A probiotic complex composition having functions of immunomodulation and immune homeostasis

Abstract

This application relates to a probiotic mixed strain having an immunomodulatory function and a composition containing the same. A composition containing, as an active ingredient, a probiotic mixed strain having an immunomodulatory function according to an embodiment of the present application has an immunomodulatory function by increasing the amount of NO production or suppressing excessive NO production induced by inflammatory substances, and the strain can be applied to food compositions, health functional food compositions, pharmaceutical compositions, and the like.

Description

Technical Field

[0001] The present application relates to a probiotic mixed strain having an immunomodulatory function and a composition containing the same.

Background Art

[0002] Many bacteria inhabit the digestive tract of the human body. While there are about 10 trillion normal cells in the human body, the number of bacteria is about 100 trillion, which is about 10 times more. Most of these bacteria form the gut microbiota and affect the immune regulation of the human body through interaction with various immune cells present in the intestine. Also, these bacteria are classified into beneficial bacteria that are useful for the health of the human intestine and harmful bacteria that are harmful to health. The human body can maintain health when beneficial bacteria occupy the dominant bacteria over harmful bacteria such as Escherichia coli (E. coli), Veillonella, and Clostridium perfringens, and various microorganisms inhabiting the digestive tract maintain a balance.

[0003] The immune cells of the human body maintain health by controlling antigens (including microorganisms) that invade from the outside and abnormal cells that occur internally. More than 70% of these immune cells are located in the digestive tract and control microorganisms that already inhabit the digestive tract or microorganisms that invade from the outside. However, when the activation of the immune response by such immune cells is excessive, diseases such as allergies, rheumatism, atopic dermatitis, asthma, rheumatoid arthritis, lupus, or inflammatory bowel diseases (ulcerative colitis, Crohn's disease, diverticulosis, etc.), diabetes, etc. may be induced. Therefore, the immunomodulatory ability to suppress the overly activated immune response and maintain immune homeostasis plays an important physiological function as an immune system that prevents the occurrence of the above diseases. Among the immune cells, T cells can directly remove host cells infected with bacteria or viruses, and among them, helper T cells promote the differentiation of other immune cells and induce activation (cellular and humoral immune responses). Such an activated immune response is suppressed by regulatory T cells (Tregs) and is known to play a central role in maintaining immune homeostasis.

[0004] Intestinal bacteria cause an immune response as an antigen in the human body. However, in order to coexist with the human body, they also have a mechanism of immune tolerance to reduce the immune response. A part of this mechanism is known to be involved in the differentiation process of T cells and induce the differentiation and proliferation of regulatory T cells in the intestine. As an example of developing a composition for immunomodulation using such microorganisms, there are a novel lactic acid bacterium derived from the human digestive tract having an immunomodulatory effect and its use (Republic of Korea Registered Patent No. 10-1862051). However, in fact, there is still a need for in-depth development and various studies on compositions that exhibit excellent effects related to immunomodulation.

[0005] Therefore, as a result of diligent efforts to develop a composition with excellent immunomodulatory function, the present inventors have developed a mixed strain capable of modulating immunity by increasing the amount of NO production or suppressing excessive NO production induced by inflammatory substances, and a composition containing the same, thus completing the present invention.

Prior Art Documents

Patent Documents

[0006]

Patent Document 1

Summary of the Invention

Problems to be Solved by the Invention

[0007] The present application provides a probiotic mixed strain having an immunomodulatory function and a composition containing the same.

[0008] However, the problems to be solved by the present application are not limited to the problems described above, and other problems not mentioned should be clearly understood by those skilled in the art from the following description.

Means for Solving the Problems

[0009] The first aspect of the present application provides a mixed strain having an immunomodulatory function.

[0010] The second aspect of the present application provides a food composition for immunomodulation, comprising a mixed strain having an immunomodulatory function as an active ingredient.

[0011] The third aspect of the present application provides a pharmaceutical composition for preventing or treating immunomodulation-related diseases, comprising a mixed strain having an immunomodulatory function as an active ingredient.

Advantages of the Invention

[0012] A composition containing, as an active ingredient, a mixed strain having an immunomodulatory function according to an embodiment of the present application has an immunomodulatory function by increasing the amount of NO produced or suppressing excessive NO production induced by inflammatory substances, and the strain can be applied to food compositions, health functional food compositions, pharmaceutical compositions, etc.

Brief Description of the Drawings

[0013]

Figure 1

Figure 2

Figure 3

Figure 4

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Figure 6

Mode for Carrying Out the Invention

[0014] Hereinafter, with reference to the accompanying drawings, embodiments of the present application will be described in detail so that those having ordinary knowledge in the technical field to which the present application pertains can easily implement it. However, the present application can be embodied in various different forms and is not limited to the embodiments described herein. And in the drawings, for the purpose of clearly explaining the present application, parts not related to the explanation are omitted, and similar reference numerals are given to similar parts throughout the specification.

[0015] Throughout the specification of the present application, when a member is located "above" another member, this includes not only the case where a member is in contact with another member, but also the case where there are other members between the two members.

