Probiotic complex composition having immunomodulatory and immunohomeostatic functions

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] This 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 useful bacteria that are beneficial to human intestinal health and harmful bacteria that are harmful to health. The human body can maintain health when useful bacteria occupy as dominant bacteria over harmful bacteria such as Escherichia coli (E. coli), Veillonella, and Clostridium perfringens, and various microorganisms inhabiting the digestive tract maintain 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 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 (Treg) and is known to play a central role in maintaining immune homeostasis.

[0004] Intestinal bacteria, while acting as antigens and triggering immune responses in the human body, also possess mechanisms of immune tolerance to reduce these responses in order to coexist with the body. Some of these mechanisms are known to be involved in the differentiation process of T cells, inducing the differentiation and proliferation of regulatory T cells in the gut. An example of developing compositions for immunomodulation using such microorganisms is a novel lactic acid bacterium derived from the human gastrointestinal tract with immunomodulatory effects and its uses (Registered Patent No. 10-1862051, Republic of Korea). However, in-depth development and diverse research on compositions that exhibit excellent effects related to immunomodulation remain necessary.

[0005] Therefore, the present inventors made diligent efforts to develop a composition with excellent immunomodulatory function, and as a result, developed a mixed bacterial strain and a composition containing the same that can regulate immunity by increasing NO production or suppressing excessive NO production induced by inflammatory substances, thereby achieving the present invention. [Prior art documents] [Patent Documents]

[0006] [Patent Document 1] Korean Registered Patent No. 10-2224027 [Overview of the project] [Problems that the invention aims to solve]

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

[0008] However, the problems that this application seeks to solve are not limited to those mentioned above, and other problems not mentioned should be clearly understood by those skilled in the art from the following description. [Means for solving the problem]

[0009] The first aspect of this application is to provide a mixed bacterial strain having immunomodulatory function.

[0010] A second aspect of the present application is to provide an immunomodulatory food composition containing a mixed bacterial strain having immunomodulatory function as an active ingredient.

[0011] A third aspect of the present application provides a pharmaceutical composition for the prevention or treatment of immunomodulation-related diseases, comprising a mixed bacterial strain having immunomodulatory function as an active ingredient. [Effects of the Invention]

[0012] A composition containing a mixed bacterial strain having immunomodulatory function as an active ingredient, as embodied in this application, has immunomodulatory function by increasing NO production or suppressing excessive NO production induced by inflammatory substances, and the bacterial strain can be applied to food compositions, health functional food compositions, pharmaceutical compositions, and the like. [Brief explanation of the drawing]

[0013] [Figure 1] This figure shows the amount of NO produced (confirmation of immunostimulatory ability) when (a) LM1071 strain and (b) LM1001 strain are treated individually, according to one embodiment of the present invention. [Figure 2] This figure shows the suppression of NO production induced by LPS (confirmation of anti-inflammatory activity) when (a) LM1071 strain and (b) LM1001 strain are treated individually, according to one embodiment of the present invention. [Figure 3] This figure shows the amount of NO produced by the LM1071 strain and the LM1001 strain in a mixed ratio, according to one embodiment of the present invention ((a) confirmation of immune-enhancing ability), and the suppression of NO production induced by LPS ((b) confirmation of anti-inflammatory ability). [Figure 4] This figure shows the amount of NO produced (confirmation of immune-enhancing ability) by five bacterial strains ((a) LM1009, (b) LM1012, (c) LM1017, (d) LM1024, (e) LM1004 dead cells) related to one embodiment of the present application. [Figure 5]This figure shows the suppression of NO production induced by LPS (confirmation of anti-inflammatory activity) when five bacterial strains ((a) LM1009, (b) LM1012, (c) LM1017, (d) LM1024, (e) LM1004 dead cells) related to one embodiment of the present invention are treated individually. [Figure 6] This figure shows the amount of NO produced ((a) confirmation of immunostimulatory ability) or the suppression of NO production induced by LPS ((b) confirmation of anti-inflammatory ability) of a mixture of seven bacterial strains relating to one example of the present invention, a mixture of three immunostimulatory strains (dead cells of LM1001, LM1012, and LM1004), and a mixture of four immunosuppressive strains (LM1071, LM1009, LM1017, and LM1024). [Modes for carrying out the invention]

