WO2022149851A1 - Composition for preventing or treating inflammatory bowel diseases - Google Patents

Abstract

A novel strain according to the present invention has tolerance to anaerobic conditions and culture conditions containing an excessive amount of hydrogen peroxide, and thus has resistance to active oxygen conditions, that is, a protective mechanism against oxidative stress, and thus can be useful for inflammatory diseases.

Description

Composition for preventing or treating inflammatory bowel disease

The present invention relates to the use of a novel symbiotic microorganism strain that has resistance to active oxygen conditions and can produce beneficial effects while maintaining a high survival rate even in an inflammatory environment.

Reactive oxygen species (ROS) or reactive oxygen species refers to chemically reactive molecules including oxygen atoms. The reactive oxygen species is a compound of oxygen generated in the living body. Because it contains peroxygen ions and hydrogen peroxide and has unpaired electrons, it has a very high reactivity, so it has a very strong oxidative power that can attack biological tissues and damage cells. corresponds to oxygen. Such reactive oxygen species can also be generated in normal metabolic processes, and has been reported to play a role in regulating cell signaling and homeostasis. However, there is a risk that the concentration of active oxygen increases rapidly in an individual as an environmental stress such as exposure to ultraviolet rays or high heat, and thus may cause various diseases by damaging the cell structure.

In the presence of chemical oxidative stress, the production of free radicals increases or the antioxidant defense effect such as glutathione is reduced to a significant level. The effect of oxidative stress can depend on the magnitude of these changes, and if the level of perturbation is low, the cell can overcome this state on its own and return to its original state. However, severe levels of perturbation by oxidative stress can result in cell death, and even moderate levels of oxidative stress can trigger apoptosis. The reactive oxygen species generated by the oxidative stress may directly damage DNA present in an individual, and accordingly, various diseases such as aging, diabetes, hyperlipidemia, obesity, colon cancer, rheumatism or asthma may be induced.

On the other hand, intestinal microorganisms are composed of strains and various microorganisms that live together on the epithelial barrier of the host, and these symbiotic strains are responsible for maintaining the health and survival of the individual, and the overall body metabolism, inflammation, immunity, blood production, etc. It has been reported to affect the physiological function of The symbiotic microflora (sum of symbiotic microorganisms) is composed of various species, and has various genes (microbiome), so it responds to external stimuli and microenvironmental changes and is very dynamic with the human body. interact A healthy human body with a normal symbiotic microflora can overcome the invasion of pathogens through various antibacterial actions when pathogenic bacteria are infected in the intestine. On the other hand, it is known that the immune system against pathogenic bacteria does not work normally when taking broad-spectrum antibiotics continuously or exposed to oxidative stress conditions.

Accordingly, there is a need for research on novel strains that maintain a symbiotic microbial flora, have resistance to active oxygen conditions that can survive even under oxidative stress conditions, and can treat inflammatory diseases of individuals.

One object of the present invention is to prevent or treat an inflammatory disease comprising a novel strain contained in the genus Bifidobacterium having resistance to active oxygen conditions, a culture thereof, a culture solution or an extract obtained therefrom as an active ingredient To provide a pharmaceutical composition.

Another object of the present invention is to provide a food composition for the prevention or improvement of inflammatory diseases comprising the novel strain, its culture, a culture medium, or an extract obtained therefrom as an active ingredient.

Another object of the present invention is to prevent an inflammatory disease comprising administering to a subject in need of administration a composition comprising the novel strain, its culture, a culture medium, or an extract obtained therefrom as an active ingredient in a pharmaceutically effective amount , to provide a method of improvement or treatment.

However, the technical task to be achieved by the present invention is not limited to the tasks mentioned above, and other tasks not mentioned will be clearly understood by those of ordinary skill in the art from the following description.

BRIEF DESCRIPTION OF THE DRAWINGS Various embodiments described herein are described below with reference to the drawings. In the following description, various specific details are set forth, such as specific forms, compositions and processes, and the like, for a thorough understanding of the present invention. However, certain embodiments may be practiced without one or more of these specific details, or in conjunction with other known methods and forms. In other instances, well-known processes and manufacturing techniques have not been described in specific detail in order not to unnecessarily obscure the present invention. Reference throughout this specification to “one embodiment” or “an embodiment” means that a particular feature, form, composition, or characteristic described in connection with the embodiment is included in one or more embodiments of the invention. Thus, references to "in one embodiment" or "an embodiment" in various places throughout this specification do not necessarily refer to the same embodiment of the invention. Additionally, the particular features, forms, compositions, or characteristics may be combined in any suitable manner in one or more embodiments.