[0016] Throughout the specification of this application, when a part includes a certain component, this means that, unless otherwise stated to the contrary, it does not exclude other components, but may further include other components. Terms such as "about" and "substantially" used throughout the specification of this application are used in the sense of the numerical value itself or a value close to it when manufacturing and material tolerances inherent in the stated meaning are presented, and are used to prevent unscrupulous infringers from misusing the disclosure where exact or absolute numerical values are mentioned to assist in the understanding of this application. The terms "step of ~(performing)" or "step of ~" used throughout the specification of this application do not mean "step for ~".

[0017] Throughout the specification of this application, the term "these combination(s)" included in the Markush-type expression means one or more mixtures or combinations selected from the group consisting of the components described in the Markush-type expression, and means including one or more selected from the group consisting of the above components.

[0018] Throughout the specification of this application, the description of "A and / or B" means "A or B, or A and B".

[0019] Hereinafter, embodiments and examples of this application will be described in detail with reference to the attached drawings. However, this application is not limited to such embodiments, examples, and drawings.

[0020] The first aspect of this application provides a mixed strain having an immunomodulatory function.

[0021] The "immunomodulatory function" used throughout the specification of this application means the ability to enhance immunity and the anti-inflammatory ability. That is, in the specification of this application, having an "immunomodulatory function" means having both the "ability to enhance immunity" and the "anti-inflammatory ability".

[0022] In one embodiment of the present application, the mixed strain may include, but is not limited to, Lactobacillus reuteri LM1071 strain (KCCM12650P) and Lactobacillus plantarum LM1001 strain (KCCM42959).

[0023] In one embodiment of the present application, in addition to Lactobacillus reuteri LM1071 strain (KCCM12650P) and Lactobacillus plantarum LM1001 strain (KCCM42959), the mixed strain may further include, but is not limited to, Bifidobacterium animalis subsp. lactis LM1017 strain (KCCM12629P), Bifidobacterium longum LM1024 strain (KCCM12919P), Lactococcus lactis LM1009 strain (KCCM80146), Streptococcus thermophilus LM1012 strain (KFCC11771P), and Lactobacillus plantarum LM1004 strain (KCCM43246).

[0024] In one embodiment of the present application, the mixed strain can regulate the immune function by increasing the NO production or suppressing the excessive NO production induced by inflammatory substances, thereby maintaining immune homeostasis.

[0025] The term "NO (nitric oxide)" used throughout the specification of this application refers to "nitrogen oxide" or "nitric oxide", which is a compound in a form where nitrogen is oxidized. It is formed from the amino acid arginine inside cells and is involved in various physiological activities such as immune function, vasodilation, and signal transduction as a kind of signaling substance. It is also known to promote the secretion of inflammatory cytokines such as TNF-α and IL-6 and induce inflammation and pain (Hu et al., 2020).

[0026] In one embodiment of this application, the inflammatory substance may be one or more selected from the group consisting of LPS, zymosan, Concanavalin A, bacteria, viruses, parasites, fungi, and allergy-inducing antigens, but is not limited thereto.

[0027] In one embodiment of this application, the mixed strain regulates immune function. Specifically, the strain may be included in various compositions such as food compositions, health functional food compositions, and pharmaceutical compositions.

[0028] The second aspect of this application provides a food composition for immune regulation containing a mixed strain having an immune regulatory function as an active ingredient. The content overlapping with the first aspect is also applied to the food composition of the second aspect.

[0029] In one embodiment of this application, the composition regulates immune function. Specifically, it regulates immune function by increasing the amount of NO produced or suppressing excessive NO production induced by inflammatory substances.

[0030] In one embodiment of this application, the composition contains a mixed strain, its live cells, its dead cells, its culture, its disrupted product, and / or its extract.

[0031] In one embodiment of the present application, the composition may include, but is not limited to, Lactobacillus reuteri strain LM1071 (KCCM12650P) and Lactobacillus plantarum strain LM1001 (KCCM42959).

[0032] In one embodiment of the present application, in addition to Lactobacillus reuteri strain LM1071 (KCCM12650P) and Lactobacillus plantarum strain LM1001 (KCCM42959), the composition may further include, but is not limited to, Bifidobacterium animalis subsp. lactis strain LM1017 (KCCM12629P), Bifidobacterium longum strain LM1024 (KCCM12919P), Lactococcus lactis strain LM1009 (KCCM80146), Streptococcus thermophilus strain LM1012 (KFCC11771P), and Lactobacillus plantarum strain LM1004 (KCCM43246).

[0033] The term "dead bacterial cells" used throughout the specification of this application is the opposite concept of live bacteria, and means a form in which live bacteria obtained by fermentation and metabolites are made unable to grow by heat treatment or the like. The dead bacterial cells may contain cytoplasm, cell wall, antibacterial substances such as bacteriocin, polysaccharide, organic acid, etc. Products using the dead bacterial cells have high safety compared to live bacteria products. In particular, they are excellent in heat resistance, have high safety against the external environment, are easier to store than existing live bacteria products, and have the advantage of being able to extend the distribution period. Also, due to the strengthening of regulations on the use of antibiotics, their usability as substitutes and the fact that there are only a few companies that have really entered the production of dead bacterial cell products, the marketability and growth potential are very high.

