KR20240141642A - Lactobacillus strain and composition for preventing and treating inflammatory diseases using the same - Google Patents

Abstract

The present invention provides a pharmaceutical composition for treating or preventing an inflammatory disease, an antimicrobial composition, a composition for probiotics and a composition for food additives, comprising as an active ingredient at least one of a Lactobacillus animalis strain having uric acid concentration controllability, a Rimolactobacillus lutei strain, a mixed strain of the Lactobacillus animalis strain and the Rimolactobacillus lutei strain (Accession No.: KCTC 15190BP), a culture solution of the Lactobacillus animalis strain, a culture solution of the Rimolactobacillus lutei strain and a culture solution of the mixed strain (Accession No.: KCTC 15190BP).

Description

{LACTOBACILLUS STRAIN AND COMPOSITION FOR PREVENTING AND TREATING INFLAMMATORY DISEASES USING THE SAME}

The present invention relates to a Lactobacillus strain and a pharmaceutical composition containing the same for preventing or treating inflammatory diseases.

Inflammation is a mechanism that protects mammals from invading pathogens. However, uncontrolled inflammation can cause tissue damage and can be the cause of many diseases. That is, chronic or acute inflammatory diseases present a significant problem in the medical industry. Here, diseases and disorders with a significant inflammatory component include intestinal disorders, skin disorders, certain degenerative neurological disorders, arthritis, and autoimmune diseases, as well as hyperuricemia.

In some patients, dietary or environmental factors may trigger an autoimmune or inflammatory response. In other cases, genetic factors may play a role in the disease.

Meanwhile, conventional anti-inflammatory ingredients such as aspirin and indomethacin, which are widely used for treating inflammation, are known to have side effects such as gastrointestinal disorders and hypertensive vasodilation. In addition, antagonists for neuropeptide components including substance P, antagonists for kinins such as bradykinin, and cyclooxygenase inhibitors are being used to alleviate inflammation, but long-term use may be difficult due to side effects.

For this reason, there is a continuous demand for the development of new therapeutic compositions that have a therapeutic effect on inflammatory diseases and can reduce the use of antibiotics.

The background technology of the invention has been written to facilitate understanding of the present invention. It should not be understood that the matters described in the background technology of the invention are recognized as prior art.

Uric acid is the final metabolic product that is produced when purine, a type of nucleic acid, is broken down in the liver. Uric acid is produced during the metabolic process of certain foods, enters the bloodstream, and is mainly excreted by the kidneys and discharged in urine, with the remainder being discharged in feces.

Therefore, the blood uric acid concentration is maintained at a certain level due to homeostasis between uric acid formation and uric acid excretion. At this time, if uric acid production is excessive or excretion is insufficient, it accumulates in the body and increases the blood concentration. If it becomes supersaturated, it forms crystals and is deposited in the tissues. If excessive uric acid accumulates in the joints, it can cause gout, and if it is deposited in the kidneys, it can cause kidney disease.

In addition, if the blood uric acid concentration remains high, urate microcrystals are deposited in the tissue, and the deposited urate microcrystals can form nodules (tophi), and the formed nodules can cause acute and chronic inflammation and tissue damage. Accordingly, the inventors of the present invention focused on the relationship between uric acid and inflammatory reactions.

More specifically, the inventors of the present invention infected mice with the Salmonella enterica Serovar Typhimurium (STM) strain, an enterobacteriaceae, to induce an inflammatory response, and then collected feces. Subsequently, qualitative and quantitative analyses were performed to confirm that xanthosine was decomposed and the levels of uric acid and xanthine significantly increased.

That is, the inventors of the present invention were able to confirm that the level of uric acid significantly increased due to an intestinal inflammatory reaction.

Accordingly, the inventors of the present invention expected a therapeutic effect on inflammatory diseases by maintaining the blood uric acid concentration at a certain level by limiting the production of uric acid.

In particular, the inventors of the present invention discovered new Lactobacillus strains that control the concentration of uric acid, and in particular, they were able to recognize that a mixed strain of these (Accession Number: KCTC 15190BP) can be used to treat and prevent inflammatory diseases by controlling the concentration of uric acid to a certain level.

As a result, the inventors of the present invention have developed Lactobacillus strains having uric acid concentration-regulating ability and mixed strains thereof (Accession Number: KCTC 15190BP), and a pharmaceutical composition containing them for preventing or treating inflammatory diseases, an antibacterial composition for inflammation-causing bacteria, a probiotic composition, and a composition for food additives.

Accordingly, the problem to be solved by the present invention is to provide a pharmaceutical composition for preventing or treating an inflammatory disease, comprising Lactobacillus strains having uric acid concentration-regulating ability and mixed strains thereof (Accession Number: KCTC 15190BP) and these strains having uric acid concentration-regulating ability.

Accordingly, the problem to be solved by the present invention is to provide a Lactobacillus strain with uric acid concentration controllability and a mixed strain thereof (Accession Number: KCTC 15190BP) and an antibacterial composition for inflammation-causing bacteria containing the strain with uric acid controllability.

Another problem to be solved by the present invention is to provide a probiotic composition comprising Lactobacillus strains having uric acid concentration-regulating ability and mixed strains thereof (Accession Number: KCTC 15190BP) and these strains having uric acid concentration-regulating ability.

Another problem to be solved by the present invention is to provide Lactobacillus strains having uric acid concentration controllability and mixed strains thereof (Accession Number: KCTC 15190BP) and a composition for food additives containing these strains having uric acid controllability.

Another problem that the present invention seeks to solve is to provide an animal model of inflammatory disease, particularly an animal model of hyperuricemia (high uric acid level).

The tasks of the present invention are not limited to the tasks mentioned above, and other tasks not mentioned will be clearly understood by those skilled in the art from the description below.

To solve the aforementioned problems, the present invention provides a Lactobacillus animalis strain and a Limosilactobacillus reuteri strain having uric acid concentration-regulating ability, and a mixed strain thereof (Accession Number: KCTC 15190BP).