Unless otherwise defined in the specification, all scientific and technical terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

In one embodiment of the present invention, a pharmaceutical composition for preventing or treating an inflammatory disease comprising at least one selected from the group consisting of Bifidobacterium sp. strain, its culture, culture medium, and extracts obtained therefrom as an active ingredient do.

The Bifidobacterium sp. strain of the present invention may be Bifidobacterium longum.

The Bifidobacterium longum strain of the present invention may preferably be Bifidobacterium longum YMC_19_03_38 (Bifidobacterium longum YMC_19_03_38) or Bifidobacterium longum YMC_19_03_2 (Bifidobacterium longum YMC_19_03_2). The Bifidobacterium longum strain according to the present invention has excellent activity for alleviating or improving symptoms of inflammatory diseases, particularly inflammatory bowel disease, and the strain is deposited at the Korean Culture Center of Microorganisms (KCCM). (Strain name: Bifidobacterium longum YMC_19_03_38, accession number: KFCC11835P, deposit date: August 6, 2019 / strain name: Bifidobacterium longum YMC_19_03_2, deposit number: KFCC11834P, deposit date: August 6, 2019).

The strains included in the genus Bifidobacterium of the present invention are selected and isolated from strains obtained from the feces of healthy people, and only strains resistant to anaerobic conditions and a culture environment containing an excess of hydrogen peroxide. It was confirmed that the selected strain belongs to the genus Bifidobacterium through phylogenetic analysis using the genomic sequence and the base sequence of the 16s rRNA gene. Furthermore, as a result of analyzing the genome sequence of each of the two deposited strains (YMC_19_03_38 and YMC_19_03_2), it was confirmed once again that the microorganism belongs to the genus Bifidobacterium containing the novel genome sequence.

The "culture" of the present invention refers to a product obtained by culturing a microorganism in a known liquid or solid medium, and refers to a medium containing the microorganism.

The culture of the present invention is obtained after culturing the strain of the present invention in a medium, and the medium may be selected from a known liquid medium or solid medium used in the culture of the Bifidobacterium sp. strain, e.g. For example, it may be GYSM medium, humic acid agar medium, Bennett's agar medium, or malt extract agar medium, but is not limited thereto.

The "culture solution" of the present invention refers to a product obtained by culturing microorganisms in a known liquid or solid medium, and refers to a concept that does not contain microorganisms. The culture medium of the present invention means a liquid product from which the strain itself is removed through a method such as filtration or centrifugation after culturing a predetermined strain in a liquid medium, and more specifically, the culture medium of the present invention is Bifidobacterium (Bifidobacterium) refers to the removal of the strain from the culture of the strain included in the genus.

The filtration method of the present invention is not particularly limited, and the number of times the filtration is performed may be, for example, 1 to 10 times, 1 to 5 times, or 1 to 3 times. In addition, when a filter paper or a filter is used for the filtration, the pore size of the filter paper may be 5 to 10 μm filter paper or a filter having a pore size of 0.1 to 1.0 μm, but is not limited thereto.

The "extract" of the present invention may be an extract of a culture or a culture solution of a strain included in the genus Bifidobacterium of the present invention, and a conventional extraction method known in the art, for example, a solvent extraction method, Extraction by supercritical fluid extraction using carbon dioxide, extraction by extraction using ultrasound, separation using an ultrafiltration membrane with a constant molecular weight cut-off value, or various chromatography (size, charge, hydrophobicity or affinity) It can be prepared using a method such as separation by separation or a combination thereof).

The extraction solvent used in the solvent extraction method of the present invention is water, a lower alcohol having 1 to 4 carbon atoms (eg, methanol, ethanol, propanol, and butanol) or a mixture thereof, which is a hydrous lower alcohol, propylene glycol, 1,3 -Butylene glycol, glycerin, acetone, diethyl ether, ethyl acetate, butyl acetate, dichloromethane, chloroform, hexane and mixtures thereof may be selected from the group consisting of, among which water, alcohol, hydrous alcohol, diethyl ether , ethyl acetate, butyl acetate, chloroform or hexane may be selected, but is not limited thereto.

The strain of the present invention, its culture, its culture medium or extract obtained therefrom has resistance to active oxygen conditions, so that the strain maintains a high survival rate even in an inflammatory environment, in particular, an inflammatory environment in the large intestine, and can produce beneficial effects.

The "resistance ability in active oxygen conditions" of the present invention is an environment in which active oxygen is inevitably generated in the process of electron transfer for energy production in all organisms that breathe oxygen, or is generated by an inflammatory reaction caused by infection, etc., That is, it means the ability to survive without being killed in an oxidative stress environment. For the purpose of the present invention, the novel strain has resistance to anaerobic conditions and culture conditions containing an excess of hydrogen peroxide, so it is resistant to active oxygen conditions, that is, a defense mechanism against oxidative stress (resistance to oxidative stress). may have Furthermore, since the novel strain of the present invention has resistance to active oxygen conditions, it is possible to maintain a normal microbial flora present in a target organ.