[0014] In the following, embodiments of the present application will be described in detail with reference to the attached drawings, so that they can be easily implemented by a person with ordinary skill in the art to which the present application pertains. However, the present application can be embodied in various different forms and is not limited to the embodiments described herein. Furthermore, in order to clearly illustrate the present application, parts unrelated to the description have been omitted from the drawings, and similar parts throughout the specification are denoted by similar reference numerals.

[0015] Throughout the specification of this application, when a member is described as being "on top of" another member, this includes not only cases where the member is in contact with another member, but also cases where there is yet another member between the two members.

[0016] Throughout the specification of this application, when a part states that a certain component "includes", this means, unless otherwise stated to the contrary, that it may further include other components rather than excluding other components. Terms such as "about" and "substantially" used throughout the specification of this application are used with the meaning 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 improperly using the disclosure where exact or absolute numerical values are mentioned to assist in the understanding of this application. The terms "step of ~(doing)" 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 combinations" included in the Markush-form expression means one or more mixtures or combinations selected from the group consisting of the components described in the Markush-form 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 invention, the mixed strain may include, but is not limited to, Lactobacillus reuteri strain LM1071 (KCCM12650P) and Lactobacillus plantarum strain LM1001 (KCCM42959).

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

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

[0025] The term "NO (nitric oxide)" used throughout this specification is also called "nitric oxide" or "nitric monoxide," and refers to a compound in which nitrogen has been oxidized. It is formed from the amino acid arginine within cells and is involved in various physiological activities as a type of signaling molecule, including immune function, vasodilation, and signal transduction. It is also known to promote the secretion of inflammatory cytokines such as TNF-α and IL-6, thereby inducing inflammation and pain (Hu et al., 2020).

[0026] In one embodiment of the present invention, 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 the present invention, the mixed bacterial strain modulates immune function, and specifically, the bacterial 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 an immunomodulatory food composition containing a mixed bacterial strain having immunomodulatory function as an active ingredient. Content that overlaps with the first aspect also applies to the food composition of the second aspect.

[0029] In one embodiment of the present application, the composition modulates immune function, specifically by increasing NO production or suppressing excessive NO production induced by inflammatory substances.

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

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

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

[0033] The term "dead bacterial cells," as used throughout this specification, is the opposite of "live bacteria," and refers to a form in which live bacteria and metabolites obtained by fermentation are rendered unable to grow through heat treatment or other means. Dead bacterial cells may contain cytoplasm, cell walls, antimicrobial active substances such as bacteriocins, polysaccharides, organic acids, etc. Products utilizing the aforementioned dead bacterial cells have higher safety compared to live bacterial products, and in particular, they have the advantages of being highly heat-resistant, highly safe from the external environment, easier to store than existing live bacterial products, and having a longer distribution period. Furthermore, given the strengthening of regulations on the use of antibiotics, there is high potential for use as an alternative, and because there are still only a handful of companies that have seriously entered the production of dead bacterial cell products, the market potential and growth potential are very high.

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

[0035] As used throughout the specification of this application, the term "food" includes all foods in the ordinary sense, such as meats, sausages, bread, chocolates, candies, snacks, confectionery, pizzas, ramen and other noodle products, gums, dairy products including ice cream, various soups, beverages, teas, energy drinks, alcoholic beverages, multivitamins, functional foods, and health foods.