The term "mixed strain" as used herein may mean, but is not limited to, a strain in which a Lactobacillus animalis strain and a Rimolactobacillus lutei strain are mixed in a ratio of 1:1. For example, the ratio may be set variously depending on the culture conditions, intended use, etc.

As used herein, the term "antibacterial" may mean antibacterial activity against pathogens, particularly inflammatory bacteria, inhibition of the activity or growth of inflammatory bacteria, or even killing of inflammatory bacteria. However, without limitation, within the present specification, "antibacterial" may mean inhibition of any phenomenon associated with an inflammatory response caused by inflammatory bacteria.

According to a feature of the present invention, the inflammation-causing bacteria may be at least one selected from the group consisting of Salmonella enteritidis , Vibrio cholerae, Salmonella gallinarum , Salmonella typhimurium , Salmonella cholerasuis , Salmonella derby , Staphylococcus aureus , enterotoxigenic E. coli , Listeria monocytogenes , Campylobacter jejuni , and Clostridium perfringens .

In order to solve the aforementioned problems, the present invention provides a pharmaceutical composition for preventing or treating an inflammatory disease, comprising a Lactobacillus animalis strain and a Rimolactobacillus lutei strain having uric acid concentration-regulating ability and a mixed strain thereof (Accession Number: KCTC 15190BP) or a culture solution thereof as an effective ingredient.

According to a feature of the present invention, the Lactobacillus animalis strain and the Rimolactobacillus lutei strain having the uric acid concentration controllability of the present invention and a mixed strain thereof (Accession Number: KCTC 15190BP) have the effect of suppressing the production of uric acid or maintaining the balance of intestinal microorganisms to maintain the blood uric acid concentration at a certain level.

Therefore, the term "uric acid control" or "uric acid concentration control ability" used in this specification means maintaining or lowering the blood uric acid concentration at a certain level by suppressing the production of uric acid or promoting the excretion of uric acid, and in one embodiment, it may mean maintaining or lowering the blood uric acid concentration at a certain level by suppressing the production of uric acid, but is not limited thereto.

According to another feature of the present invention, the Lactobacillus animalis strain and the Rimolactobacillus lutei strain having the uric acid concentration regulating ability of the present invention and the mixed strain thereof (Accession Number: KCTC 15190BP) can suppress the production of uric acid by inhibiting the activity of xanthine oxidase involved in the production of uric acid. However, the mechanism of inhibiting uric acid production of the strains is not limited to that described above.

According to another feature of the present invention, the Lactobacillus animalis strain and the Rimolactobacillus lutei strain and their mixed strain (Accession Number: KCTC 15190BP) having the ability to control uric acid concentration can inhibit the activity of xanthine oxidase involved in the production of uric acid, thereby inhibiting the production of uric acid. Since hyperuricemia is defined as a case where a blood uric acid level exceeds 6.8 to 7.0 mg/dL in a man or exceeds 6 mg/dL in a woman, the certain level of uric acid here may be, but is not limited to, 2.4 to 7.0 mg/DL.

Furthermore, according to the characteristic of the present invention, since uric acid is accumulated by an inflammatory reaction, the blood uric acid concentration can be maintained at a certain level or lowered by using the Lactobacillus animalis strain and the Rimolactobacillus lutei strain having the uric acid concentration control ability of the present invention and the mixed strain thereof (Accession No.: KCTC 15190BP). Accordingly, the Lactobacillus animalis strain and the Rimolactobacillus lutei strain having the uric acid concentration control ability of the present invention and the mixed strain thereof (Accession No.: KCTC 15190BP), particularly the mixed strain (Accession No.: KCTC 15190BP), can treat or prevent not only diseases related to the accumulation of uric acid but also inflammatory diseases.

According to a feature of the present invention, each of the Lactobacillus animalis strain and Rimolactobacillus lutei strain having the ability to control uric acid concentration in a pharmaceutical composition can be provided alone as an active ingredient.

Preferably, a mixed strain of Lactobacillus animalis and Rimolactobacillus lutei (Accession Number: KCTC 15190BP) may be provided as an active ingredient in the pharmaceutical composition, but is not limited thereto.

At this time, the mixed strain of Lactobacillus animalis and Rimolactobacillus lutei (Accession Number: KCTC 15190BP) and its culture solution may have excellent bile resistance and acid resistance, and when administered, may reach the intestines to balance the intestinal microbial community or substantially inhibit the activity of bacteria that cause inflammation, and may be effective in preventing or treating inflammatory responses caused by them and, further, inflammatory diseases.

Meanwhile, the term "culture solution" used in the present specification may mean a liquid culture containing the Lactobacillus animalis strain and the Rimolactobacillus lutei strain and the mixed strain thereof (Accession No.: KCTC 15190BP) having the uric acid concentration controllability of the present invention and a medium component necessary for the growth thereof, or a culture filtrate from which the Lactobacillus animalis strain and the Rimolactobacillus lutei strain and the mixed strain thereof (Accession No.: KCTC 15190BP) are removed from the culture. In this case, the culture filtrate may contain an active ingredient that lowers the concentration of uric acid secreted from the strain during the culturing process.

In the present specification, the novel Lactobacillus strains including the mixed strain (Accession Number: KCTC 15190BP) can be cultured through a conventional Lactobacillus strain culture method. A natural medium or a synthetic medium can be used as a medium. As a carbon source of the medium, for example, glucose, sucrose, dextrin, glycerol, starch, etc. can be used, and as a nitrogen source, peptone, meat extract, yeast extract, dried yeast, soybean, ammonium salt, nitrate, and other organic or inorganic nitrogen-containing compounds can be used, but are not limited to these components. As an inorganic salt included in the medium, magnesium, manganese, calcium, iron, potassium, etc. can be used, but are not limited to these. In addition to the carbon source, nitrogen source, and inorganic salt components, amino acids, vitamins, nucleic acids, and compounds related thereto can also be added to the medium. The culture temperature conditions of the Lactobacillus strains according to various embodiments of the present invention are 20 to 40°C, and can be cultured for 12 hours to 4 days within this temperature range.