Inflammation of the "inflammatory disease" of the present invention corresponds to a protective response involving immune cells, blood vessels, and inflammatory mediators as one of the biological responses to harmful stimuli. Substances that cause an inflammatory response include pathogens, damaged cells, and irritants. Inflammation itself is not a disease, but rather has a defense system necessary for living things, that is, a protective function to heal infection or promote tissue regeneration, but at the same time Inflammation can result in tissue damage or disease. Inflammatory disease refers to a disease that occurs when the action of inhibiting cell damage at an early stage, the action of removing the destroyed tissue and necrotic cells of the wounded part, and the action of regenerating the tissue at the same time are disturbed. Examples of inflammatory diseases include inflammatory bowel disease, atopic dermatitis, allergic diseases such as asthma, dermatitis, rhinitis, otitis media, sore throat, tonsillitis, conjunctivitis, cataract, periodontitis, pneumonia, sepsis, gastric ulcer, gastritis, Crohn's disease, hemorrhoids, autologous There are immune diseases, fibromyalgia, arthritis, myositis, hepatitis, cystitis, nephritis, multiple sclerosis, and degenerative neurodegenerative diseases.

The "inflammatory bowel disease (IBD)" of the present invention is a disease that induces chronic inflammation in the gastrointestinal tract, and its symptoms include abdominal pain, fever, diarrhea, and bleeding. In general, inflammatory bowel disease can be classified into two types: ulcerative colitis (UC) and Crohn's disease (CD). Ulcerative colitis is a type of diffuse nonspecific inflammation of unknown cause of the large intestine that forms pimples or ulcers, and is accompanied by various systemic symptoms including bloody diarrhea. In addition, Crohn's disease is a granulomatous inflammatory lesion of unknown cause in which ulcers, fibrosis, stenosis and lesions progress from the mucosa to the entire intestinal tract with discontinuity in the entire digestive tract from the oral cavity to the anus. accompanied by systemic symptoms of The incidence of such inflammatory bowel disease was known to be high among Westerners in the past, but the number of patients is increasing rapidly in Asian countries including Korea due to changes in lifestyle such as eating habits. Although the cause or pathophysiology of the inflammatory bowel disease of the present invention has not yet been clearly known, it is assumed that genetic factors, environmental factors such as intestinal bacteria or food, and immunological factors are involved in a complex manner. have. One of the causes of this occurrence is an imbalance between oxidative stress and antioxidant defense mechanisms that occur in the intestine. Specifically, the production of oxidants and free radicals can not only activate signaling mechanisms mediating the expression of inflammatory genes, but also promote the expression of genes regulating cell division, differentiation and death. However, when these oxidizing agents and free radicals are produced in excess, inflammation, cell damage and apoptosis can be induced in intestinal epithelial cells (IEC), as well as dysfunction of the mucosal barrier. It is hypothesized that chronic inflammation may be induced. For the purpose of the present invention, the strain has resistance to anaerobic conditions and culture conditions containing an excessive amount of hydrogen peroxide, so it has resistance to active oxygen conditions, that is, a defense mechanism against oxidative stress, so inflammatory diseases, preferably inflammatory bowel It may be used for the treatment of diseases, but is not limited thereto.

The inflammatory bowel disease of the present invention may be at least one selected from the group consisting of ulcerative colitis, Crohn's disease, enteric Behcet's disease, hemorrhagic rectal ulcer, and pouchitis, but is not limited thereto. .

In the present invention, the "prevention" refers to any act of using a therapy that protects against the onset of a disease so that the clinical symptoms of an inflammatory disease do not spread using the composition of the present invention, and more specifically, the treatment of symptoms of inflammatory bowel disease. It refers to suppressing the expression, recurrence or spread.

In the present invention, the "treatment" may include, without limitation, any action that improves or benefits an inflammatory disease, more specifically, an inflammatory bowel disease using the composition of the present invention.

In the present invention, the pharmaceutical composition may be characterized in the form of capsules, tablets, granules, injections, ointments, powders or drinks, and the pharmaceutical composition may be characterized in that it is targeted to humans.

