brown fat.

[0060] In one specific example, the first active ingredient and the second active ingredient may be administered concurrently, sequentially, or in reverse order. For example, the anti-obesity activity of the first active ingredient may exhibit a synergistic effect when administered concurrently with the second active ingredient, further inducing succinic acid production.

[0061] As used herein, the terms "combination therapy," "combined treatment," "combination administration," or "in combination" refer to any form of simultaneous or concurrent treatment using at least two separate agents useful for treating a disease. The components of the combination therapy may be administered simultaneously, sequentially, in reverse order, or in any order. The components may be administered in different dosages, at different administration frequencies, or via different routes, as appropriate.

[0062] As used herein, the term "administration" means introducing a given substance into a subject in an appropriate manner, and "subject" refers to any living organism, including humans, rats, mice, and livestock, that may have a metabolic disease. A specific example may be a mammal, including humans.

[0063] The term "administered simultaneously" as used herein is not particularly limited and means that the components of the combination therapy are administered substantially simultaneously, for example, as a mixture or in an immediately subsequent sequence.

[0064] The term "administered sequentially" as used herein is not particularly limited and means that the components of the combination therapy are administered not simultaneously, but one after another or in clusters with a specific time interval between administrations. The time intervals may be the same or different between the administrations of each of the components of the combination therapy, and may be selected, for example, from the range of 2 minutes to 96 hours, 1 day to 7 days, or 1 week, 2 weeks, or 3 weeks. Typically, the time interval between administrations may range from several minutes to several hours, for example, from 2 minutes to 72 hours, from 30 minutes to 24 hours, or from 1 to 12 hours. Additional examples include time intervals ranging from 24 to 96 hours, from 12 to 36 hours, from 8 to 24 hours, and from 6 to 12 hours.

[0065] The above combination administration may mean administering the Rimosylactobacillus fermentum strain and tartaric acid or a substance containing tartaric acid, or citric acid or a substance containing citric acid, simultaneously, sequentially, or separately, and in any order. Specifically, the combination administration may mean administering simultaneously a first active substance containing the Rimosylactobacillus fermentum strain and a second active substance containing tartaric acid or a substance containing tartaric acid, or citric acid or a substance containing citric acid, or administering the Rimosylactobacillus fermentum strain and then administering the second active substance.

[0066] A combination therapy according to the present invention may be defined as providing a synergistic effect if the efficacy, as measured by, for example, the degree of response, the rate of response, the time to disease progression, or the duration of survival, is therapeutically superior to the efficacy obtained by administering one or more of the components of the combination therapy at their usual doses. For example, the efficacy of the combination therapy is synergistic if the efficacy is therapeutically superior to the efficacy obtained by using each of the components alone. In particular, a synergistic effect is considered to exist if the usual dose of the strain of Rimosylactobacillus fermentum and tartaric acid or a substance comprising tartaric acid, or citric acid or a substance comprising citric acid, can be reduced without compromising one or more of the degree of response, the rate of response, the time to disease progression, and the duration of the response, and in particular without compromising the duration of the response, and with reduced and / or fewer problematic side effects than would occur when each component is used at its usual doses.

[0067] The term "pharmaceutical composition" as used herein means a mixture comprising one or more active ingredients and various ingredients that deliver or assist them for a specific therapeutic, preventive or diagnostic purpose.

[0068] The pharmaceutical composition may additionally 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, a disintegrant, a binder, a glidant, or a combination thereof. The excipient may be microcrystalline cellulose, lactose, low-substituted hydroxycellulose, or a combination thereof. The disintegrant may be calcium carboxymethylcellulose, sodium starch glycolate, calcium dihydrogen phosphate anhydrous, or a combination thereof. The binder may be polyvinylpyrrolidone, low-substituted hydroxypropyl cellulose, hydroxypropyl cellulose, or a combination thereof. The glidant may be magnesium stearate, silicon dioxide, talc, or a combination thereof.

[0069] When the above pharmaceutical composition is formulated, it can be prepared using diluents or excipients such as lubricants, sweeteners, flavoring agents, emulsifiers, suspending agents, preservatives, fillers, bulking agents, binders, wetting agents, disintegrating agents, and surfactants that are commonly used. Solid preparations for oral administration may include tablets, pills, powders, granules, capsules, etc., and such solid preparations can be prepared by mixing at least one excipient, such as starch, calcium carbonate, sucrose or lactose, gelatin, etc., with the above composition. In addition to simple excipients, lubricants such as magnesium stearate and talc can also be used. Liquid preparations for oral administration include suspensions, solutions, emulsions, and syrups. In addition to commonly used simple diluents such as water and liquid paraffin, they may contain various excipients such as wetting agents, sweeteners, fragrances, and preservatives. Preparations for parenteral administration may include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, and suppositories. Non-aqueous solvents and suspensions may include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate. Suppository bases may include witepsol, macrogol, tween 61, cacao butter, laurin butter, and glycerogelatin. When manufacturing in the form of eye drops, known diluents or excipients may be used.