[0036] The term "health functional food" as used throughout the specification of this application means a food manufactured and processed using raw materials or components that have functional properties useful to the human body, as defined in Act No. 6727 on Health Functional Foods. "Functionality" means obtaining effects useful for health purposes, such as regulating nutrients or physiological effects on the structure and function of the human body.

[0037] The food product of this invention can be manufactured by methods commonly used in the industry, and during the manufacturing process, raw materials and components commonly added in the industry may be added. Furthermore, the dosage form of the food product is not limited as long as it is a dosage form recognized as a food product. The food composition of the present invention can be manufactured in various dosage forms, and unlike general pharmaceuticals, it is made from food as a raw material, so it has the advantage of not having side effects that can occur with long-term use of pharmaceuticals, and it is highly portable, so the food product of the present invention can be taken as an adjunct to enhance the effect of improving the intestinal environment.

[0038] The term "health food" refers to foods that have a more active effect on maintaining or promoting health compared to general foods, while "health supplement food" refers to foods intended to supplement health. Depending on the context, the terms "health functional food," "health food," and "health supplement food" may be used interchangeably. Specifically, the term "health functional food" refers to foods in which the mixed bacterial strain of this application is added to food materials such as beverages, teas, spices, gums, and confectionery, or manufactured in the form of capsules, powders, or suspensions, and which, when consumed, produce specific health effects. However, unlike general medicines, it uses food as a raw material, and therefore has the advantage of not having the side effects that can occur with long-term use of medicines.

[0039] Since the food composition of this invention can be consumed on a daily basis, it can be expected to have a high effect in improving depression and can therefore be used very effectively.

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

[0041] Furthermore, the food composition may contain additional ingredients commonly used in food compositions that can improve smell, taste, 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), and chromium (Cr). In addition, it may contain amino acids such as lysine, tryptophan, cysteine, and valine.

[0042] Furthermore, the food composition may also contain food additives such as preservatives (potassium sorbate, sodium benzoate, salicylic acid, sodium dehydroacetate, etc.), disinfectants (bleaching powder and high-grade bleaching powder, sodium hypochlorite, etc.), antioxidants (butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), etc.), colorants (tar dyes, etc.), color fixatives (sodium nitrite, sodium nitrite, etc.), bleaching agents (sodium sulfite), seasonings (MSG, monosodium glutamate, etc.), sweeteners (dulcin, cyclamate, saccharin, sodium, etc.), flavorings (vanillin, lactones, etc.), leavening agents (alum, potassium bitartrate, etc.), fortifiers, emulsifiers, thickeners, coating agents, gum bases, antifoaming agents, solvents, and improvers. The additives may be selected according to the type of food and used in appropriate amounts.

[0043] The mixed bacterial strain of this invention may be added as is, used together with other foods or food components, or used appropriately by conventional methods. The amount of active ingredient mixed may be appropriately determined according to its intended use (prevention, health, or therapeutic treatment). Normally, when manufacturing food or beverages, the food composition of this invention may be added to the food or beverage in an amount of 50 parts by weight or less, specifically 20 parts by weight or less. However, when ingested for a long period of time for health and hygiene purposes, the content may be less than the above range, and there are no safety concerns, so the active ingredient may be used in amounts greater than the above range.

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

[0045] In addition to the above, the health beverage composition may contain various nutrients, vitamins, electrolytes, flavorings, colorings, pectin, salts of pectin, alginic acid, salts of alginic acid, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohol, or carbonation agents. It may also contain fruit pulp for producing natural fruit juice, fruit juice beverages, or vegetable beverages. These components may be used individually or in combination. The proportion of such additives is not particularly important, but it is generally selected in the range of 0.01 to 0.1 parts by weight per 100 parts by weight of the health beverage composition of the present invention.

[0046] The food composition of the present invention may contain the mixed bacterial strain of the present invention in various weight percentages, as long as it can produce the effect of improving the intestinal environment. Specifically, the mixed bacterial strain of the present invention may be contained in an amount of 0.00001 to 100% by weight, or 0.01 to 80% by weight, relative to the total weight of the food composition, but is not limited thereto.