That is, the composition of the culture medium may include not only the components necessary for the usual Lactobacillus culture, but also additional components that act synergistically on the growth of Lactobacillus, and the composition accordingly may be easily selected by a person skilled in the art. In addition, the state of the strain may be a liquid state or a dry state, and the drying method may include, but is not limited to, ventilation drying, natural drying, spray drying, and freeze drying.

In various embodiments, the concentration of each Lactobacillus strain or mixture in the pharmaceutical composition is not limited thereto, but is 10 ³ CFU/ml to 10 ¹⁰ CFU/ml, 10 ³ CFU/ml to 10 ⁹ CFU/ml, 10 ³ CFU/ml to 10 ⁸ CFU/ml, 10 ³ CFU/ml to 10 ⁷ CFU/ml, 10 ³ CFU/ml to 10 ⁶ CFU/ml, 10 ³ CFU/ml to 10 ⁵ CFU/ml, 10 ³ CFU/ml to 10 ⁴ CFU/ml, 10 ⁴ CFU/ml to 10 ¹⁰ CFU/ml, 10 ⁵ CFU/ml to 10 ¹⁰ CFU/ml, 10 ⁶ CFU/ml to 10 ¹⁰ CFU/ml, 10 ⁷ CFU/ml to It can be 10 ¹⁰ CFU/ml, 10 ⁸ CFU/ml to 10 ¹⁰ CFU/ml, 10 ⁹ CFU/ml to 10 ¹⁰ CFU/ml, 10 ⁴ CFU/ml to 10 ⁹ CFU/ml, 10 ⁵ CFU/ml to 10 ⁸ CFU/ml, or 10 ⁶ CFU/ml to 10 ⁷ CFU/ml.

In various embodiments, the pharmaceutical composition may further comprise a cryoprotectant or excipient. The cryoprotectant or excipient may be, but is not limited to, a non-naturally occurring substance or a naturally occurring substance. In another specific example, the cryoprotectant or excipient may be, but is not limited to, a substance with which the novel strain does not naturally come into contact, or a substance that is not naturally co-incorporated with the novel strain. In another specific example, the composition may further comprise one or more cryoprotectants selected from the group consisting of glycerol, trehalose, maltodextrin, skimmed milk powder, and starch; and/or one or more excipients selected from the group consisting of glucose, dextrin, and skimmed milk.

Here, the cryoprotectant may be included in an amount of 0.01 wt% to 20 wt%, or 0.01 wt% to 10 wt%, based on the total weight of the composition, glycerol may be included in an amount of 5 wt% to 20 wt%, trehalose may be included in an amount of 2 wt% to 10 wt%, maltodextrin may be included in an amount of 2 wt% to 10 wt%, skimmed milk powder may be included in an amount of 0.5 wt% to 2 wt%, and starch may be included in an amount of 0.1 wt% to 1 wt%. In addition, the excipient may be included in an amount of 75 wt% to 95 wt%, or 85 wt% to 95 wt%, based on the total weight of the composition.

In addition, a method for preparing a pharmaceutical composition comprising a Lactobacillus animalis strain and a Rimolactobacillus lutei strain having uric acid concentration-regulating ability and a mixed strain thereof (Accession No.: KCTC 15190BP), a lysate thereof, a culture thereof, a concentrate thereof, or a dried product thereof may include a step of mixing a Lactobacillus animalis strain and a Rimolactobacillus lutei strain having uric acid concentration-regulating ability and a mixed strain thereof (Accession No.: KCTC 15190BP), a lysate thereof, a culture thereof, a concentrate thereof, or a dried product thereof, with an additive. The additive may be the above-mentioned cryoprotectant or excipient.

As used herein, the term “prevention” may mean any action to suppress or delay the onset of a disease (particularly an inflammatory disease) by administering the pharmaceutical composition, and “treatment” may mean any action to improve or beneficially change the symptoms of the disease by administering the pharmaceutical composition.

According to a feature of the present invention, the inflammatory disease may be at least one selected from the group consisting of hyperuricemia (high uric acid level), stroke, food poisoning, typhoid fever, peritonitis, ulcerative colitis, Crohn's disease, Behcet's disease of the intestine, infectious diarrhea, gastroenteritis, inflammatory bowel disease, neurogenic enterocolitis syndrome, small intestinal microbiota overgrowth, intestinal infectious diarrhea, and ischemic enteritis.

Preferably, the inflammatory disease may be, but is not limited to, hyperuricemia.

In various embodiments, the inflammatory disease may include any inflammatory response caused by an infection with a pathogen. For example, the inflammatory response may include an inflammatory response based on the inflammatory enteric pathogenic bacteria Salmonella sp., Enterococcus faecalis , Escherichia coli O157:H7, and Vancomycin resistant Enterococci (VRE).

As used herein, the term "antibacterial" refers to the property of inhibiting viral infections, particularly infections caused by bacteria that cause inflammation. In various embodiments, the pharmaceutical composition may have antibacterial activity against bacteria that cause inflammatory diseases. More specifically, the pharmaceutical composition may inactivate bacteria that cause inflammation by itself, or may enhance the immunity of an individual infected with bacteria through immunoactivation, thereby enabling the individual to resist infection and inflammation.

That is, in various embodiments of the present invention, a pharmaceutical composition containing a mixed strain of Lactobacillus animalis and Rimolactobacillus lutei (Accession No.: KCTC 15190BP) or a culture solution thereof can be provided as an antibacterial composition against inflammation-causing bacteria.