[0070] The pharmaceutical composition may contain carbohydrates such as glucose, sucrose or dextran, antioxidants such as ascorbic acid or glutathione, chelating agents, low-molecular-weight proteins or other stabilizers to increase stability or absorbability.

[0071] In one specific embodiment, the pharmaceutical composition may be formulated as an oral or parenteral dosage form. Oral dosage forms may include granules, powders, liquids, tablets, capsules, dry syrups, or combinations thereof. Parenteral administration may be administered topically, or by intraperitoneal injection, intrarectal injection, subcutaneous injection, intravenous injection, intramuscular injection, or intrathoracic injection.

[0072] In one specific example, the route of administration of the pharmaceutical composition includes, but is not limited to, oral, intravenous, intramuscular, intraarterial, intramedullary, intrathecal, intracardiac, transdermal, subcutaneous, intraperitoneal, intranasal, enteral, topical, sublingual, or rectal. Specifically, the first active ingredient comprising a Lactobacillus fermentum strain and the second active ingredient comprising tartaric acid or a substance comprising tartaric acid, or citric acid or a substance comprising citric acid may be administered orally in the form of granules, powders, liquids, tablets, capsules, dry syrups, or a combination thereof, or may be administered parenterally by topical application to the skin or by intraperitoneal injection, intrarectal injection, subcutaneous injection, intravenous injection, intramuscular injection, or intrathoracic injection.

[0073] In one specific example, the pharmaceutical composition comprises a first oral formulation comprising the first active substance and a second oral formulation comprising the second active substance, wherein the first and second oral formulations can be administered orally.

[0074] In one specific example, the pharmaceutical composition may be administered orally by including the first active substance and the second active substance in a single oral formulation.

[0075] The above pharmaceutical composition is administered in a pharmaceutically effective amount. The term "pharmaceutically effective amount" means an amount sufficient to treat a disease with a reasonable benefit / risk ratio applicable to medical treatment. The effective dosage level may be determined based on the type and severity of the patient's disease, the activity and sensitivity of the drug, the time of administration, the route of administration, and the excretion rate, the duration of treatment, concomitant medications, and other factors well known in the medical field.

[0076] According to one specific example, the composition may include a strain of Lactobacillus fermentum in an amount of 0.001 wt% to 80 wt% based on the total weight of the composition. In addition, the dosage of the strain of Lactobacillus fermentum may be 0.01 mg to 10,000 mg, 0.1 mg to 1000 mg, 1 mg to 100 mg, 0.01 mg to 1000 mg, 0.01 mg to 100 mg, 0.01 mg to 10 mg, or 0.01 mg to 1 mg. The strain is included in the composition in a therapeutically effective amount or nutritionally effective concentration, for example, the strain is included in an amount of 10 3 10 inland 16 CFU / g, 10 3 10 inland 15 CFU / g, 10 3 10 inland 14 CFU / g, 10 3 10 inland 13 CFU / g, 10 3 10 inland 12 CFU / g, 10 4 10 inland 16 CFU / g, 10 4 10 inland 15 CFU / g, 10 4 10 inland 14 CFU / g, 10 4 10 inland 13 CFU / g, 10 4 10 inland 12 CFU / g, 10 5 10 inland 16 CFU / g, 10 510 inland 15 CFU / g, 10 5 10 inland 14 CFU / g, 10 5 10 inland 13 CFU / g, 10 5 10 inland 12 CFU / g, 10 6 10 inland 13 CFU / g, 10 6 10 inland 12 CFU / g, 10 7 10 inland 13 CFU / g, 10 7 10 inland 12 CFU / g, 10 8 10 inland 13 CFU / g or 10 8 10 inland 12 It may be included in the composition as a content of CFU / g or as a culture of an equivalent number of live or dead cells. Specifically, for adult patients, 1X10 3 Inside 1X10 16 Live or killed bacteria at a concentration of CFU / g may be administered once or in multiple doses. However, the dosage may vary depending on factors such as the formulation method, administration method, patient age, weight, sex, pathological condition, food, administration time, administration route, excretion rate, and response sensitivity, and those skilled in the art can appropriately adjust the dosage considering these factors. In one embodiment, the composition comprises killed dried strains, and may be administered at a dose of 1 g to 10 g, 0.5 g to 1.5 g, 2.5 g to 3.5 g, or 4.5 g to 5.5 g.

[0077] In one aspect, the pharmaceutical composition may be administered once a day or in several divided doses. Specifically, the pharmaceutical composition may be administered in a cycle of 6 days of administration followed by 1 day of rest, 5 days of administration followed by 2 days of rest, or 4 days of administration followed by 3 days of rest, based on a 7-day schedule. More specifically, the pharmaceutical composition may be administered in a cycle of 5 days of administration followed by 2 days of rest.