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

[0048] The third aspect of this application provides a pharmaceutical composition for the prevention or treatment of immunomodulatory diseases, comprising a mixed bacterial strain having immunomodulatory function as an active ingredient. The content that overlaps with the first and second aspects also applies to the pharmaceutical composition of the third aspect.

[0049] In one embodiment of the present application, the composition may include the mixed bacterial strain of the present application, its live cells, its dead cells, its culture, its crushed products and / or extracts thereof.

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

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

[0052] As used throughout the specification of this application, the term "treatment" means all actions that improve or alleviate the symptoms of an immune-related disease by administering a pharmaceutical composition containing the mixed bacterial strain of this application as an active ingredient to an individual who has developed an immune-related disease.

[0053] In one embodiment of the present application, the composition prevents or treats immune-related diseases, specifically by increasing NO production or suppressing excessive NO production induced by inflammatory substances.

[0054] The term "immune-related disease" as used throughout the specification of this application refers to all diseases related to immunity, including various diseases caused by bacterial and viral infections that are likely to appear when immunity is weakened, and autoimmune diseases and allergic reactions that are likely to appear due to an excessive immune response.

[0055] The term "immune deficiency" as used throughout the specification of this application refers to a general term for diseases resulting from dysfunction of components of the immune system, including severe combined immunodeficiency (SCID) and acquired immunodeficiency syndrome (AIDS).

[0056] The term "hyperimmune disease" as used throughout the specification of this application is a general term for diseases that occur due to the overexpression of the immune system, and includes autoimmune diseases such as rheumatoid arthritis, allergies, and atopic dermatitis.

[0057] In one embodiment of the present application, the pharmaceutical composition may be formulated by conventional methods into oral dosage forms such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, topical preparations, suppositories, or sterile injection solutions for use, but is not limited thereto.

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

[0059] In one embodiment of the present invention, the solid preparation for oral administration includes tablets, pills, powders, granules, or capsules, and such solid preparations may be prepared by mixing the dead bacterial cells of the strain with at least one excipient, such as starch, calcium carbonate, sucrose, lactose, or gelatin. In addition to mere excipients, lubricants such as magnesium stearate or talc may also be used, but are not limited thereto.

[0060] In one embodiment of the present invention, the liquid formulation for oral administration includes suspensions, oral solutions, emulsions, syrups, etc., and may include, but is not limited to, various excipients such as water and liquid paraffin, which are commonly used diluents, as well as humectants, sweeteners, fragrances, and preservatives.

[0061] In one embodiment of the present invention, the formulation for parenteral administration may include, but is not limited to, sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized formulations, and suppositories. For example, the non-aqueous solvent or suspension may include, but is not limited to, propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate. For example, the suppository may include, but is not limited to, witepsol, macrogol, tween 61, cocoa butter, lauric butter, and glycerol gelatin.

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

[0063] The term "quasi-drug" as used throughout this specification refers to articles used for the purpose of diagnosing, treating, improving, alleviating, managing, or preventing diseases in humans or animals, which have a milder effect than pharmaceuticals. For example, according to the Pharmaceutical Affairs Law, quasi-drugs are articles other than those used for pharmaceutical purposes, and include products used for the treatment or prevention of diseases in humans and animals, and products that have a mild or no direct effect on the human body.