The pharmaceutical composition of the present invention is not limited thereto, but each can be formulated in the form of oral dosage forms such as powders, granules, capsules, tablets, aqueous suspensions, external preparations, suppositories, and sterile injection solutions according to conventional methods. can The pharmaceutical composition of the present invention may include a pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers may include binders, lubricants, disintegrants, excipients, solubilizers, dispersants, stabilizers, suspending agents, coloring agents, fragrances, etc., for oral administration, and in the case of injections, buffers, preservatives, pain-freezing agents A topical agent, solubilizer, isotonic agent, stabilizer, etc. may be mixed and used. For topical administration, a base, excipient, lubricant, preservative, etc. may be used. The dosage form of the pharmaceutical composition of the present invention can be prepared in various ways by mixing with a pharmaceutically acceptable carrier as described above. For example, in the case of oral administration, tablets, troches, capsules, elixirs, suspensions, syrups, wafers, etc. can be prepared in the form of, and in the case of injection, it can be prepared in the form of unit dose ampoules or multiple doses. have. In addition, it can be formulated as a solution, suspension, tablet, capsule, sustained release formulation, and the like.

Meanwhile, examples of suitable carriers, excipients and diluents for formulation include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, malditol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, Cellulose, methyl cellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate or mineral oil may be used. In addition, it may further include a filler, an anti-agglomeration agent, a lubricant, a wetting agent, a flavoring agent, an emulsifier, a preservative, and the like.

The route of administration of the pharmaceutical composition according to the present invention is not limited thereto, but oral, intravenous, intramuscular, intraarterial, intramedullary, intrathecal, intracardiac, transdermal, subcutaneous, intraperitoneal, intranasal, intestinal, topical , sublingual or rectal. Oral or parenteral administration is preferred.

As used herein, "parenteral" includes subcutaneous, intradermal, intravenous, intramuscular, intraarticular, intrasynovial, intrasternal, intrathecal, intralesional and intracranial injection or infusion techniques. The pharmaceutical composition of the present invention may also be administered in the form of a suppository for rectal administration.

The pharmaceutical composition of the present invention depends on several factors including the activity of the specific compound used, age, weight, general health, sex, formula, administration time, administration route, excretion rate, drug formulation, and the severity of the specific disease to be prevented or treated. The dosage of the pharmaceutical composition may vary depending on the patient's condition, weight, disease severity, drug form, administration route and period, but may be appropriately selected by those skilled in the art, and 0.0001 to 50 mg per day It may be administered at /kg or 0.001 to 50 mg/kg. Administration may be administered once a day, or may be administered in several divided doses. The above dosage does not limit the scope of the present invention in any way. The pharmaceutical composition according to the present invention may be formulated as pills, dragees, capsules, solutions, gels, syrups, slurries, and suspensions.

According to another embodiment of the present invention, an inflammatory disease comprising administering to a subject in need of administration a composition comprising the novel strain, a culture thereof, a culture medium or an extract obtained therefrom as an active ingredient in a pharmaceutically effective amount It relates to a method of prevention or treatment of

In the present invention, descriptions regarding the strain, its culture, culture medium or extract obtained therefrom, inflammatory diseases, etc. are the same as those described in the composition for the prevention or treatment of inflammatory diseases, and are omitted to avoid excessive complexity of the present specification.

In the present invention, "administration" means providing a predetermined composition of the present invention to a subject by any suitable method.

In the present invention, the "subject" in need of such administration may include both mammals and non-mammals. Here, examples of such mammals include humans, non-human primates such as chimpanzees, other apes or monkey species; livestock animals such as cattle, horses, sheep, goats, pigs; domestic animals such as rabbits, dogs or cats; laboratory animals such as rodents such as rats, mice or guinea pigs, but are not limited thereto. In addition, examples of the non-mammal in the present invention may include, but are not limited to, birds or fish.

The formulation of the composition administered as described above in the present invention is not particularly limited, and may be administered as a solid formulation, a liquid formulation, or an aerosol formulation for inhalation, and a liquid formulation for oral or parenteral administration immediately before use. It may be administered in a solid form preparation intended to be converted into However, the present invention is not limited thereto.

In addition, during the administration in the present invention, a pharmaceutically acceptable carrier may be additionally administered together with the composition of the present invention. Here, the pharmaceutically acceptable carrier may include a binder, a lubricant, a disintegrant, an excipient, a solubilizer, a dispersing agent, a stabilizer, a suspending agent, a colorant, a flavoring agent, etc., in the case of oral administration, and in the case of an injection, a buffer, Preservatives, analgesics, solubilizers, isotonic agents, stabilizers, etc. can be mixed and used. For topical administration, bases, excipients, lubricants, preservatives, etc. can be used. The formulation of the compound of the present invention can be prepared in various ways by mixing with the pharmaceutically acceptable carrier as described above. For example, in the case of oral administration, it can be prepared in the form of tablets, troches, capsules, elixirs, suspensions, syrups, wafers, etc., and in the case of injections, it can be prepared in the form of unit dose ampoules or multiple doses. have. In addition, it can be formulated as a solution, suspension, tablet, capsule, sustained release formulation, and the like.