[0078] In addition, the pharmaceutically effective amount and effective dosage of the pharmaceutical composition may vary depending on the method of formulation, administration method, administration time, and / or administration route of the pharmaceutical composition. In addition, the type and degree of the response to be achieved by administration of the pharmaceutical composition, the type, age, weight, general health condition, symptoms or degree of the disease, sex, diet, excretion, drugs used simultaneously or simultaneously in the subject, other components of the composition, etc., may vary depending on various factors, and similar factors well known in the pharmaceutical field. A person having ordinary skill in the art can easily determine and prescribe an effective dosage for the desired treatment. The pharmaceutical composition according to the present invention may be administered once a day or divided into several times. Therefore, the above dosage does not limit the scope of the present invention in any way. The dosage of the pharmaceutical composition may be 1 ug / kg / day to 1,000 mg / kg / day.

[0079] The subject may be a mammal, such as a human, cow, horse, pig, dog, sheep, goat, or cat. The subject may be an individual in need of treatment for side effects resulting from taking a metabolic disease treatment drug.

[0080] In one specific example, the pharmaceutical composition for preventing or treating a metabolic disease may include a first active substance including a strain expressing citrate lyase, a culture of the strain, a lysate of the strain, or a mixture thereof; and a second active substance including tartaric acid or a substance including tartaric acid, or citric acid or a substance including citric acid, as active ingredients; or may include the first active substance as an active ingredient and the second active substance may be administered in combination.

[0081] The term "treatment" herein may refer to the healing of metabolic diseases and the like in a shorter period of time compared to natural healing. Such treatment may include improvement and / or alleviation of the metabolic disease. Furthermore, such treatment may refer to the healing and / or recovery of symptoms resulting from the metabolic disease.

[0082] As used herein, the term "prevention" refers to a method of partially or completely delaying or preventing the onset or recurrence of a disease, disorder, or its associated symptoms, preventing the acquisition or reacquisition of a disease or disorder, or reducing the risk of acquiring a disease or disorder. For example, the term "prevention" refers to any action that inhibits or delays the onset of a metabolic disease by administering a composition according to the present invention.

[0083] In this specification, the term "including as an active ingredient" means that a strain of Rimosyl Lactobacillus fermentum, a vesicle derived from the strain, a lysate of the strain, a culture medium, or an extract of the culture medium thereof, or tartaric acid or citric acid are added as a main ingredient, and includes formulation in various forms by adding various ingredients as auxiliary ingredients for drug delivery and stabilization, etc.

[0084] The term "obesity" herein refers to a state of excessive body fat. Obesity can be clinically defined as a body mass index (BMI) of 25 or higher in Korea and 30 or higher according to the World Health Organization (WHO). Generally, obesity refers to a case in which body weight is higher than the normal range, but even if the body weight is not high, obesity can occur if the proportion of body fat among the body composition is high. Obesity can occur in both adults and children. Obesity can cause not only weight gain but also obesity-related metabolic diseases such as overeating, excessive drinking, and bulimia, hypertension, diabetes, increased plasma insulin concentration, 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 for the prevention or treatment of obesity as well as obesity-related metabolic diseases. Furthermore, the composition can be used in individuals who desire to reduce body weight or body fat, even if they are not obese.

[0085] The aforementioned obesity can be caused by various factors. For example, the causes may include a high-fat diet, reduced exercise, genetics, psychological factors, endocrine disorders, metabolic disorders, and social and environmental factors. In particular, the aforementioned obesity may be induced by a high-fat diet.

[0086] The term "metabolic disease" used herein refers to a general term for various disorders that occur due to abnormalities in the body's metabolic processes, also called metabolic disorders. Generally, they are caused by imbalances in carbohydrates, lipids, proteins, vitamins, electrolytes, and water. Specifically, the metabolic diseases include, but are not limited to, metabolic syndrome, type 1 diabetes, type 2 diabetes, hypertension, hyperlipidemia, obesity, fatty liver, insulin resistance, glucose tolerance syndrome, angina pectoris, coronary arteriosclerosis, and arteriosclerosis.

[0087] According to one 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, impaired fasting blood glucose, dyslipidemia, lipid metabolism disorder, obesity, fatty liver, insulin resistance syndrome, and glucose tolerance syndrome.

[0088]

[0089] Another aspect is to provide a health functional food for preventing or improving metabolic diseases, comprising a first active substance including a strain expressing tartrate dehydratase, a culture of the strain, a lysate of the strain, or a mixture thereof; and a second active substance including tartaric acid or a substance including tartaric acid, or citric acid or a substance including citric acid, as active ingredients; or comprising the first active substance as an active ingredient and the second active substance being administered in combination.