[0034] The term "culture" used throughout the specification of this application means a single product obtained by culturing the strain of this application in a known liquid medium or solid medium, and may be used interchangeably with "culture broth".

[0035] The term "food" used throughout the specification of this application includes meats, sausages, bread, chocolate, candies, snacks, confectioneries, pizza, ramen, other noodles, gums, dairy products including ice creams, various soups, beverages, tea, drink agents, alcoholic beverages, compound vitamins, health functional foods and health foods, etc., and includes all foods in the ordinary sense.

[0036] The term "health functional food" used throughout the specification of this application means a food manufactured and processed using raw materials and ingredients having useful functions for the human body according to Law No. 6727 regarding health functional foods. "Functionality" means obtaining useful effects for health purposes such as regulating nutrients with respect to the structure and function of the human body and physiological actions.

[0037] The food of the present application can be manufactured by methods commonly used in the industry. During the above manufacturing process, raw materials and components commonly added in the industry may be added for manufacturing. Also, the dosage form of the food may be manufactured without limitation as long as it is a dosage form recognized as a food. The food composition of the present invention may be manufactured in various dosage forms. Different from general drugs, since it uses food as a raw material, it has the advantage of having no side effects that may occur during long-term use of drugs and is excellent in portability. Therefore, the food of the present invention can be ingested as an adjuvant for enhancing the effect of improving the intestinal environment.

[0038] The "health food" means a food having a positive health maintenance or promotion effect compared to general food, and the "health supplement food" means a food for health supplement purposes. In some cases, the terms "functional food", "health food", and "health supplement food" may be used interchangeably. Specifically, the functional food is a food in which the mixed strains of the present application are added to food materials such as beverages, teas, spices, gums, and confectioneries, or manufactured into capsules, powders, suspensions, etc., and means a food that brings specific health effects when ingested. Different from general drugs, since it uses food as a raw material, it has the advantage of having no side effects that may occur during long-term use of drugs.

[0039] Since the food composition of the present application can be ingested daily, a high effect can be expected for improving depression, and thus it can be used very usefully.

[0040] The food composition may further contain a physiologically acceptable carrier, but the type of the carrier is not particularly limited, and any carrier commonly used in the technical field can be used.

[0041] In addition, the food composition may contain additional ingredients that are commonly used in food compositions and can improve odor, taste, visual appearance, etc. For example, it may contain vitamins A, C, D, E, B1, B2, B6, B12, niacin, biotin, folate, pantothenic acid, etc. It may also contain minerals such as zinc (Zn), iron (Fe), calcium (Ca), chromium (Cr), magnesium (Mg), manganese (Mn), copper (Cu), chromium (Cr), etc. Furthermore, it may contain amino acids such as lysine, tryptophan, cysteine, valine, etc.

[0042] In addition, the food composition may contain food additives such as preservatives (potassium sorbate, sodium benzoate, salicylic acid, sodium dehydroacetate, etc.), bactericides (sun-dried powder and highly sun-dried powder, sodium hypochlorite, etc.), antioxidants (butylhydroxyanisole (BHA), butylhydroxytoluene (BHT), etc.), colorants (tar dyes, etc.), color developers (sodium nitrite, sodium nitrite, etc.), bleaching agents (sodium sulfite), seasonings (sodium glutamate such as MSG, etc.), sweeteners (dulcin, cyclamate, saccharin, sodium, etc.), flavors (vanillin, lactones, etc.), swelling agents (alum, potassium hydrogen D-tartrate, etc.), fortifiers, emulsifiers, thickeners (paste), film-forming agents, gum bases, foam suppressants, solvents, improvers, etc. The additives may be selected according to the type of food and used in appropriate amounts.

[0043] The mixed strain of the present application may be added as it is, or used together with other foods or food ingredients, and may be appropriately used by ordinary methods. The mixing amount of the active ingredient may be appropriately determined according to its purpose of use (prevention, health or therapeutic treatment). Usually, when manufacturing food or beverages, the food composition of the present invention may be added in an amount of 50 parts by weight or less, specifically 20 parts by weight or less, based on the food or beverage. However, when ingested for a long time for the purpose of health and hygiene, it may contain a content below the above range, and since there are no problems from the safety aspect, the active ingredient may be used in an amount above the above range.

[0044] As an example of the food composition of the present application, it may be used as a health beverage composition, and in that case, it may contain various flavoring agents or natural carbohydrates, etc. as additional ingredients like ordinary beverages. The above-mentioned natural carbohydrates may be monosaccharides such as glucose and fructose; disaccharides such as maltose and sucrose; polysaccharides such as dextrin and cyclodextrin; sugar alcohols such as xylitol, sorbitol, and erythritol. Sweeteners may be natural sweeteners such as thaumatin and stevia extract; synthetic sweeteners such as saccharin and aspartame. The proportion of the above-mentioned natural carbohydrates may be usually about 0.01 - 0.04 g, specifically about 0.02 - 0.03 g, per 100 mL of the health beverage composition of the present invention.