Meanwhile, examples of suitable carriers, excipients and diluents for formulation include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, malditol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, Cellulose, methyl cellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate or mineral oil may be used. In addition, it may further include a filler, an anti-agglomeration agent, a lubricant, a wetting agent, a flavoring agent, an emulsifier, a preservative, and the like.

The route of administration of the composition according to the present invention is, but not limited to, oral, intravenous, intramuscular, intraarterial, intramedullary, intrathecal, intracardiac, transdermal, subcutaneous, intraperitoneal, intranasal, intestinal, topical, sublingual or work. Oral or parenteral administration is preferred.

As used herein, "parenteral" includes subcutaneous, intradermal, intravenous, intramuscular, intraarticular, intrasynovial, intrasternal, intrathecal, intralesional and intracranial injection or infusion techniques. The pharmaceutical composition of the present invention may also be administered in the form of a suppository for rectal administration.

As used herein, a "pharmaceutically effective amount" refers to an amount sufficient of an agent to provide a desired biological result. The result may be reduction and/or alleviation of the signs, symptoms or causes of a disease, or any other desirable change in the biological system. For example, an "effective amount" for therapeutic use is the amount of a composition disclosed herein required to provide a clinically significant effect in a disease. An appropriate "effective" amount in any individual case can be determined by one of ordinary skill in the art using routine experimentation. Accordingly, the expression "effective amount" generally refers to the amount in which the active substance has a therapeutic effect. In the case of the present invention, the active substance is an agent for preventing or treating an inflammatory disease.

The composition of the present invention depends on several factors including the activity of the specific strain used, the age, weight, general health, sex, diet, administration time, administration route, excretion rate, drug formulation, and severity of the specific disease to be prevented or treated of the subject. It can be variously changed, and the dosage of the composition varies depending on the patient's condition, body weight, degree of disease, drug form, administration route and period, but may be appropriately selected by those skilled in the art, and 0.0001 to 100 mg/kg per day Or 0.001 to 100 mg/kg may be administered. Administration may be administered once a day, or may be administered in several divided doses. The above dosage does not limit the scope of the present invention in any way. The compound according to the present invention can be formulated as pills, dragees, capsules, solutions, gels, syrups, slurries, and suspensions.

The composition of the present invention may be used alone or in combination with methods using surgery, radiation therapy, hormone therapy, chemotherapy, and biological response modifiers.

In another embodiment of the present invention, a food composition for preventing or improving inflammatory diseases comprising at least one selected from the group consisting of Bifidobacterium sp. strain, its culture, culture medium, and extracts obtained therefrom as an active ingredient is provided. do.

In the food composition of the present invention, the Bifidobacterium sp. strain, culture, culture medium, extract, inflammatory disease, etc. are the same as those described in the pharmaceutical composition for preventing or treating inflammatory diseases, and thus omitted to avoid excessive complexity of the specification. do.

In the present invention, "improvement" may include, without limitation, any action in which an inflammatory disease, more specifically, an inflammatory bowel disease, is improved or beneficial by examining the composition of the present invention.

The food composition of the present invention may be prepared in the form of various foods, for example, beverages, gum, tea, vitamin complexes, powders, granules, tablets, capsules, confectionery, rice cakes, bread, and the like.

When the strain, etc. of the present invention is included in the food composition, the amount may include 0.001% to 90% by weight, preferably 0.1% to 40% by weight, and in the case of long-term ingestion use, the above It may be less than the range, but if the active ingredient has no problem in terms of safety, it may be used in an amount greater than or equal to the above range, so it is not limited thereto.

When the food composition of the present invention is prepared in the form of a beverage, there is no particular limitation other than containing the food composition in the indicated ratio, and it may contain various flavoring agents or natural carbohydrates as additional ingredients, as in a conventional beverage. That is, as natural carbohydrates, monosaccharides such as glucose, disaccharides such as fructose, polysaccharides such as sucrose, conventional sugars such as dextrin, cyclodextrin, etc., and sugar alcohols such as xylitol, sorbitol and erythritol are included. can do. Examples of the flavoring agent include natural flavoring agents (taumatin, stevia extract (eg, rebaudioside A, glycyrrhizin, etc.) and synthetic flavoring agents (saccharin, aspartame, etc.).

In the food composition of the present invention, other food compositions of the present invention include various nutrients, vitamins, minerals (electrolytes), synthetic flavoring agents and flavoring agents such as natural flavoring agents, coloring agents, pectic acid and salts thereof, alginic acid and salts thereof , organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohols, carbonation agents used in carbonated beverages, and the like.

The above components of the present invention may be used independently or in combination. The proportion of these additives is not critical, but is generally selected in the range of 0.1 to about 50 parts by weight per 100 parts by weight of the composition of the present invention.