[0064] The quasi-drug composition of this application may be manufactured in a dosage form selected from the group consisting of body cleansers, disinfectants, detergents, kitchen detergents, cleaning detergents, toothpaste, mouthwashes, wet wipes, detergents, soaps, hand washes, hair washes, hair softeners, 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 pharmacokinetically effective amount, but the term "pharmacokinetically effective amount" as used in the present application means an amount sufficient to treat or prevent a disease with a reasonable benefit / risk ratio applicable to medical treatment or prevention, and the level of the effective dose may be determined by the severity of the disease, the activity of the drug, the patient's age, weight, health, sex, the patient's drug sensitivity, the time of administration, route of administration and excretion rate of the composition of the present invention used, the duration of treatment, factors including drugs used in combination with or concurrently with the composition of the present invention used, 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 exert therapeutic effects on known intestinal diseases. Taking all of the above factors into consideration, it is important to administer an amount that provides 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 a person skilled in the art, taking into consideration the purpose of use, the degree of toxicity of the disease, the patient's age, weight, sex, medical history, or the type of substance used as the active ingredient. For example, the pharmaceutical composition of the present invention may be administered to an adult at a dose of about 0.1 ng to about 1,000 mg / kg, preferably 1 ng to about 100 mg / kg, and the frequency of administration of the composition of the present application is not particularly limited thereto, but may be administered once a day or divided into several doses. The above dosage or frequency of administration does not limit the scope of the present application in any way.

[0067] The pharmaceutical compositions of this application are not limited to those described herein and may be administered via routes such as intraperitoneal administration, intravenous administration, intramuscular administration, subcutaneous administration, intradermal administration, transdermal patch administration, oral administration, intranasal administration, intrapulmonary administration, or intrarectal administration, depending on the purpose. However, in the case of oral administration, the composition may be administered in an unformulated form, and since the Lactobacillus reuteri LM1071 strain may be denatured or degraded by gastric acid, the oral composition may be administered orally in a form coated with the active agent, or in a form that is formed to protect it from degradation in the stomach, or in the form of an oral patch. Furthermore, the composition may be administered by any device that allows the active substance to move to target cells. [Examples]

[0068] The present invention will be described in more detail below with reference to the embodiments of this application, but the embodiments described below are for illustrative purposes only to aid in understanding this application, and the content of this application is not limited to the embodiments described below.

[0069] [Examples] Example 1. Experiment to confirm the immunostimulatory and anti-inflammatory effects of LM1071 and LM1001 when treated individually.

[0070] (1) Confirmation of immune-enhancing ability For culturing Raw264.7 cells, a mouse-derived macrophagous cell line, a DMEM culture medium (Welgene, Korea) containing 10% fetal bovine serum (Welgene, Korea) and penicillin-streptomycin (Welgene, Korea) was used. Raw264.7 cells were placed in 3x10⁶ well plates in a 96-well plate. 4 After dispensing at the cells / well concentration, the cells were cultured overnight in a 37°C CO2 incubator. After removing the culture medium from each well, LM1071 or LM1001 was diluted in the culture medium according to the number of cells and treated for 24 hours. LPS was used as a positive control group during this time. To measure the amount of NO produced, Promega's Griess Reagent system was used. 50 μl of cultured medium was transferred to a new 96-well plate. At this time, NO standard solution was used to dilute it to 1.56-100 μM for the NO standard curve and added. 50 μl of sulfanilamide solution was added to each well, and the reaction was allowed to proceed at room temperature for 5 minutes in the dark. After adding another 50 μl of N-1-napthylethylenediamine dihydrochloride (NED) solution to each well and allowing the reaction to proceed 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 1).

[0071] The experimental results confirmed that both strains LM1071 and LM1001 increased NO production.

[0072] (2) Confirmation of anti-inflammatory activity For culturing Raw264.7 cells, a mouse-derived macrophagous cell line, a DMEM culture medium (Welgene, Korea) containing 10% fetal bovine serum (Welgene, Korea) and penicillin-streptomycin (Welgene, Korea) was used. Raw264.7 cells were placed in 3x10⁶ well plates in a 96-well plate. 4 After dispensing at the cells / well concentration, the cells were cultured overnight in a 37°C CO2 incubator. After removing the medium from each well, LM1071 or LM1001 was diluted in the medium according to the number of cells and treated for 2 hours. To induce inflammation in Raw264.7 cells, LPS was treated in each well, and then the cells were cultured for another 24 hours in a 37°C CO2 incubator. To measure the amount of NO produced, the Promega Griess Reagent system was used. 50 μl of cultured medium was transferred to a new 96-well plate. At this time, a 1.56–100 μM NO standard solution was used to create the NO standard curve and added. 50 μl of sulfanilamide solution was added to each well, and the reaction was allowed to proceed at room temperature for 5 minutes in the dark. After adding another 50 μl of NED solution to each well and allowing the reaction to proceed 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 2).