[0045] In addition to the above, the health beverage composition may contain various nutrients, vitamins, electrolytes, flavoring agents, coloring agents, pectic acid, salts of pectic acid, alginic acid, salts of alginic acid, organic acids, protective colloid thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohol, or carbonating agents, etc. In addition, it may contain natural fruit juice, fruit juice beverage, or pulp for manufacturing vegetable beverages. Such ingredients may be used independently or in combination. The proportion of such additives is not particularly important, but it is generally selected in the range of 0.01 - 0.1 parts by weight per 100 parts by weight of the health beverage composition of the present invention.

[0046] As long as the food composition of the present application can achieve the effect of improving the intestinal environment, it may contain the mixed strains of the present application in various weight percentages. Specifically, the mixed strains of the present application may be contained in an amount of 0.00001 to 100% by weight, or 0.01 to 80% by weight based on the total weight of the food composition, but is not limited thereto.

[0047] In one embodiment of the present application, the food composition may be a health functional food composition.

[0048] The third aspect of the present application provides a pharmaceutical composition for preventing or treating immune regulation-related diseases containing a mixed strain having an immune regulation function as an active ingredient. The contents overlapping with the first aspect and the second aspect are also commonly applied to the pharmaceutical composition of the third aspect.

[0049] In one embodiment of the present application, the composition may contain the mixed strains of the present application, their viable cells, their dead cells, their cultures, their disrupted products, and / or their extracts.

[0050] In one embodiment of the present application, the composition may contain Lactobacillus reuteri LM1071 strain (KCCM12650P), Lactobacillus plantarum LM1001 strain (KCCM42959), but is not limited thereto.

[0051] In one embodiment of the present application, the composition may further include, but is not limited to, Lactobacillus reuteri strain LM1071 (KCCM12650P), Lactobacillus plantarum strain LM1001 (KCCM42959), Bifidobacterium animalis subsp. lactis strain LM1017 (KCCM12629P), Bifidobacterium longum strain LM1024 (KCCM12919P), Lactococcus lactis strain LM1009 (KCCM80146), Streptococcus thermophilus strain LM1012 (KFCC11771P), and Lactobacillus plantarum strain LM1004 (KCCM43246).

[0052] As used throughout the specification of the present application, the term "treatment" means all acts of alleviating or improving the symptoms of an immune-related disease by administering a pharmaceutical composition containing the mixed strains of the present application as an active ingredient to an individual suffering from the immune-related disease.

[0053] In one embodiment of the present application, the composition is for preventing or treating immune-related diseases. Specifically, it prevents or treats immune-related diseases by increasing the amount of NO produced or suppressing excessive NO production induced by inflammatory substances.

[0054] As used throughout the specification of the present application, the term "immune-related disease" refers to all diseases related to immunity, such as various diseases caused by infections of bacteria and viruses that are likely to occur due to a decrease in immunity, and autoimmune diseases and allergic reactions that are likely to occur due to excessive immune responses.

[0055] As used throughout the specification of this application, the term "immunodeficiency disease" generally refers to diseases caused by dysfunction of components of the immune system, such as severe combined immunodeficiency (SCID), acquired immunodeficiency syndrome (AIDS), and the like.

[0056] As used throughout the specification of this application, the term "hyperimmune disease" generally refers to diseases caused by overexpression of immunity, such as autoimmune diseases like rheumatoid arthritis, allergies, atopy, and the like.

[0057] In one embodiment of this application, the pharmaceutical composition may be formulated and used in the form of oral dosage forms such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, etc., external preparations, suppositories, or sterile injection solutions by ordinary methods, but is not limited thereto.

[0058] In one embodiment of this application, when formulating the pharmaceutical composition, it may be prepared using diluents or excipients such as commonly used fillers, extenders, binders, wetting agents, disintegrants, or surfactants, but is not limited thereto.

[0059] In one embodiment of this application, solid preparations for oral administration include tablets, pills, powders, granules, capsules, etc. Such solid preparations may be prepared by mixing at least one excipient, such as starch, calcium carbonate, sucrose, lactose, or gelatin, with the killed cells of the strain. Also, for example, in addition to simple excipients, lubricants such as magnesium stearate and talc may be used, but is not limited thereto.

[0060] In one embodiment of this application, liquid preparations for oral administration include suspensions, oral solutions, emulsions, syrups, etc. In addition to commonly used simple diluents such as water and liquid paraffin, various excipients, such as wetting agents, sweeteners, fragrances, preservatives, etc., may be included, but is not limited thereto.

[0061] In one embodiment of the present application, the formulations for parenteral administration may include, but are not limited to, sterilized aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized formulations, and suppositories. For example, as the non-aqueous solvent or suspension, propylene glycol, polyethylene glycol, vegetable oils such as olive oil, injectable esters such as ethyl oleate, etc. may be used, but are not limited thereto. For example, as the suppository, witepsol, macrogol, tween 61, cacao butter, laurin fat, glycerogelatin, etc. may be used, but are not limited thereto.

[0062] The pharmaceutical composition according to one embodiment of the present application may be a pharmaceutical composition or a quasi-drug composition.