According to another feature of the present invention, the novel strain can be effective in improving inflammatory diseases by lowering the concentration of uric acid in the body. For example, when a pharmaceutical composition containing a mixed strain of Lactobacillus animalis and Rimolactobacillus lutei (Accession No.: KCTC 15190BP) or a culture solution thereof is administered, the concentration of uric acid caused by pathogenic bacteria can be reduced compared to before administration.

Meanwhile, the pharmaceutical composition according to one embodiment of the present invention may be formulated in various forms. For example, when the pharmaceutical composition is manufactured as a solid preparation, it may be manufactured in the form of tablets, pills, powders, granules, and capsules. Such a solid preparation may further include one or more excipients, for example, starch, calcium carbonate, sucrose, lactose, or gelatin. In addition, the composition of the solid preparation may further include lubricants such as magnesium stearate, talc, and the like in addition to simple excipients. Furthermore, when the pharmaceutical composition according to one embodiment of the present invention is manufactured as a liquid preparation, it may be manufactured in the form of an aqueous solution, a suspension, and a pharmaceutically acceptable fat and oil, alcohol, or organic solvent, as well as an ester, an emulsion, a syrup, or an elixir, a suspension reconstituted from non-effervescent granules, and an effervescent preparation reconstituted from effervescent granules. At this time, the composition of the liquid preparation can be orally administered as a liquid dosage form by combining with an inert carrier. Suitable inert carriers may include pharmaceutically acceptable inert carriers such as ethanol, glycerol, and water. Furthermore, the composition of the liquid preparation can further include a solvent, a preservative, an emulsifier, a suspending agent, a diluent, a sweetener, a thickener, and a melting agent. In addition, the composition of the liquid preparation can further include a colorant and a flavoring agent to increase patient acceptance.

The pharmaceutical composition according to one embodiment of the present invention can be administered to a subject via oral administration or parenteral administration. More specifically, parenteral administration methods may include intravenous administration, intraperitoneal administration, intramuscular administration, subcutaneous administration, or local administration.

Meanwhile, the appropriate formulation, dosage, and administration method of the pharmaceutical composition may vary depending on factors such as the age, weight, sex, degree of disease symptoms, food consumed, administration time, administration route, excretion rate, and reaction sensitivity of the subject. In addition, a skilled physician or veterinarian can easily determine and prescribe the dosage and administration method of the pharmaceutical composition that is effective for the intended treatment.

The term "Lactobacillus" used in this specification refers to a microorganism of the genus Lactobacillus, which is aerobic or facultative anaerobic, gram-positive bacilli, widely distributed in nature. Microorganisms belonging to the genus Lactobacillus include Lactobacillus animalis and Rimolactobacillus lutei. In this specification, a mixed strain comprising Lactobacillus animalis and Rimolactobacillus lutei is deposited and provided under the deposit number: KCTC 15190BP. This corresponds to a probiotic strain, is harmless to the human body, and can be used without side effects.

The term "probiotics" as used herein refers to live bacteria that enter the body and have a beneficial effect on health. Most probiotics known to date have been consumed as fermented milk products made using lactic acid bacteria such as Lactobacillus, but probiotics can also be provided in the form of fermented milk, granules, powder, etc. that include some strains other than Lactobacillus, such as Bifidobacterium and Enterococcus. In the present specification, the Lactobacillus animalis strain and the Rimolactobacillus lutei strain having uric acid concentration controllability and the mixed strain KCTC 15190BP thereof can also be used in the form of fermented milk, granules, powder, etc., but are not limited thereto.

At this time, the probiotic composition based on the Lactobacillus animalis strain and the Rimolactobacillus lutei strain having the ability to control uric acid concentration and the mixed strain thereof (Accession Number: KCTC 15190BP) can settle on the digestive tract wall in the intestines, prevent harmful bacteria from settling, and inhibit the proliferation of viruses. In particular, it can control uric acid concentration, and the beneficial digestive enzymes produced by the probiotic composition can facilitate the absorption and utilization of nutrients.

In various embodiments of the present invention, a method for preparing a probiotic composition may include a step of culturing a Lactobacillus animalis strain, a Rimolactobacillus lutei strain, or a mixed strain thereof (Accession No.: KCTC 15190BP) having uric acid concentration-regulating ability, and a step of mixing the recovered strain with an additive.

In a specific example, the additive is a cryoprotectant, and a freeze-drying step may be additionally performed after the mixing step.

At this time, the strain in the freeze-dried composition may be in a live state.

As another specific example, the above-described manufacturing method may include a step of packaging in a predetermined amount after the mixing step. In this case, the step of packaging in a predetermined amount may be packaging so that the total amount of strains including the new strain is 10 ⁶ CFU/g or more.

In various embodiments of the present invention, a food composition comprising a culture solution of Lactobacillus animalis strain and Rimolactobacillus lutei strain having uric acid concentration-regulating ability and a mixed strain thereof (Accession Number: KCTC 15190BP) as an active ingredient includes all forms of a functional food, a nutritional supplement, a health food, and a food additive. In this case, the food composition can be manufactured in various forms according to a conventional method known in the art.

According to the feature of the present invention, when the Lactobacillus animalis strain and the Rimolactobacillus lutei strain having the ability to regulate uric acid concentration and the mixed strain thereof (Accession No.: KCTC 15190BP) are used as a food additive, the strains may be added as they are or used together with other foods or food ingredients, and may be used appropriately according to a conventional method. The mixing amount of the effective ingredients may be suitably determined depending on the purpose of use (prevention, health, or therapeutic treatment). In general, when manufacturing a food or beverage, the Lactobacillus animalis strain and the Rimolactobacillus lutei strain having the ability to regulate uric acid concentration and the mixed strain thereof (Accession No.: KCTC 15190BP) are added in an amount of 0.0001 to 1 wt%, specifically, 0.001 to 0.1 wt%, to the raw material composition. However, in the case of long-term intake for the purpose of health and hygiene or health control, the amount may be used below the above-mentioned range.