[0090] In the above health functional food, the “strain”, “first active substance”, “tartaric acid”, “citric acid”, “second active substance”, “combined administration”, etc. may be within the above-mentioned range.

[0091] The term "health functional food" as used herein refers to a food manufactured or processed for the purpose of health supplementation using a specific ingredient as a raw material or a specific ingredient contained in a food raw material through extraction, concentration, purification, mixing, etc., and refers to a food designed and processed so that the above-mentioned ingredient can sufficiently exert bioregulatory functions on the body, such as biological defense, biological rhythm regulation, disease prevention, and recovery. The above-mentioned health functional food can perform functions related to the prevention and improvement of metabolic diseases, etc.

[0092] There are no specific restrictions on the types of the above foods. Examples of foods to which the above extract can be added include formulations selected from the group consisting of powders, granules, tablets, capsules, pills, gels, jellies, suspensions, emulsions, syrups, tea bags, infused teas, gums, candies, and health drinks, and include all health foods in the conventional sense.

[0093] The above health functional food may include food additives that are food-related and acceptable, and may include an appropriate carrier commonly used in the manufacture of health functional foods.

[0094] As used herein, the term "food-acceptable" means that the compound exhibits the property of being non-toxic to cells or humans exposed to it.

[0095] The above health functional food may be formulated into one selected from the group consisting of tablets, pills, powders, granules, powders, capsules, and liquid formulations, further comprising one or more of a carrier, diluent, excipient, and additive. Foods to which compounds according to one aspect may be added include various foods, powders, granules, tablets, capsules, syrups, beverages, gum, tea, vitamin complexes, and health functional foods.

[0096] In addition to containing the above-mentioned effective ingredient, the above-mentioned health functional food may contain other ingredients as essential ingredients without special restrictions. For example, it may contain various flavoring agents or natural carbohydrates as additional ingredients, as in conventional beverages. Examples of the above-mentioned natural carbohydrates may include conventional sugars such as monosaccharides, such as glucose, fructose, etc.; disaccharides, such as maltose, sucrose, etc.; and polysaccharides, such as dextrin, cyclodextrin, etc.; and sugar alcohols, such as xylitol, sorbitol, and erythritol. As flavoring agents other than those described above, natural flavoring agents (thaumatin, stevia extracts (e.g., rebaudioside A, glycyrrhizin, etc.)) and synthetic flavoring agents (saccharin, aspartame, etc.) may be advantageously used. The proportion of the above-mentioned natural carbohydrates may be appropriately determined by a person skilled in the art.

[0097] In addition to the above, health functional foods according to the aspect may contain various nutrients, vitamins, minerals (electrolytes), flavoring agents such as synthetic flavoring agents and natural flavoring agents, coloring agents and thickening agents (cheese, chocolate, etc.), pectic acid and its salts, alginic acid and its salts, organic acids, protective colloid thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohol, carbonating agents used in carbonated beverages, etc. These ingredients may be used independently or in combination, and the ratio of these additives may also be appropriately selected by those skilled in the art.

[0098] In the above health functional food, the active ingredient can be added directly to the food or used in combination with other foods or food ingredients, and can be used appropriately according to conventional methods. The amount of the active ingredient mixed can be appropriately determined depending on the intended use (prevention or improvement). Generally, when manufacturing a food or beverage, the health functional food can be added in an amount of about 15% by weight or less, more specifically about 10% by weight or less, based on the raw material. However, in the case of long-term intake for the purpose of health and hygiene or health control, the amount may be below the above range.

[0099] The above health functional food may be provided in combination with a health functional food for preventing or improving a metabolic disease known in the past or with another existing health functional food, and the other health functional food for preventing or improving a metabolic disease may be a health functional food for preventing or improving a metabolic disease known in the past, an existing health functional food, or a newly developed health functional food.

[0100] When the above health functional food includes another health functional food that has a preventive or improving effect on metabolic diseases, it is important to mix the two in an amount that can achieve the maximum effect with the minimum amount without causing side effects, and this can be easily determined by a person skilled in the art.

[0101]

[0102] Another aspect provides a composition for improving, preventing or treating metabolic diseases, comprising as active ingredients a 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 a second active substance comprising tartaric acid or a substance comprising tartaric acid, or citric acid or a substance comprising citric acid.

[0103] Another aspect is to provide a composition for improving, preventing or treating metabolic diseases, comprising as an active ingredient a 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 co-administering tartaric acid or a substance containing tartaric acid, or citric acid or a substance containing citric acid.

[0104] Another aspect provides a method for improving, preventing or treating a metabolic disease, comprising administering to a subject in need thereof a composition comprising as active ingredients a 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 a second active substance comprising tartaric acid or a material comprising tartaric acid, or citric acid or a material comprising citric acid.