[0063] The term "quasi-drug" used throughout the specification of the present application means an article having a milder action than a pharmaceutical among articles used for the purpose of diagnosing, treating, improving, alleviating, treating, or preventing diseases in humans and animals. For example, according to the Pharmaceutical Affairs Law, a quasi-drug excludes articles used for the purpose of pharmaceuticals and includes products used for the treatment or prevention of human and animal diseases, products having a mild or no direct action on the human body, etc.

[0064] The quasi-drug composition of the present application may be manufactured into a dosage form selected from the group consisting of body cleansers, disinfectant cleaners, detergents, kitchen detergents, cleaning detergents, toothpastes, gargles, wet tissues, detergents, soaps, hand washes, hair shampoos, hair conditioners, humidifier fillers, masks, ointments, and filter fillers, but is not limited thereto.

[0065] In one embodiment of the present application, the pharmaceutical composition may be administered in a pharmaceutically effective amount. However, the term "pharmaceutically effective amount" in the present application means an amount sufficient to treat or prevent a disease at a reasonable benefit / risk ratio applicable to medical treatment or prevention. The level of the effective dose may be determined by factors such as the severity of the disease, the activity of the drug, the age, weight, health, gender of the patient, the drug sensitivity of the patient, the administration time, administration route and excretion rate of the composition of the present invention used, the treatment period, factors including drugs formulated or used simultaneously with the composition of the present invention, and other factors well known in the medical field. The pharmaceutical composition of the present application may be administered alone or in combination with components known to have a therapeutic effect on known intestinal diseases. It is important to consider all the above factors and administer an amount that can obtain the maximum effect with the minimum amount without side effects.

[0066] In one embodiment of the present application, the dosage of the pharmaceutical composition may be determined by those skilled in the art considering factors such as the purpose of use, the toxicity of the disease, the age, weight, gender, medical history of the patient, or the type of substance used as the active ingredient. For example, the pharmaceutical composition of the present invention may be administered at about 0.1 ng to about 1,000 mg / kg, preferably 1 ng to about 100 mg / kg per adult. The dosing frequency of the composition of the present application is not particularly limited thereto, but it may be administered once a day or the dose may be divided and administered several times. The above dosage or dosing frequency does not limit the scope of the present application in any way.

[0067] The pharmaceutical composition of the present application is not particularly limited thereto, but may be administered through routes such as intraperitoneal administration, intravenous administration, intramuscular administration, subcutaneous administration, intradermal administration, transdermal patch administration, oral administration, intranasal administration, pulmonary administration, rectal administration, etc. depending on the purpose. However, in the case of oral administration, it may be administered in a non-formulated form. Since the Lactobacillus reuteri LM1071 strain may be denatured or decomposed by gastric acid, the oral composition may be administered orally in a form that coats the active drug or is formulated to be protected from degradation in the stomach or in the form of an oral patch. Further, the composition may be administered by any device capable of moving the active substance to the target cells.

Example

[0068] Hereinafter, the present invention will be described in more detail with reference to the examples of the present application. However, the following examples are only illustrative for helping the understanding of the present application, and the content of the present application is not limited to the following examples.

[0069] [Example] Example 1. Experiment for confirming the immune enhancing ability and anti-inflammatory ability during single treatment of LM1071 and LM1001

[0070] (1) Confirmation of immune enhancement ability For the culture of Raw264.7 cells, a macrophage cell line derived from mice, DMEM culture medium (Welgene, Korea) containing 10% fetal bovine serum (Welgene, Korea) and penicillin-streptomycin (Welgene, Korea) was used. After dispensing Raw264.7 cells into a 96-well plate at a concentration of 3x10 4 cells / well, they were cultured in a 37 °C CO 2 incubator overnight. After removing the medium from each well, LM1071 or LM1001 was diluted in the medium according to the number of bacteria and treated for 24 hours. At this time, LPS was used as a positive control group. To measure the production amount of NO, the Griess Reagent system of Promega was used. 50 μl of the cultured medium was transferred to a new 96-well plate. At this time, for the NO standard curve, a NO standard solution was used to dilute it to 1.56 - 100 μM and added. After adding 50 μl of sulfanilamide solution to each well, the light was blocked for 5 minutes and the reaction was carried out at room temperature. After further adding 50 μl of N-1-napthylethylenediamine dihydrochloride (NED) solution to each well and reacting for 5 minutes at room temperature while blocking the light, the absorbance was measured at 540 nm using a microplate reader, and the NO production amount was calculated using this NO standard curve (see Figure 1).

[0071] As a result of the experiment, it was confirmed that both the LM1071 strain and the LM1001 strain increased the amount of NO production.