There are no special restrictions on the types of food. Examples of foods to which substances can be added include meat, sausage, bread, chocolate, candy, snacks, confectionery, pizza, ramen, other noodles, gum, dairy products including ice cream, various soups, beverages, tea, drinks, alcoholic beverages, and vitamin complexes, and all health foods in the conventional sense are included.

In various embodiments, when the food composition is applied to a health beverage, various flavoring agents or natural carbohydrates may be contained as additional ingredients, just like in a regular beverage. The above-mentioned natural carbohydrates are monosaccharides such as glucose and fructose, disaccharides such as maltose and sucrose, polysaccharides such as dextrin and cyclodextrin, and sugar alcohols such as xylitol, sorbitol and erythritol. As a sweetener, a natural sweetener such as thaumatin and stevia extract, or a synthetic sweetener such as saccharin and aspartame may be used. At this time, the proportion of the additional ingredient may be 0.01 to 0.04 parts by weight, specifically 0.02 to 0.03 parts by weight per 100 parts by weight of the composition.

In addition to the above, the food composition according to various embodiments of the present invention may contain various nutrients, vitamins, electrolytes, flavoring agents, coloring agents, pectic acid and its salts, alginic acid and its salts, organic acids, protective colloid thickeners, pH regulators, stabilizers, preservatives, glycerin, alcohol, carbonating agents used in carbonated beverages, etc. The proportion of these additives is not particularly important, but is generally selected in the range of 0.01 to 0.1 parts by weight per 100 parts by weight of the composition.

In addition, the food composition according to various embodiments of the present invention may contain fruit pulp for producing natural fruit juice, fruit juice beverage and vegetable beverage. It is generally selected in the range of 0.01 to 10 parts by weight per 100 parts by weight of the composition of such fruit pulp. These components may be used independently or in combination.

In various embodiments of the present invention, an inflammatory disease animal model, particularly a hyperuricemia animal model, is provided.

At this time, the development of animal models of hyperuricemia may be very important for the development of treatments for stroke and kidney stones. In particular, the metabolic pathway of uric acid in animal models (e.g., mice) may be different from that of humans, so complex procedures may be required to simultaneously treat inducers and inhibitors to induce uric acid production. Furthermore, even if an animal model is established, symptoms associated with the disease may disappear very quickly or may not be reproduced.

The inflammatory disease animal model provided in various embodiments of the present invention can be provided, in particular, as a disease model of hyperuricemia.

The term "animal model" as used herein refers to an animal model of a disease. Specifically, the animal model may be an animal model that is caused by a disease similar to a human disease or is congenitally caused by the disease. The animal model in the present specification may be an inflammatory disease animal model, preferably a hyperuricemia animal model. In addition, an animal that may be used as an animal model of the present invention may be a non-human animal such as a monkey, a dog, a cat, a rabbit, a guinea pig, a rat, a mouse, a cow, a sheep, a pig, or a goat. Preferably, the hyperuricemia animal model may be a mouse, but is not limited thereto.

According to a feature of the present invention, the animal model may be a 7 to 8 week old C57Bl/c SPF mouse.

According to another feature of the present invention, an animal model can be produced through a step of infecting a mouse with 10 ⁷ CFU of STM strain, and a step of determining a mouse 5 days or more after infection as an animal model, but is not limited thereto.

Specific details of other embodiments are included in the detailed description and drawings.

The present invention can provide a novel strain that alleviates inflammation by suppressing the production of uric acid.

At this time, the strain can substantially lower the uric acid concentration caused by a pathogen that causes an inflammatory response due to infection, and thus can be used as a pharmaceutical composition for prevention or treatment of diseases caused by inflammation.

Accordingly, the present invention can contribute to reducing the use of antibiotics and solving problems arising from the use of antibiotics.

Furthermore, the present invention has the effect of providing an effective amount of the strain in a pharmaceutical composition.

The present invention can provide an animal model capable of evaluating the uric acid regulating ability of a novel strain and applicable as an animal model for hyperuricemia.

The effects according to the present invention are not limited to those exemplified above, and more diverse effects are included in this specification.

Figures 1a to 1e illustrate the manufacturing procedure of an inflammatory disease animal model used in various embodiments of the present invention and the evaluation results thereof.
Figures 2a to 2c illustrate the results of antibacterial activity evaluation for novel Lactobacillus strains used in various embodiments of the present invention.
Figures 3a and 3b illustrate the results of activity evaluation according to the culture environment for novel Lactobacillus strains used in various embodiments of the present invention.
Figures 4a to 4d illustrate the results of evaluating the uric acid regulating ability of novel Lactobacillus strains using an inflammatory disease animal model used in various embodiments of the present invention.
FIG. 5 illustrates the results of evaluating uric acid concentration control for novel Lactobacillus strains used in various embodiments of the invention.

The advantages and features of the present invention, and the method for achieving them, will become clear with reference to the embodiments described in detail below together with the accompanying drawings. However, the present invention is not limited to the embodiments disclosed below, but may be implemented in various different forms, and these embodiments are provided only to make the disclosure of the present invention complete and to fully inform a person having ordinary skill in the art to which the present invention belongs of the scope of the invention, and the present invention is defined only by the scope of the claims.

Hereinafter, with reference to Examples 1 to 5, the animal model of an inflammatory disease used in various embodiments of the present invention and the uric acid regulating ability of a novel strain using the same are specifically described.

At this time, the uric acid regulating ability of the novel strain is explained by application to the inflammatory response following infection with Salmonella sp. , Enterococcus faecalis , E. coli O157:H7, and VRE ( Vancomycin resistant Enterococci ), which are inflammatory enteropathogenic bacteria. However, the efficacy of the strain used in various embodiments of the present invention should not be interpreted as being limited thereto.