Since the novel strain of the present invention is resistant to anaerobic conditions and culture conditions containing an excess of hydrogen peroxide, it has resistance to active oxygen conditions, that is, a defense mechanism against oxidative stress. Accordingly, it is possible to maintain a normal microbial flora even in an inflammatory environment in which free radicals in a target organ exist, and thus it can be usefully used for effectively preventing, improving or treating inflammatory diseases by producing beneficial effects.

1 is a view showing the result of measuring the OD600 value in order to confirm the culture ability in the culture medium containing active oxygen of the selected strain according to an embodiment of the present invention.

2 is a diagram showing the results of measuring the survival rate of mice by the log-rank test of each of the colitis-inducing group (DSS+PBS) and the B. longum administration group (DSS+B.longum) according to an embodiment of the present invention. .

3 is a DAI score of each of the normal control group (control), colitis-induced group (DSS+PBS), and B. longum administration group (DSS+B.longum) in a DSS-induced model according to an embodiment of the present invention. It is a diagram showing disease activity index score).

4 is a view showing the measurement of the colon length of each group of mice in the DSS-induced model of colitis according to an embodiment of the present invention.

5 and 6 show PAS staining on each of the normal control group (control), the colitis-inducing group (DSS+PBS) and the B. longum administration group (DSS+B.longum) according to an embodiment of the present invention. and Image J ((NIH Image, Bethesda, MD, USA) is a diagram showing the results of quantifying PAS staining.

In one embodiment of the present invention, a pharmaceutical composition for preventing or treating an inflammatory disease comprising at least one selected from the group consisting of Bifidobacterium sp. strain, its culture, culture medium, and extracts obtained therefrom as an active ingredient it's about

In another embodiment of the present invention, a food composition for preventing or improving inflammatory diseases comprising at least one selected from the group consisting of Bifidobacterium sp. strain, its culture, culture medium, and extracts obtained therefrom as an active ingredient it's about

In another embodiment of the present invention, a composition comprising at least one selected from the group consisting of a Bifidobacterium sp. strain, a culture thereof, a culture solution and an extract obtained therefrom as an active ingredient to a subject in need of administration is pharmaceutically effective It relates to a method for preventing, improving or treating an inflammatory disease, comprising administering in an amount.

Hereinafter, the present invention will be described in more detail through examples. These examples are only for illustrating the present invention in more detail, and it will be apparent to those skilled in the art that the scope of the present invention is not limited by these examples according to the gist of the present invention. .

Example 1: Confirmation of culture ability in a medium containing active oxygen of the strain

A stool sample for obtaining the strain was diluted 10 ^-1 to 10 ^-10 times with saline. Among the diluted samples, the samples diluted 10 ^-1 , 10 ^-2 , and 10 ^-10 times contain 2 mM hydrogen peroxide (H ₂ O ₂ ) containing a strain-specific culture medium of the genus Bifidobacterium. Each was spread on a Petri disk, and incubated for 16 to 18 hours in anaerobic conditions at 37 °C. Then, strains were selected as shown in Table 1 below, which are colonies generated on the Petri disk, and put into skim milk until used in the following experiments and stored at -20 °C.

division	Bacteria name
S2	Bifidobacterium longum
S10	Bifidobacterium longum
S38	Bifidobacterium longum

Each of the strains selected in Example 1 was dispensed and cultured for 24 hours under anaerobic conditions at 37° C., and then the OD ₆₀₀ value was checked, and the results are shown in FIG. 1 . Here, as a control, the reference strain Bifidobacterium longum (Ref) and Typical E.Coli (tEc) were used. As shown in FIG. 1, in the case of the reference strain (Ref), hydrogen peroxide It was confirmed that the OD ₆₀₀ value was 0 in the condition containing , whereas the OD ₆₀₀ value of S2, S10 and S38 was 0.4 to 0.6 in the condition containing 2 mM hydrogen peroxide.

Through the above results, S2 (Accession No.: KFCC11834P ( Bifidobacterium longum YMC_19_03_2)), S10 and S38 (Accession No.: KFCC11835P ( Bifidobacterium longum YMC_19_03_38)) according to the present invention are active oxygen completely different from the conventional Bifidobacterium longum. It can be seen that it is a novel strain having resistance to the conditions. Accordingly, the following experiments were performed to further confirm the effect on inflammatory bowel disease because it has resistance to reactive oxygen species.

Example 2: Evaluation of Colitis Treatment Effect in Colitis Mouse Model

1. Preparation of Colitis Mouse Model

The inventors of the present invention performed all experiments with the approval of the Yonsei University College of Medicine Evaluation Committee (Institutional Review Board, IRB), and in the following experiments, 8-week-old C57BL/6 male mice were purchased from Orient Bio and used in the experiments . The laboratory environment was acclimatized by supplying sufficient feed and water for 1 week under the conditions of temperature 21±2 ℃, humidity 50±10 %, and 12 hours/12 hours (dark/light cycle).