[0072] (2) Confirmation of anti-inflammatory ability For the culture of Raw264.7 cells, a mouse-derived macrophage cell line, DMEM culture medium (Welgene, Korea) containing 10% fetal bovine serum (Welgene, Korea) and penicillin-streptomycin (Welgene, Korea) was used. After dispensing Raw264.7 cells into a 96-well plate at a concentration of 3x10 4 cells / well, they were cultured in a 37°C CO 2 incubator overnight. After removing the medium from each well, LM1071 or LM1001 was diluted in the medium according to the number of bacteria and treated for 2 hours. After treating each well with LPS to induce inflammation in Raw264.7 cells, they were further cultured in a 37°C CO 2 incubator for 24 hours. To measure the amount of NO production, the Griess Reagent system from Promega was used. 50 μl of the cultured medium was transferred to a new 96-well plate. At this time, for the NO standard curve, a NO standard solution was used to dilute it to 1.56 - 100 μM and added. After adding 50 μl of sulfanilamide solution to each well, the light was blocked for 5 minutes and the reaction was allowed to proceed at room temperature. After further adding 50 μl of NED solution to each well and reacting for 5 minutes at room temperature with the light blocked, the absorbance was measured at 540 nm using a microplate reader, and the amount of NO production was calculated using the NO standard curve (see Figure 2).

[0073] As a result of the experiment, it was confirmed that only the LM1071 strain suppressed the excessive NO production induced by LPS, an inflammatory substance.

[0074] Example 2. Experiment for confirming the immune enhancing ability and anti-inflammatory ability of the mixed strain of LM1071 and LM1001

[0075] (1) Confirmation of immune-enhancing ability For the culture of Raw264.7 cells, a macrophage cell line derived from mice, DMEM culture medium (Welgene, Korea) containing 10% fetal bovine serum (Welgene, Korea) and penicillin-streptomycin (Welgene, Korea) was used. Raw264.7 cells were dispensed into a 96-well plate at a concentration of 3x10 4 cells / well, and then cultured in a 37 °C CO 2 incubator overnight. After removing the medium from each well, a mixture of LM1071 and LM1001 (LM1071:LM1001 = 8:1, 4:1, 1:1, 1:4, 1:8) was diluted in the medium and treated for 24 hours. At this time, Lactobacillus rhamnosus GG (hereinafter, L. rhamnosus GG) was used as a positive control group. To measure the production amount of NO, the Griess Reagent system from Promega was used. 50 μl of the cultured medium was transferred to a new 96-well plate. At this time, for the NO standard curve, a NO standard solution was used and diluted to 1.56 - 100 μM and added. After adding 50 μl of sulfanilamide solution to each well, the light was blocked for 5 minutes and the reaction was carried out at room temperature. After further adding 50 μl of NED solution to each well and reacting for 5 minutes at room temperature while blocking the light, the absorbance was measured at 540 nm using a microplate reader, and the production amount of NO was calculated using this NO standard curve (see Figure 3(a)).

[0076] As a result of the experiment, it was confirmed that the mixture of LM1071 strain and LM1001 strain had a higher NO production amount than L. rhamnosus GG in the range of 8:1 to 1:8.

[0077] (2) Confirmation of anti-inflammatory ability For the culture of Raw264.7 cells, a macrophage cell line derived from mice, DMEM culture medium (Welgene, Korea) containing 10% fetal bovine serum (Welgene, Korea) and penicillin-streptomycin (Welgene, Korea) was used. Raw264.7 cells were dispensed into a 96-well plate at a concentration of 3x104 After dispensing at a concentration of cells / well, it was cultured in a 37°C CO 2 incubator overnight. After removing the medium from each well, a mixture of LM1071 and LM1001 (LM1071:LM1001 = 8:1, 4:1, 1:1, 1:4, 1:8) was diluted in the medium and treated for 2 hours. After treating each well with LPS to induce inflammation in Raw264.7 cells, it was further cultured in a 37°C CO 2 incubator for 24 hours. To measure the production amount of NO, the Griess Reagent system from Promega was used. 50 μl of the cultured medium was transferred to a new 96-well plate. At this time, for the NO standard curve, a NO standard solution was used to dilute it to 1.56 - 100 μM and added. After adding 50 μl of sulfanilamide solution to each well, the light was blocked for 5 minutes and reacted at room temperature. After further adding 50 μl of NED solution to each well and reacting for 5 minutes at room temperature with the light blocked, the absorbance was measured at 540 nm using a microplate reader, and the production amount of NO was calculated using this NO standard curve (see Figure 3(b)).

[0078] As a result of the experiment, it was confirmed that when a mixture of LM1071 strain and LM1001 strain was used in combination with LPS, the NO production induced by LPS was suppressed in the range of 8:1 to 1:4.

[0079] Overall, the mixture of LM1071 strain and LM1001 strain is excellent in immunopotentiating ability in the range of LM1071:LM1001 mixing ratios of 8:1 to 1:8, and excellent in anti-inflammatory ability in the range of 8:1 to 1:4. Therefore, it can be seen that the mixture of LM1071 strain and LM1001 strain is excellent in immunomodulatory functions (immunopotentiating ability and anti-inflammatory ability) in the range of 8:1 to 1:4.