Example 1: Preparation of an animal model of inflammatory disease

Figures 1a to 1e illustrate the manufacturing procedure of an inflammatory disease animal model used in various embodiments of the present invention and the evaluation results thereof.

First, referring to Figure 1a, uric acid is produced in the body largely through two pathways and can be produced using purine as a precursor. At this time, xanthosine, a purine metabolite, is broken down into xanthine in the animal's liver and then produces uric acid again.

Referring together to FIG. 1b, a procedure for producing a mouse model capable of evaluating the efficacy of Lactobacillus animalis strains and Rimolactobacillus lutei strains having uric acid concentration controllability according to various embodiments of the present invention, and particularly capable of providing a hyperuricemia animal model, is illustrated. More specifically, 7-8 week old C57Bl/c SPF mice are divided into an infected group (n=5) and a non-treated group (n=4), and the infected group is infected with 10 ⁷ CFU of Salmonella enterica Serovar Typhimurium (hereinafter referred to as STM) strain. Then, feces of the infected group and the non-treated group are collected on the 5th day of infection for evaluation.

Next, referring to Fig. 1c, about 50 mg of the collected feces were subjected to qualitative and quantitative analysis using CE-TOFMS (Capillary electrophoresis time-of-flight mass spectrometry), and as a result, it was shown that xanthosine was completely decomposed in the infected group, and the concentrations of xanthine and uric acid significantly increased.

This may mean that the concentration of uric acid in the intestines increased due to inflammation induced by infection with the STM strain.

Next, referring to Figs. 1d and 1e, the results of quantitative analysis of fecal samples collected immediately before infection and 1 and 5 days after infection to observe the change in uric acid concentration over time are shown for each individual. More specifically, the STM infection group showed a maximum 5.66-fold increase in uric acid concentration compared to the non-infection group. Here, in order to observe whether STM infection effectively induces inflammation in the mouse body, the concentration of LCN-2 (Lipocalin-2), a representative inflammatory factor, was measured, and the results showed that a very high level of inflammatory response was induced.

At this time, LCN-2 is distributed in various tissues including the lungs, heart, and kidneys and is known to be up-regulated by the representative immune response NF-κERK and JAK-STAT signaling pathways, so it may be a major biomarker of kidney damage.

That is, these results may imply that a mouse model has been established that can evaluate the activity of strains with uric acid regulation ability and can be applied as an animal model of hyperuricemia, as enteric bacterial infections including STM can induce an inflammatory response and consequently regulate the production of high levels of uric acid.

Example 2: Antibacterial activity against novel strains

Hereinafter, the antibacterial activity of Lactobacillus animalis (UM1) and Limosilactobacillus reuteri (UM3) against enterobacteria, such as Efs ( Enterococcus faecalis ), EHEC ( E. coli O157:H7), and VRE ( Vancomycin resistant Enterococcus ) , an antibiotic-resistant bacterium, is described with reference to FIGS. 2a to 2c.

Here, Lactobacillus animalis (UM1) and Limosilactobacillus reuteri may be a component of a mixed strain (Accession Number: KCTC 15190BP) provided in various embodiments of the present invention.

At this time, for Efs, VRE, and EHEC, TSB liquid medium was cultured for 18 hours, and for UM1 and UM3, MRS liquid medium was cultured, and after the OD value was adjusted to 1, the inoculum was prepared. Next, MRS liquid medium and TSB liquid medium were mixed 1:1 to prepare a culture medium, and the prepared inoculum was inoculated into 10 ml of mixed medium at 2.5% (v/v) each and cultured for 18 hours. After culture, it was diluted stepwise with PBS and spread on Enterococcal agar medium (EC, Difco) for Efs, Chrom agar medium for VRE, and LB agar medium for EHEC, and cultured for 24 hours.

Referring to Figures 2a and 2b, the greatest inhibitory effect was shown in the mixed culture of Lactobacillus animalis strain (UM1) and Rimolactobacillus lutei strain (UM3) for Efs and VRE.

Referring to Fig. 2c together, in the case of EHEC, the greatest inhibitory effect was observed when Lactobacillus animalis strain (UM1) was treated alone, which may imply that the inhibitory effects of representative Gram-positive pathogenic bacteria and Gram-negative bacteria may occur through different mechanisms.

That is, a mixed strain of two types of strains, Lactobacillus animalis and Rimolactobacillus lutei (Accession Number: KCTC 15190BP), can be provided as an effective ingredient in a composition according to various embodiments of the present invention, but is not limited thereto.

Example 3: Cultivation of new strains

Hereinafter, the activity evaluation results according to the culture of Lactobacillus animalis strain (UM1) and Rimolactobacillus lutei strain (UM3) are described with reference to FIGS. 3a and 3b.

First, referring to Fig. 3a, the results of co-cultivation of Lactobacillus animalis strain (UM1) and Rimolactobacillus lutei strain (UM3) on MRS (De Man - Rogosa - Sharpe) agar medium containing 0.6% CaCO ₃ are shown. More specifically, Lactobacillus animalis strain (UM1) produces acid, but Rimolactobacillus lutei strain (UM3) does not produce acid.

Referring further to Fig. 3b, the strains were individually cultured in a liquid medium and the number of each strain was measured by co-cultivating them, and the results showed that both the Lactobacillus animalis strain (UM1) and the Rimolactobacillus lutei strain (UM3) showed increased growth when cultured together compared to when cultured alone.

At this time, the count result of each strain is expressed as (Log CFU)/ml (Only UM1; Lactobacillus animalis strain (UM1) single culture, Only UM3; Rimolactobacillus lutei strain (UM3) single culture, UM1+UM3; co-culture of Lactobacillus animalis strain (UM1) and Rimolactobacillus lutei strain (UM3))

That is, a mixed strain of two types of strains, Lactobacillus animalis and Rimolactobacillus lutei (Accession Number: KCTC 15190BP), may be provided as an effective ingredient in a composition according to various embodiments of the present invention, but is not limited thereto, and each single strain may be provided.