Normal control group (control, n=5), colitis inducing group (DSS+PBS, n=8), B. longum S2 administration group (DSS+B.longum, n=8) For DSS (MPbio product, catalog No. 0216011080) was used. More specifically, the colitis-inducing group (DSS+Veh) and the B. longum-administered group (DSS+B.longum) were supplied with drinking water containing 2.5% (W/V) DSS for 7 consecutive days, and colitis induced from the 8th day. The group stopped DSS administration and replaced with sterilized general drinking water until the mouse was sacrificed, and the B. longum administration group orally administered 200 μl of B. longum selected in Example 1 at a concentration of 1 x 10 ^-9 CFU daily for 7 days. did.

2. Assessing Mouse Survival Rate

In order to confirm the therapeutic effect of B. longum for inflammatory bowel disease according to the present invention, the colitis-inducing group (DSS+PBS) obtained in 1. above and the B. longum administration group (DSS+B.longum) died due to colonic inflammation Based on the day, the survival rate of mice was measured by the log-rank test, and the results are shown in FIG. 2 . At this time, the B. longum administration group was administered with B. longum (S2) of Table 1.

As shown in FIG. 2, the survival rate in the colitis-induced group decreased by 15% on the 10th day, and decreased by more than 40% from the 11th day, but the B. longum-administered group (S2) of the present invention showed a significantly higher survival rate than the colitis-induced group. increase was confirmed. Through this, it can be confirmed that when B. longum is administered, the therapeutic effect of inflammatory bowel disease appears.

3. DAI (Disease activity index) measurement and evaluation

The normal control group, colitis-inducing group, and B. longum-administered mice obtained in 1. were measured daily from the start date to the end date of the experiment, and the change in body weight, bloody stool, and stool viscosity were measured, and disease activity index (DAI) was analyzed. It was scored according to Table 1 below. More specifically, the body weight of the mice in each group was measured daily for 14 days to confirm the change in body weight. In addition, three factors including weight loss, stool viscosity, and bleeding were determined from 0 to 4 based on the criteria shown in Table 2 below. After scoring with points, the activity of colitis was calculated by summing them up, and the results are shown in FIG. 3 . *** in FIG. 3 means a significant difference (P < 0.001) between the colitis-inducing group and the B. longum administration group, respectively.

score	weight loss	bloody stool	stool viscosity
0	(doesn't exist)	normal	normal
One	More than 1% - 5% or less	red bloody stool		thinness
2	More than 5%-10% or less	dark red blood	very thin
3	More than 10% -20% or less	severe bleeding	diarrhea
4	over 20%	-	-

Referring to FIG. 3 , the colitis-inducing group started to show red bloody stools from the 7th day, and the DAI score was increased. It can be seen that the DAI is mitigated.

Through the above results, it can be seen that since the intestinal epithelial barrier can be maintained by B. longum S2, the intestinal epithelial response can be improved as the first defense mechanism against pathogens.

4. Measurement and Results of Changes in Colon Length

In order to confirm the change in the length of the intestine in each group of mice obtained in 1. above, the mice were sacrificed, and the length of the large intestine was measured after colonectomy, and the results are shown in FIG. 4 . In FIG. 4, * means P < 0.05, and *** means P < 0.001.

As shown in FIG. 4 , compared to the normal control group, it was confirmed that the colon length of the colitis-induced group was significantly reduced by about 30%. From this, it can be confirmed that colitis was induced by DSS. However, the B. longum administration group (S2) of the present invention showed an increase in the length of the colon compared to the colitis-induced group, through which the B. longum of the present invention minimizes or shortens the change in the length of the colon due to colitis. It was confirmed that there is an effect of inhibiting the

Through the above results, it can be seen that the B. longum strain according to the present invention is very excellent in alleviating or improving the symptoms of individuals with inflammatory bowel disease.

5. PAS staining analysis and quantification

Mucin protects the intestinal mucosa (intestinal epithelial cells) from irritating food or harmful substances ingested from the large intestine, acts as a lubricant for intestinal peristalsis, and helps settle lactic acid bacteria to reduce harmful bacteria in the gut It corresponds to a substance known to help the proliferation of lactic acid bacteria that serve as food and play a beneficial role in the intestine. In order to measure the degree of mucin secretion, the colon tissue of each group obtained in 1. above was taken, followed by PAS staining, and PAS staining was quantified through Image J (NIH Image, Bethesda, MD, USA), and colon tissue The degree of damage was confirmed and the results are shown in FIG. 5 . * in FIG. 5 indicates P < 0.05, and *** indicates P < 0.001.