[0105] Another aspect provides a method for improving, preventing or treating a metabolic disease, comprising the steps of: administering to a subject in need thereof a 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 administering a second active substance comprising tartaric acid or a substance comprising tartaric acid, or citric acid or a substance comprising citric acid.

[0106] Another aspect provides the use of a composition comprising as active ingredients a 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, for use in the manufacture of a medicament for improving, preventing, or treating a metabolic disease; and a second active substance comprising tartaric acid or a material comprising tartaric acid, or citric acid or a material comprising citric acid.

[0107] Another aspect provides the use of a composition comprising a first active substance as an active ingredient, which comprises a strain expressing tartrate dehydratase or citrate lyase, a culture of the strain, a lysate of the strain, or a mixture thereof, for use in the manufacture of a medicament for improving, preventing, or treating a metabolic disease, and wherein tartaric acid or a substance comprising tartaric acid, or citric acid or a substance comprising citric acid, is co-administered.

[0108] The terms and methods described for the above inventions apply equally to each invention.

[0109] When a strain of Rimosyl Lactobacillus fermentum expressing tartaric acid dehydratase according to the daily pattern is administered in combination with tartaric acid, citric acid or a substance containing them respectively, it is very effective in the prevention or treatment of metabolic diseases due to the synergistic effect of anti-obesity activity.

[0110] Figure 1 is a table showing the change in the content of intermediates of the energy metabolism pathway among metabolites in the culture supernatants of GB102, GB103, and L. fermentum strains and the MRS culture supernatant (control group) as fold change based on the overall average.

[0111] Figure 2 shows the distribution of dicarboxylic acid metabolism-related genes in the genomes of Rimosylactobacillus fermentum strains.

[0112] Figure 3 is a graph showing the results of confirming the effect of adding tartarate or citrate on the change in the content of succinic acid in the strain culture medium.

[0113] Figure 4 is a graph measuring the body weight gain of high-fat diet mice when GB102, tartrate, or citrate were administered separately, and when GB102 and tartrate, or GB102 and citrate were administered together.

[0114] Hereinafter, preferred embodiments are presented to aid understanding of the present invention. However, the following embodiments are provided solely to facilitate a better understanding of the present invention and are not intended to limit the scope of the present invention. The embodiments are susceptible to various modifications, and thus the embodiments are not limited to the embodiments disclosed below and may be implemented in various forms.

[0115]

[0116] Example 1. Isolation and identification of Limosilactobacillus fermentum strains

[0117] Isolation and identification of Limosilactobacillus fermentum GB102 strain was performed using the method described in Republic of Korea Patent Application No. 10-2022-0080568, which is incorporated herein by reference in its entirety.

[0118] Briefly, the Limosilactobacillus fermentum strain of the present invention was isolated from a vaginal sample of a healthy woman who visited a hospital for the purpose of a health checkup. Specifically, a vaginal sample was collected using a cotton swab, inoculated onto a Rogosa SL (MRS) plate medium, and cultured in an anaerobic chamber at 37°C for 48 hours. When bacterial colonies grew, single colonies were subcultured onto new MRS plate medium for pure isolation. After pure isolation, the strain was cultured using MRS medium. The identification of the finally selected Limosilactobacillus fermentum GB102 strain was analyzed using the 16S rRNA gene sequence. The 16S rRNA gene sequence obtained through PCR using 27F and 1492R primers targeting the 16S rRNA gene of bacteria was analyzed by Sanger sequencing, and the 16S rRNA sequence of Lactobacillus fermentum GB102 is shown in Sequence Listing 1. The present inventors named the GB102 strain "Lactobacillus fermentum GB102" (Accession No.: KCTC 14105BP) and deposited it in the Korean collection for type cultures (KCTC) at the Korea Research Institute of Bioscience and Biotechnology on January 14, 2020.

[0119] Meanwhile, the strain name of Lactobacillus fermentum was changed to Limosilactobacillus fermentum. In the experimental examples below, the original strain name and the changed strain name are described interchangeably.

[0120]

[0121] Experimental Example 1. Confirmation of changes in the content of energy metabolism-related intermediates in the GB102 strain.

[0122] Analysis of energy metabolism-related intermediates was performed in the MRS culture supernatant (control), GB102, GB103, and L. fermentum culture supernatants. Specifically, changes in the contents of lactic acid, pyruvic acid, fumaric acid, succinic acid, and malic acid were measured in the culture supernatants of each strain.

[0123] The results are shown in Fig. 1.

[0124] Figure 1 is a table showing the change in the content of intermediates of the energy metabolism pathway among metabolites in the culture supernatants of GB102, GB103, and L. fermentum strains and the MRS culture supernatant (control group) as fold change based on the overall average.

[0125] As shown in Fig. 1, succinic acid, known to be involved in the energy consumption activity of brown fat, was confirmed to be significantly increased during the culture of GB102.