Example 4: Uric acid regulation ability of novel strains (mouse model based)

Hereinafter, with reference to FIGS. 4a to 4d, the results of evaluating the uric acid regulation of a mixed strain of Lactobacillus animalis and Rimolactobacillus lutei (Accession Number: KCTC 15190BP) (LAB) using a hyperuricemia mouse model induced by STM (Salmonella enterica Serovar Typhimurium UK-1) infection are described.

First, referring to Fig. 4a, the production of uric acid was increased using a Salmonella infection model, and mixed strains (LAB) were treated in the pre-infection model (LAB+STM) and the post-infection model (STM+LAB).

Referring to Figures 4b and 4c, in the case of the mouse model that was not treated with the mixed strain (LAB), a 10% weight loss was observed after infection, and all mice died on the 11th day. On the other hand, in the case of the mixed strain (LAB) treatment, both the pre-treatment and post-treatment models showed a tendency of gradual recovery after weight loss after infection. In particular, in the case of the survival rate of the mice, a significant increase in the survival rate was observed in both the pre-treatment model before infection (LAB+STM) and the post-treatment model after infection (STM+LAB) compared to the control group, and in the case of the post-treatment model, a survival rate of more than 50% was observed even at the end of the experiment.

Referring to Fig. 4d, the uric acid concentration in the feces is illustrated. More specifically, in the case of the control group, the uric acid concentration increased by 1.5 to 1.8 times 2 days after infection. On the other hand, in the case of the mixed strain (LAB) treatment group, no significant increase in the uric acid concentration was observed in either the pre-treatment model of the pre-infection model (LAB+STM) or the post-treatment model of the post-infection model (STM+LAB). These results may imply that the mixed strain (LAB) treatment alleviates the inflammatory response of the mouse model and suppresses the production of uric acid in the body.

At this time, the mixed strain (LAB) can suppress the production of uric acid by inhibiting the activity of xanthine oxidase enzyme, which is mainly involved in the production of uric acid, but is not limited thereto.

Example 5: Uric acid regulation ability of novel strains (mouse liver cell-based)

Hereinafter, with reference to FIG. 5, the results of evaluating the production of uric acid in liver cells and the control of uric acid concentration according to treatment with a mixed strain of Lactobacillus animalis and Rimolactobacillus lutei (Accession Number: KCTC 15190BP) (LAB) are described.

In this evaluation, AML-12 (alpha mouse liver-12) cells were used. These cells are liver cells with typical hepatocyte characteristics, so they can be used to measure the production and suppression efficacy of uric acid produced in the liver.

First, AML-12 cells were seeded at ^1x105 cells per well in a 24-well plate and cultured. After 72 h of culture, the medium was replaced with fresh DMEM/F-12 medium without FBS (fetal bovine serum), and the culture plate was cultured for 24 h under the same conditions as described above. At this time, AML-12 cells were treated with 100 uM each of ATP (Adenosine 5′ disodium salt trihydrate) and GTP (Guanosine 5′ sodium salt hydrate), which are precursors of uric acid, to induce the production of uric acid (A+G). Here, ATP and GTP can act as precursors of uric acid. Next, the culture solution and bacterial lysate of Lactobacillus animalis strain (LA) and Rimolactobacillus lutei strain (LR), which constitute the mixed strain (Accession Number: KCTC 15190BP) provided in various embodiments of the present invention, were treated to measure the efficacy in inhibiting uric acid production. Here, allopurinol, a drug that inhibits urea production by blocking xanthine oxidase, i.e., the pathway through which xanthine is converted to uric acid, was used as a positive control.

Referring to Figure 5, A+G treatment effectively induced the production of uric acid (A+G), and the production of uric acid was inhibited by allopurinol ( A+G +All). Furthermore, when the culture medium (AR sup) and cell lysate (AR lysate) in which Lactobacillus animalis strain and Rimolactobacillus lutei strain were co-cultured for 18 hours were treated, the production of uric acid was significantly reduced.

According to the results of Examples 2 to 5 above, a mixed strain of Lactobacillus animalis and Rimolactobacillus lutei (Accession Number: KCTC 15190BP) can be provided as a strain with uric acid regulation ability used in various embodiments of the present invention.

In particular, since the strain can suppress the production of uric acid and thus alleviate the inflammatory response due to pathogen infection, it can be provided as an effective ingredient of a pharmaceutical composition for preventing or treating inflammatory diseases or an antibacterial composition for inflammation-causing bacteria.

However, the present invention is not limited thereto and the strains may also be provided as probiotic compositions and compositions for food additives.

In various embodiments, the composition may contain the strain in an amount of 10 ⁵ to 10 ¹² cfu/g relative to the total weight of the composition, or contain the culture medium of the strain in an amount of 10 ⁵ to 10 ¹² ml/g. However, the content of the strain or the culture medium of the strain is not limited thereto.

Although the embodiments of the present invention have been described in more detail with reference to the attached drawings, the present invention is not necessarily limited to these embodiments, and various modifications may be made without departing from the technical idea of the present invention. Accordingly, the embodiments disclosed in the present invention are not intended to limit the technical idea of the present invention, but to explain it, and the scope of the technical idea of the present invention is not limited by these embodiments. Therefore, it should be understood that the embodiments described above are exemplary in all aspects and not restrictive. The protection scope of the present invention should be interpreted by the following claims, and all technical ideas within a scope equivalent thereto should be interpreted as being included in the scope of the rights of the present invention.