As shown in FIG. 5, in the colitis-inducing group, it was confirmed that the crypts were damaged throughout the colon tissue and inflammatory cells were infiltrated into the colonic mucosa in a wide range, whereas B. In the longum administration group (S2), relatively normal crypts were observed, and it was confirmed that goblet cells were increased.

In addition, referring to FIG. 6 , as a result of analyzing PAS staining, it was confirmed that mucin secretion was significantly increased in the B. longum administration group (S2) of the present invention compared to the colitis-induced group, and in mice and mice not induced by DSS. It was confirmed that it appeared at an equivalent level or an increased level than that, confirming the increase in mucin secretion.

Summarizing the above results, the Bifidobacterium longum strain according to the present invention is a novel strain having resistance to active oxygen conditions completely different from the conventional Bifidobacterium longum, and increases the secretion of mucin to enhance the intestinal immunity of the individual. It can be very effectively improved, and it can be seen that it is very effective in the treatment of inflammatory diseases, particularly inflammatory bowel diseases.

As described above in detail a specific part of the present invention, for those of ordinary skill in the art, this specific description is only a preferred embodiment, and it is clear that the scope of the present invention is not limited thereto. Accordingly, the substantial scope of the present invention will be defined by the appended claims and their equivalents.

[accession number]

Deposited institution name: Korea Microorganism Conservation Center

Accession number: KCCM 11835P

Deposit date: 2019. 08. 06

Deposited institution name: Korea Microorganism Conservation Center

Accession number: KCCM 11834P

Deposit date: 2019. 08. 06

The novel strain of the present invention can maintain a normal microbial flora even in an inflammatory environment in which active oxygen in a target organ exists, and thus has a beneficial effect, so that it can be effectively used for preventing, improving or treating inflammatory diseases. can

Claims (12)

1. A pharmaceutical composition for preventing or treating an inflammatory disease comprising at least one selected from the group consisting of Bifidobacterium sp. strain, its culture, culture solution, and extract obtained therefrom as an active ingredient.
2. The method of claim 1,

   The Bifidobacterium sp. strain is a Bifidobacterium longum ( Bifidobacterium longum ) strain, the composition.
3. 3. The method of claim 2,

   The name of the Bifidobacterium longum strain is Bifidobacterium longum YMC_19_03_38 (Accession No.: KFCC11835P) or Bifidobacterium longum YMC_19_03_2 (Accession No.: KFCC11834P), the composition.
4. The method of claim 1,

   The strain will have resistance to active oxygen conditions, the composition.
5. The method of claim 1,

   The inflammatory disease is inflammatory bowel disease, atopic dermatitis, allergic diseases such as asthma, dermatitis, rhinitis, otitis media, sore throat, tonsillitis, conjunctivitis, cataract, periodontitis, pneumonia, sepsis, gastric ulcer, gastritis, Crohn's disease, hemorrhoids, autoimmune disease , Fibromyalgia, arthritis, myositis, hepatitis, cystitis, nephritis, multiple sclerosis, at least one selected from the group consisting of degenerative neurodegenerative diseases, the composition.
6. 6. The method of claim 5,

   The inflammatory bowel disease is at least one selected from the group consisting of ulcerative colitis, Crohn's disease, enteric Behcet's disease, hemorrhagic rectal ulcer and ileal capsuleitis (pouchitis).
7. A food composition for preventing or improving inflammatory diseases comprising at least one selected from the group consisting of Bifidobacterium sp. strains, cultures thereof, culture solutions and extracts obtained therefrom as an active ingredient.
8. 8. The method of claim 7,

   The Bifidobacterium sp. strain is a Bifidobacterium longum ( Bifidobacterium longum ) strain, the composition.
9. 9. The method of claim 8,

   The name of the Bifidobacterium longum strain is Bifidobacterium longum YMC_19_03_38 (Accession No.: KFCC11835P) or Bifidobacterium longum YMC_19_03_2 (Accession No.: KFCC11834P), the composition.
10. 8. The method of claim 7,

    The strain will have resistance to active oxygen conditions, the composition.
11. 8. The method of claim 7,

    The inflammatory disease is inflammatory bowel disease, atopic dermatitis, allergic diseases such as asthma, dermatitis, rhinitis, otitis media, sore throat, tonsillitis, conjunctivitis, cataract, periodontitis, pneumonia, sepsis, gastric ulcer, gastritis, Crohn's disease, hemorrhoids, autoimmune disease , Fibromyalgia, arthritis, myositis, hepatitis, cystitis, nephritis, multiple sclerosis, at least one selected from the group consisting of degenerative neurodegenerative diseases, the composition.
12. 12. The method of claim 11,

    The inflammatory bowel disease is at least one selected from the group consisting of ulcerative colitis, Crohn's disease, enteric Behcet's disease, hemorrhagic rectal ulcer and ileal capsuleitis (pouchitis).

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