[0126] This means that GB102 is effective in preventing or treating metabolic diseases such as obesity.

[0127]

[0128] Experimental Example 2. Identification of genes encoding tartaric acid dehydrogenase and citrate lyase in the GB102 strain.

[0129] 2.1. Cultivation and genome extraction of isolated strains of Lactobacillus fermentum

[0130] To determine the genomic characteristics of the GB102 strain, the genome sequence of the Rimosylactobacillus fermentum GB102 strain was completely sequenced using next-generation sequencing technology (NGS) and bioinformatics technology.

[0131] 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 read data with an average length of 10 kb or more, sequence analysis was performed using the PacBio RS II instrument. To produce short-term sequence data with a sequence length of less than 500 bp with high accuracy to supplement the long read data with low accuracy, sequence analysis was performed using the NovaSeq 6000 instrument. The long read data were assembled into a high-fidelity genome draft using the HGAP2 pipeline of the SMRT Analysis server. SNP and InDel errors that may exist in the assembled genome draft sequence were corrected using the long read data and short-term sequence data. The Prodigal program was used to predict the coding DNA sequence (CDS) from the completed genome sequence, and the RFAM tool was used to predict rRNA and tRNA. The predicted CDSs were predicted to have functions by performing homology searches (using the BLAST algorithm) based on the publicly available UniProt database, GenBank nr database, Subsystem database, PFAM database, and COG database.

[0132] 2.2. Clustering of orthologous genes

[0133] The complete genome sequences of 34 Lactobacillus fermentum strains published in GenBank were compared with the genome sequence of strain GB102 extracted by the method of Experimental Example 2.1.

[0134] Specifically, ortholog clustering of protein-coding genes present in the genomes of the strains was performed using the orthoMCL program. The clustering results confirmed differences in the gene lists between the GB102 strain, which accumulates succinate, and the isolated strain that does not accumulate succinate. Unlike other Lactobacillus fermentum strains, the GB102 strain was confirmed to contain genes encoding both tartrate dehydrogenase and citrate lyase, which are genes related to tricarboxylic / dicarboxylic acid metabolism.

[0135] Figure 2 shows the distribution of orthologs in the genomes of Rimosylactobacillus fermentum strains, as compared with the distribution of orthologs in the genome sequence of the GB102 strain extracted and assembled using the method of Experimental Example 2.1, showing the distribution of genes related to dicarboxylic acid metabolism in the genomes of Rimosylactobacillus fermentum strains.

[0136] As shown in Fig. 2, among the genomes of Rimosylactobacillus fermentum published in GenBank, only the HFD1 strain was confirmed to possess both genes encoding citrate lyase and tartrate dehydrogenase, similar to the GB102 strain. Additionally, strains possessing tartrate dehydrogenase were confirmed to be limited to the B44 and HFD1 strains.

[0137] These results imply that it is unusual for Rimosylactobacillus fermentum strains to contain genes encoding both tartrate dehydrogenase and citrate lyase.

[0138]

[0139] Experimental Example 3. Comparison of succinic acid content in strain culture supernatants when tartaric acid or citric acid was added.

[0140] The effect of tartarate or citrate addition on the change in succinic acid content in the strain culture medium was confirmed.

[0141] Specifically, 200 μL of each strain culture supernatant was extracted with cold methanol for 10 min in a mixer mill (MM400; Retsch®, Haan, Germany) at a frequency of 30 Hz, followed by sonication for 10 min. Then, the supernatant was centrifuged (Centrifuge 5427R; Eppendorf, Hamburg, Germany) at 12,000 rpm and 4 °C for 10 min, and the supernatant was filtered through a 0.2 μm PTFE (polytetrauoroethylene) filter (Chromdisc, Daegu, Korea) and evaporated using a high-speed vacuum concentrator (Labex, Gimpo, Korea). For metabolite analysis, each dried sample was redissolved in 80% methanol to a final concentration of 20,000 ppm. For GC-MS analysis, 100 μL of each redissolved sample was dried using a high-speed vacuum concentrator and derivatized as follows.

[0142] First, 50 μL of methoxyamine hydrochloride (20 mg / mL in pyridine) was added to the dried samples and incubated in a thermomixer at 30°C for 90 min. Then, 50 μL of MSTFA, used as a silylating agent, was added and incubated at 37°C for 30 min. 1 μL of each derivatized sample was injected into a CHroZen GC system (Young In Chromass; Anyang, Korea) equipped with an L-PAL3 autosampler system and a YL9600 mass scan spectrometer. Metabolites were separated on a DB-5MS column (30 m length × 0.25 mm inner diameter × 0.25 μm particle size; Agilent, Santa Clara, USA) using helium as a carrier gas at a constant flow rate of 1.0 mL / min. The GC oven temperature was maintained at 60°C for 3 minutes, then increased by 10°C per minute to reach 320°C, and this final temperature was maintained for an additional 3 minutes.