[0080] Example 3. Experiment for confirming the immune enhancing ability and anti-inflammatory ability of five strains

[0081] (1) Confirmation of immunopotentiating ability For the culture of Raw264.7 cells, a mouse-derived macrophage cell line, DMEM culture medium (Welgene, Korea) containing 10% fetal bovine serum (Welgene, Korea) and penicillin-streptomycin (Welgene, Korea) was used. Raw264.7 cells were cultured in a 96-well plate at 3 × 10 4 After dispensing at a concentration of 100 cells / well, incubate at 37°C in CO for O / N. 2 The wells were cultured in an incubator. After removing the medium from each well, the killed bacteria of LM1009, LM1012, LM1071, LM1024, and LM1004 were diluted in medium according to the number of bacteria and treated for 24 hours. At this time, LPS was treated as a positive control. Promega's Griess Reagent system was used to measure the amount of NO produced. 50 μl of the culture medium was transferred to a new 96-well plate. At this time, NO standard solution was diluted to 1.56-100 μM for the NO standard curve. 50 μl of sulfanilamide solution was added to each well and reacted at room temperature for 5 minutes in the dark. 50 μl of NED solution was further added to each well and reacted at room temperature for 5 minutes in the dark, and the absorbance was measured at 540 nm using a microplate reader, and the amount of NO produced was calculated using the NO standard curve (see Figure 4).

[0082] As a result of the experiment, it was confirmed that all of the killed cells of LM1009, LM1012, LM1017, LM1024, and LM1004 increased the amount of NO produced.

[0083] (2) Confirmation of anti-inflammatory activity For the culture of Raw264.7 cells, a mouse-derived macrophage cell line, DMEM culture medium (Welgene, Korea) containing 10% fetal bovine serum (Welgene, Korea) and penicillin-streptomycin (Welgene, Korea) was used. Raw264.7 cells were cultured in a 96-well plate at 3 × 10 4 After dispensing at a concentration of 100 cells / well, incubate at 37°C in CO for O / N. 2After removing the medium from each well, killed bacteria of LM1009, LM1012, LM1071, LM1024, and LM1004 were diluted in the medium according to the number of bacteria and treated for 2 hours. To induce inflammation in Raw264.7 cells, LPS was treated in each well, and the cells were incubated at 37°C for another 24 hours in CO 2 The cells were cultured in an incubator. Promega's Griess Reagent system was used to measure the amount of NO produced. 50 μl of the culture medium was transferred to a new 96-well plate. At this time, NO standard solution was diluted to 1.56-100 μM for the NO standard curve. 50 μl of sulfanilamide solution was added to each well, and the cells were reacted at room temperature for 5 minutes in the dark. 50 μl of NED solution was further added to each well, and the cells were reacted at room temperature for 5 minutes in the dark. The absorbance was measured at 540 nm using a microplate reader, and the amount of NO produced was calculated using the NO standard curve (see Figure 5).

[0084] As a result of the experiment, it was confirmed that the LM1009, LM1017, and LM1024 strains had anti-inflammatory properties by suppressing NO production induced by LPS.

[0085] Based on the experimental results of Examples 1 to 3, three strains with excellent immune-enhancing ability (LM1001, LM1012, LM1004) and four strains with excellent anti-inflammatory ability (LM1071, LM1009, LM1017, LM1024) were selected, and used to conduct the experiment of Example 4.

[0086] Example 4. Experiment for confirming the immune enhancing ability and anti-inflammatory ability of seven mixed strains

[0087] (1) Confirmation of immune enhancing ability For the culture of Raw264.7 cells, a mouse-derived macrophage cell line, DMEM culture medium (Welgene, Korea) containing 10% fetal bovine serum (Welgene, Korea) and penicillin-streptomycin (Welgene, Korea) was used. Raw264.7 cells were cultured in a 96-well plate at 3 × 10 4 After dispensing at a concentration of 100 cells / well, incubate at 37°C in CO for O / N. 2 The wells were cultured in an incubator. After removing the medium from each well, the mixture of strains (a mixture of seven strains, a mixture of three immune-enhancing strains, and a mixture of four immune-suppressing strains) was diluted in the medium and treated for 24 hours. At this time, L. rhamnosus GG was treated as a positive control. Promega's Griess Reagent system was used to measure the amount of NO produced. 50 μl of the culture medium was transferred to a new 96-well plate. At this time, a NO standard solution was used to dilute the NO standard curve to 1.56-100 μM. 50 μl of sulfanilamide solution was added to each well, and the wells were reacted at room temperature for 5 minutes in the dark. 50 μl of NED solution was further added to each well and the wells were reacted at room temperature for 5 minutes in the dark, and the absorbance was measured at 540 nm using a microplate reader, and the amount of NO produced was calculated using the NO standard curve (see Figure 6(a)).

[0088] As a result of the experiment, it was confirmed that the mixture of the seven strains of the present invention produced more NO than the mixture of the four immunosuppressant strains and L. rhamnosus GG.