Korea Research Institute of Bioscience and Biotechnology, Biological Resource Center (KCTC) KCTC15190BP 20221110

Claims (15)

A Lactobacillus strain having uric acid concentration regulating ability, comprising at least one strain among a Lactobacillus animalis strain, a Limosilactobacillus reuteri strain, and a mixed strain of the Lactobacillus animalis strain and the Limosilactobacillus reuteri strain (Accession Number: KCTC 15190BP),
The above uric acid concentration regulating ability is defined as the activity of maintaining or lowering the blood uric acid concentration at a certain level. In the first paragraph,
The above schedule level is 2.4 to 7.0 mg/DL, strains. A pharmaceutical composition for preventing or treating an inflammatory disease, the pharmaceutical composition comprising at least one selected from among a Lactobacillus animalis strain having uric acid concentration-regulating ability, a Rimolactobacillus lutei strain, a mixed strain of the Lactobacillus animalis strain and the Rimolactobacillus lutei strain (Accession No.: KCTC 15190BP), a culture solution of the Lactobacillus animalis strain, a culture solution of the Rimolactobacillus lutei strain, and a culture solution of the mixed strain (Accession No.: KCTC 15190BP), as an active ingredient,
A pharmaceutical composition for preventing or treating an inflammatory disease, wherein the above uric acid concentration regulating ability is defined as an activity for maintaining or lowering the blood uric acid concentration at a certain level. In the third paragraph,
The above inflammatory diseases are,
A pharmaceutical composition for preventing or treating an inflammatory disease, at least one selected from hyperuricemia, stroke, food poisoning, typhoid fever, peritonitis, ulcerative colitis, Crohn's disease, Behcet's disease, infectious diarrhea, gastroenteritis, inflammatory bowel disease, neurogenic enterocolitis syndrome, small intestinal microbiota overgrowth and ischemic enteritis. In clause 3 or 4,
The above inflammatory diseases are,
A pharmaceutical composition for preventing or treating hyperuricemia and inflammatory diseases. In the third paragraph,
The above inflammatory diseases are,
A pharmaceutical composition for preventing or treating an inflammatory disease, which is an inflammatory disease induced by inflammatory bacteria. In Article 6,
The above-mentioned inflammatory bacteria are:
The inflammatory bacteria is at least one selected from the group consisting of Salmonella enteritidis , Vibrio cholerae, Salmonella gallinarum , Salmonella typhimurium , Salmonella cholerasuis , Salmonella derby , Staphylococcus aureus , enterotoxigenic E. coli , Enterococcus faecalis , E. coli O157:H7, VRE (Vancomycin resistant Enterococci ), Listeria monocytogenes , Campylobacter jejuni , and Clostridium perfringens . A pharmaceutical composition for preventing or treating an inflammatory disease. In the third paragraph,
The above pharmaceutical composition,
At least one strain of the Lactobacillus animalis strain, the Rimolactobacillus lutei strain and the mixed strain (Accession Number: KCTC 15190BP) is included in an amount of 10 ⁵ to 10 ¹² cfu/g based on the total weight of the pharmaceutical composition,
A pharmaceutical composition for preventing or treating an inflammatory disease, comprising a culture solution of the Lactobacillus animalis strain, a culture solution of the Rimolactobacillus lutei strain, and a culture solution of the mixed strain (Accession No.: KCTC 15190BP) in an amount of 10 ⁵ to 10 ¹² ml/g. In clause 6 or 7,
The above-mentioned inflammatory bacteria produce uric acid,
A pharmaceutical composition for preventing or treating an inflammatory disease, wherein at least one strain among the Lactobacillus animalis strain, the Rimolactobacillus lutei strain, and the mixed strain (Accession Number: KCTC 15190BP) inhibits uric acid production by the inflammatory bacteria. In Article 6,
A pharmaceutical composition for preventing or treating an inflammatory disease, wherein the above-mentioned inflammatory bacteria are intestinal infectious bacteria that induce inflammation. In Article 6,
A pharmaceutical composition for preventing or treating an inflammatory disease, wherein at least one strain among the Lactobacillus animalis strain, the Rimolactobacillus lutei strain, and the mixed strain (Accession Number: KCTC 15190BP) inhibits the activity of xanthine oxidase. In the third paragraph,
A pharmaceutical composition for preventing or treating an inflammatory disease, wherein the above-mentioned predetermined level is 2.4 to 7.0 mg/DL. A composition comprising at least one selected from among a Lactobacillus animalis strain having uric acid concentration regulating ability, a Rimolactobacillus lutei strain, a mixed strain of the Lactobacillus animalis strain and the Rimolactobacillus lutei strain (Accession No.: KCTC 15190BP), a culture solution of the Lactobacillus animalis strain, a culture solution of the Rimolactobacillus lutei strain, and a culture solution of the mixed strain (Accession No.: KCTC 15190BP) as an effective ingredient,
An antibacterial composition for inflammation-causing bacteria, wherein the above uric acid concentration regulating ability is defined as an activity for maintaining or lowering the blood uric acid concentration at a certain level. A composition comprising at least one selected from among a Lactobacillus animalis strain having uric acid concentration regulating ability, a Rimolactobacillus lutei strain, a mixed strain of the Lactobacillus animalis strain and the Rimolactobacillus lutei strain (Accession No.: KCTC 15190BP), a culture solution of the Lactobacillus animalis strain, a culture solution of the Rimolactobacillus lutei strain, and a culture solution of the mixed strain (Accession No.: KCTC 15190BP) as an effective ingredient,
A composition for probiotics, wherein the above uric acid concentration regulating ability is defined as an activity that maintains or lowers the blood uric acid concentration at a certain level. A composition comprising at least one selected from among a Lactobacillus animalis strain having uric acid concentration regulating ability, a Rimolactobacillus lutei strain, a mixed strain of the Lactobacillus animalis strain and the Rimolactobacillus lutei strain (Accession No.: KCTC 15190BP), a culture solution of the Lactobacillus animalis strain, a culture solution of the Rimolactobacillus lutei strain, and a culture solution of the mixed strain (Accession No.: KCTC 15190BP) as an effective ingredient,
A composition for food additive, wherein the above uric acid concentration control ability is defined as an activity for maintaining or lowering the blood uric acid concentration at a certain level.

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