[0143] The mass data acquisition rate was set to 2750 amu / s over a scan range of 50–600 amu, followed by electron ionization (EI) mode at -70 eV. The ion source and transfer line temperatures were set to 230°C and 250°C, respectively. The abundance of metabolites was expressed as the area of ​​the graph peaks or as a relative percentage to the average value of all samples.

[0144] Figure 3 is a graph showing the results of confirming the effect of adding tartarate or citrate on the change in the succinic acid content in the strain culture. In Figure 3, the culture supernatant (1) is the value of the succinic acid content measured after culturing GB102 or L. fermantum strains for 6 and 16 hours in a medium from which citrate was removed from the GIP 9 medium components; the culture supernatant (2) is the value of the succinic acid content measured after culturing GB102 or L. fermantum strains for 6 and 16 hours in a GIP 9 medium to which citrate was added; and the culture supernatant (3) is the value of the succinic acid content measured after culturing GB102 or L. fermantum strains for 6 and 16 hours in a GIP 9 medium to which tarta was added.

[0145] As shown in Fig. 3, in the culture supernatant of the L. fermantum strain, there was almost no change in the content of succinic acid in the culture supernatant depending on whether tartrate or citrate was added, and in the culture supernatant of the GB102 strain, it was confirmed that the content of succinic acid increased rapidly when tartrate or citrate was added.

[0146] This means that the characteristic of increased production of succinic acid when tartrate or citrate is a specific characteristic of GB102.

[0147]

[0148] Experimental Example 4. Changes in body weight gain in high-fat diet mice following combined administration of tartaric acid or citric acid.

[0149] When GB102 was co-administered with tartrate or citrate, changes in body weight gain in high-fat diet mice were observed.

[0150] Specifically, 5-week-old C57 / BL6 mice were acclimatized for 2 weeks, and then their body weights were measured and grouped to ensure a uniform average weight for each group. 7-week-old mice were fed a high-fat diet (HFD) and orally administered (200 ul / head) with the appropriate test substance for each group daily for 8 weeks. (G1: PBS, G2: GB102 (5x10 9 CFU / head) + PBS, G3: Tartrate (1mg / head), G4: GB102 (5x10 9 CFU / head) + Tartrate (1mg / head), G5: Citrate (1mg / head), G6: GB102 (5x10 9 CFU / head) + Citrate (1mg / head)).

[0151] Figure 4 is a graph measuring the body weight gain of high-fat diet mice when GB102, tartrate, or citrate were administered separately, and when GB102 and tartrate, or GB102 and citrate were administered together.

[0152] As shown in Fig. 4, when the body weights of each group were compared after 8 weeks, the group administered GB102 showed a significantly (p*<0.05) lower weight gain than the groups that did not. In particular, when tartrate or citrate was co-administered with GB102, the weight gain was significantly (p**<0.01) lower than the group administered PBS.

[0153] This means that even when GB102 is administered alone, it is effective in preventing or treating metabolic diseases such as obesity, but when GB102 is administered in combination with tartrate or citrate, it is even more effective in preventing or treating metabolic diseases such as obesity.

[0154]

[0155] The foregoing description of the present invention is provided for illustrative purposes only. Those skilled in the art will readily appreciate that the present invention can be readily modified into other specific forms without altering the technical spirit or essential characteristics of the present invention. Therefore, the embodiments described above should be understood as illustrative in all respects and not restrictive.

[0156]

[0157] [Accession number]

[0158] Name of depositor: Korea Research Institute of Bioscience and Biotechnology

[0159] Accession number: KCTC14105BP

[0160] Date of acceptance: 20200114

[0161]

[0162]

Claims

1. A first active substance comprising a strain expressing tartrate dehydratase, a culture of the strain, a lysate of the strain, or a mixture thereof; and A second active substance comprising tartrate or a substance containing tartaric acid, or citrate or a substance containing citric acid as an active ingredient; or Or a pharmaceutical composition for preventing or treating a metabolic disease, comprising the first active substance as an active ingredient and co-administering the second active substance.

2. A pharmaceutical composition for preventing or treating a metabolic disease, wherein the strain according to claim 1 expresses citrate lyase.

3. A pharmaceutical composition for preventing or treating a metabolic disease, wherein the strain according to claim 1 is Limosilactobacillus fermentum.

4. A pharmaceutical composition for preventing or treating a metabolic disease, wherein the strain according to claim 1 has at least 98.5% sequence identity with the 16S rRNA sequence of sequence number 1.

5. A pharmaceutical composition for preventing or treating a metabolic disease, wherein the strain in claim 1 is a strain deposited with the deposit number KCTC14105BP.