[0073] The experimental results confirmed that only the LM1071 strain could suppress the excessive NO production induced by LPS, an inflammatory substance.

[0074] Example 2. Experiment to confirm the immunostimulatory and anti-inflammatory abilities of a mixed strain of LM1071 and LM1001.

[0075] (1) Confirmation of immune-enhancing ability For culturing Raw264.7 cells, a mouse-derived macrophagous cell line, a DMEM culture medium (Welgene, Korea) containing 10% fetal bovine serum (Welgene, Korea) and penicillin-streptomycin (Welgene, Korea) was used. Raw264.7 cells were placed in 3x10⁶ well plates in a 96-well plate. 4 After dispensing at the cells / well concentration, the cells were incubated overnight in a 37°C CO2 incubator. After removing the culture 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 culture medium and treated for 24 hours. At this time, Lactobacillus rhamnosus GG (hereinafter, L. rhamnosus GG) was treated as a positive control group. To measure the amount of NO produced, Promega's Griess Reagent system was used. 50 μl of cultured medium was transferred to a new 96-well plate. At this time, NO standard solution was diluted to 1.56-100 μM using NO standard solution and added to create an NO standard curve. After adding 50 μl of sulfanilamide solution to each well, the reaction was allowed to proceed at room temperature for 5 minutes in the dark. After adding 50 μl of NED solution to each well and allowing it to react at room temperature for 5 minutes while blocking out light, 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 3(a)).

[0076] The experimental results confirmed that a mixture of LM1071 strain and LM1001 strain produced more NO than L. rhamnosus GG in a ratio ranging from 8:1 to 1:8.

[0077] (2) Confirmation of anti-inflammatory activity For culturing Raw264.7 cells, a mouse-derived macrophagous cell line, a DMEM culture medium (Welgene, Korea) containing 10% fetal bovine serum (Welgene, Korea) and penicillin-streptomycin (Welgene, Korea) was used. Raw264.7 cells were placed in 3x10⁶ well plates in a 96-well plate. 4After dispensing at the cells / well concentration, the cells were cultured overnight in a 37°C CO2 incubator. 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. To induce inflammation in Raw264.7 cells, LPS was treated in each well, and then the cells were cultured for another 24 hours in a 37°C CO2 incubator. To measure the amount of NO produced, the Promega Griess Reagent system was used. 50 μl of cultured medium was transferred to a new 96-well plate. At this time, a standard NO solution was used and diluted to 1.56–100 μM for the NO standard curve. 50 μl of sulfanilamide solution was added to each well, and the reaction was allowed to proceed at room temperature for 5 minutes in the dark. After adding 50 μl of NED solution to each well and allowing it to react at room temperature for 5 minutes while blocking out light, 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 3(b)).

[0078] The experimental results confirmed that when a mixture of LM1071 strain and LM1001 strain was treated with LPS, LPS-induced NO production was suppressed in a ratio of 8:1 to 1:4.

[0079] Overall, the mixture of LM1071 and LM1001 strains exhibits superior immunostimulatory activity in a LM1071:LM1001 ratio of 8:1 to 1:8, and superior anti-inflammatory activity in a ratio of 8:1 to 1:4. Therefore, it can be concluded that the mixture of LM1071 and LM1001 strains exhibits superior immunomodulatory function (immunostimulatory and anti-inflammatory activity) in a ratio of 8:1 to 1:4.