[0089] (2) Confirmation of anti-inflammatory activity For the culture of Raw264.7 cells, a mouse-derived macrophage cell line, DMEM culture medium (Welgene, Korea) containing 10% fetal bovine serum (Welgene, Korea) and penicillin-streptomycin (Welgene, Korea) was used. Raw264.7 cells were cultured in a 96-well plate at 3 × 10 4 After dispensing at a concentration of 100 cells / well, incubate at 37°C in CO for O / N. 2After removing the medium from each well, the mixture of strains (a mixture of seven strains, a mixture of three immune-enhancing strains, and a mixture of four immune-suppressing strains) was diluted in the medium and treated for 2 hours. To induce inflammation in Raw264.7 cells, LPS was treated in each well, and the cells were incubated at 37°C for another 24 hours in CO 2 The wells were cultured in an incubator. Promega's Griess Reagent system was used to measure the amount of NO produced. 50 μl of the culture medium was transferred to a new 96-well plate. At this time, NO standard solution was diluted to 1.56-100 μM for the NO standard curve. 50 μl of sulfanilamide solution was added to each well, and the wells were reacted at room temperature for 5 minutes in the dark. 50 μl of NED solution was further added to each well, and the wells were reacted at room temperature for 5 minutes in the dark. The absorbance was measured at 540 nm using a microplate reader, and the amount of NO produced was calculated using the NO standard curve (see Figure 6(b)).

[0090] As a result of the experiment, it was confirmed that the mixture of the seven strains of the present invention suppressed excessive NO production induced by inflammatory substances more effectively than the mixture of the three immune-enhancing strains or the LPS-only treatment group.

[0091] Overall, it is clear that the mixture of the seven strains of the present application can regulate immune function by increasing NO production (immune-enhancing ability) and suppressing excessive NO production induced by inflammatory substances (anti-inflammatory ability).

[0092] The above description of the present application is for illustrative purposes only, and a person having ordinary skill in the art to which the present application pertains should understand that the present application can be easily modified into other specific forms without changing the technical idea or essential features of the present application. Therefore, it should be understood that the above-described embodiments are illustrative in all respects and are not limiting. For example, each component described as a single type may be implemented in a distributed form, and similarly, each component described as a distributed type may be implemented in a combined form.

[0093] The scope of the present application is indicated by the claims set forth below rather than by the above detailed description, and all modifications and variations derived from the meaning and scope of the claims, as well as their equivalent concepts, should be construed as being included within the scope of the present application. [Accession number]

[0094] Depository institution: Korea Center for Culture Collection of Microorganisms (KCCM) Accession number: KCCM42959 Date of acceptance: 20101112 Depository institution: Korea Center for Culture Collection of Microorganisms (KCCM) Accession number: KCCM43246 Date of acceptance: 20161028 Depository institution: Korea Center for Culture Collection of Microorganisms (KCCM) Accession number: KCCM80146 Date of acceptance: 20170516 Depository institution: Korea Center for Culture Collection of Microorganisms (KCCM) Accession number: KFCC11771P Date of acceptance: 20180530 Depository institution: Korea Center for Culture Collection of Microorganisms (KCCM) Accession number: KCCM12629P Date of acceptance: 20191114 Depository institution: Korea Center for Culture Collection of Microorganisms (KCCM) Accession number: KCCM12650P Date of acceptance: 20191231 Depository institution: Korea Center for Culture Collection of Microorganisms (KCCM) Accession number: KCCM12919P Date of acceptance: 20201223 JPEG2025517725000002.jpg247170JPEG2025517725000003.jpg248170JPEG202 5517725000004.jpg247170JPEG2025517725000005.jpg247170JPEG20255177250 00006.jpg247170JPEG2025517725000007.jpg247170JPEG2025517725000008.j pg247170JPEG2025517725000009.jpg247170JPEG2025517725000010.jpg248170

Claims

1. A mixed strain comprising Lactobacillus reuteri LM1071 strain (KCCM12650P) and Lactobacillus plantarum LM1001 strain (KCCM42959).

2. The mixed strain according to Claim 1, wherein the ratio of Lactobacillus reuteri LM1071 strain (KCCM12650P) and Lactobacillus plantarum LM1001 strain (KCCM42959) is 8:1 to 1:

4.

3. A food composition for immunomodulation, comprising the mixed strain according to Claim 1 or Claim 2 as an active ingredient.

4. A pharmaceutical composition for preventing or treating immune-related diseases, comprising the mixed strain according to Claim 1 or Claim 2 as an active ingredient.

5. A health functional food composition for immunomodulation, comprising the mixed strain according to Claim 1 or Claim 2 as an active ingredient.

6. The mixed strain according to Claim 1, further comprising Bifidobacterium animalis subsp. lactis LM1017 strain (KCCM12629P), Bifidobacterium longum LM1024 strain (KCCM12919P), Lactococcus lactis LM1009 strain (KCCM80146), Streptococcus thermophilus LM1012 strain (KFCC11771P) and Lactobacillus plantarum strain.

7. A food composition for immunomodulation, comprising the mixed strain according to Claim 6 as an active ingredient.

8. A pharmaceutical composition for preventing or treating immune-related diseases, comprising the mixed strain according to Claim 6 as an active ingredient.

9. A health functional food composition for immunomodulation, comprising the mixed strain according to Claim 6 as an active ingredient.

10. The Lactobacillus plantarum strain of the mixed strain according to claim 6 is Lactobacillus plantarum LM1004 strain (KCCM43246).

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