6. A pharmaceutical composition for preventing or treating a metabolic disease, wherein the strain according to claim 1 comprises a mutation of a naturally occurring strain of Lactobacillus fermentum.

7. A pharmaceutical composition for preventing or treating a metabolic disease, wherein the first active ingredient according to claim 1 has anti-obesity activity.

8. In claim 7, the anti-obesity activity is a pharmaceutical composition for preventing or treating a metabolic disease, comprising at least one of the following: - Suppresses weight gain; - Increase in basal metabolic rate; - Accumulation of brown fat; - Activation of energy consumption of brown fat; - Inhibition of liver steatosis; - Reduction of blood AST (alanine aminotransferase), ALT (alanine transaminase, GPT), triglycerides, low-density cholesterol, high-density cholesterol or total cholesterol; and - Increases the expression of UCP-1 (Uncoupling protein 1).

9. A pharmaceutical composition for preventing or treating a metabolic disease, wherein the tartaric acid in claim 1 is L-tartaric acid, D-tartaric acid, meso-tartaric acid, or any combination thereof.

10. A pharmaceutical composition for preventing or treating a metabolic disease, wherein the tartaric acid and citric acid in claim 1 are included in the form of a salt.

11. In claim 1, the second active ingredient is selected from the group consisting of tamarind (Tamarindus indica), grape (Vitis vinifera), carambola (Averrhoa carambola), tamarillo (Solanum betaceum), damasonia (Hibiscus sabdariffa), citrus (Citrus spp.), apple (Malus domestica), plum (Prunus mume), date palm (Phoenix dactylifera), cherry (Prunus avium, Prunus cerasus), raspberry (Rubus idaeus), blackberry (Rubus fruticosus), redcurrant (Ribes rubrum), cranberry (Vaccinium macrocarpon), pineapple (Ananas comosus), hawthorn (Hovenia dulcis), banana (Musa spp.), pomegranate (Punica granatum), chokeberry (Aronia melanocarpa), and blueberry. A pharmaceutical composition for preventing or treating metabolic diseases, which comprises at least one extract selected from the group consisting of (Vaccinium corymbosum), (Rubus coreanus) and (Morus alba).

12. A pharmaceutical composition for preventing or treating a metabolic disease according to claim 1, wherein the metabolic disease is at least one selected from the group consisting of type 1 diabetes, type 2 diabetes, impaired glucose tolerance, impaired fasting blood glucose, dyslipidemia, lipid metabolism disorder, obesity, fatty liver, insulin resistance syndrome, and glucose tolerance syndrome.

13. A pharmaceutical composition for preventing or treating a metabolic disease according to claim 1, wherein the first active substance and the second active substance are administered simultaneously, sequentially, or in reverse order.

14. A pharmaceutical composition for preventing or treating a metabolic disease, comprising a first oral preparation containing the first active substance and a second oral preparation containing the second active substance, according to claim 1, wherein the first and second oral preparations are administered orally.

15. A first active substance comprising a strain expressing citrate lyase, a culture of the strain, a lysate of the strain, or a mixture thereof; and A second active substance comprising, as an active ingredient, citric acid or a substance containing citric acid; or Or a pharmaceutical composition for preventing or treating a metabolic disease, comprising the first active substance as an active ingredient and co-administering the second active substance.

16. A first active substance comprising a strain expressing tartrate dehydratase, a culture of the strain, a lysate of the strain, or a mixture thereof; and A second active substance comprising, as an active ingredient, citric acid or a substance containing citric acid; or Or a health functional food for preventing or improving metabolic diseases, which comprises the first active substance as an active ingredient and the second active substance is administered in combination.

17. A first active substance comprising a strain expressing tartrate dehydratase, a culture of the strain, a lysate of the strain, or a mixture thereof; and A second active substance comprising, as an active ingredient, citric acid or a substance containing citric acid; or Or, a composition containing the first active substance as an active ingredient and co-administering the second active substance for the prevention or treatment of metabolic diseases.

18. A method for improving, preventing or treating a metabolic disease, comprising administering to a subject in need thereof a composition comprising as active ingredients a 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 a second active substance comprising tartaric acid or a substance containing tartaric acid, or citric acid or a substance containing citric acid.

19. A step of administering to a subject in need thereof a 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 A method for improving, preventing or treating a metabolic disease, comprising administering a second active substance comprising tartaric acid or a substance containing tartaric acid, or citric acid or a substance containing citric acid; 20. A first active substance comprising a strain expressing tartrate dehydratase for use in the manufacture of a medicine for improving, preventing or treating metabolic diseases, a culture of the strain, a lysate of the strain, or a mixture thereof; and A second active substance comprising, as an active ingredient, citric acid or a substance containing citric acid; or Or use of a composition comprising the first active substance as an active ingredient and co-administering the second active substance.

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