[0080] Example 3. Experiment to confirm the immune-enhancing and anti-inflammatory abilities of five bacterial strains.

[0081] (1) Confirmation of immune-enhancing ability For culturing Raw264.7 cells, a mouse-derived macrophagous cell line, a DMEM culture medium (Welgene, Korea) containing 10% fetal bovine serum (Welgene, Korea) and penicillin-streptomycin (Welgene, Korea) was used. Raw264.7 cells were placed in 3x10⁶ well plates in a 96-well plate. 4 After dispensing at the cells / well concentration, the cells were cultured overnight in a 37°C CO2 incubator. After removing the culture medium from each well, dead cells of LM1009, LM1012, LM1071, LM1024, and LM1004 were diluted in culture medium according to the number of cells and treated for 24 hours. LPS was used as a positive control group during this time. Promega's Griess Reagent system was used to measure the amount of NO produced. 50 μl of cultured medium was transferred to a new 96-well plate. At this time, NO standard solution was used to dilute it to 1.56-100 μM for the NO standard curve and added. 50 μl of sulfanilamide solution was added to each well, and the reaction was allowed to proceed at room temperature for 5 minutes in the dark. After adding another 50 μl of NED solution to each well and allowing the reaction to proceed 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 4).

[0082] The experimental results confirmed that all dead bacterial cells of LM1009, LM1012, LM1017, LM1024, and LM1004 increased NO production.

[0083] (2) Confirmation of anti-inflammatory activity For culturing Raw264.7 cells, a mouse-derived macrophagous cell line, a DMEM culture medium (Welgene, Korea) containing 10% fetal bovine serum (Welgene, Korea) and penicillin-streptomycin (Welgene, Korea) was used. Raw264.7 cells were placed in 3x10⁶ well plates in a 96-well plate. 4After dispensing at the cells / well concentration, the cells were cultured overnight in a 37°C CO2 incubator. After removing the medium from each well, dead cells of LM1009, LM1012, LM1071, LM1024, and LM1004 were diluted in medium according to the number of cells and treated for 2 hours. To induce inflammation in Raw264.7 cells, LPS was treated in each well, and then the cells were cultured for another 24 hours in a 37°C CO2 incubator. To measure the amount of NO produced, the Promega Griess Reagent system was used. 50 μl of cultured medium was transferred to a new 96-well plate. At this time, a standard NO solution was used to dilute it to 1.56–100 μM for the NO standard curve and added. 50 μl of sulfanilamide solution was added to each well, and the reaction was allowed to proceed at room temperature for 5 minutes in the dark. After adding 50 μl of NED solution to each well and allowing it to react at room temperature for 5 minutes while blocking out light, 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] The experimental results confirmed that the LM1009, LM1017, and LM1024 strains suppressed LPS-induced NO production, demonstrating anti-inflammatory activity.

[0085] Based on the experimental results of Examples 1 to 3 described above, three strains with superior immune-enhancing ability (LM1001, LM1012, LM1004) and four strains with superior anti-inflammatory ability (LM1071, LM1009, LM1017, LM1024) were selected, and the experiment in Example 4 was carried out using these strains.

[0086] Example 4. Experiment to confirm the immune-enhancing and anti-inflammatory abilities of seven mixed bacterial strains.

[0087] (1) Confirmation of immune-enhancing ability For culturing Raw264.7 cells, a mouse-derived macrophagous cell line, a DMEM culture medium (Welgene, Korea) containing 10% fetal bovine serum (Welgene, Korea) and penicillin-streptomycin (Welgene, Korea) was used. Raw264.7 cells were placed in 3x10⁶ well plates in a 96-well plate. 4 After dispensing at a concentration of cells / well, the cells were cultured overnight in a 37°C CO2 incubator. After removing the culture medium from each well, a mixture of strains (a mixture of 7 strains, a mixture of 3 immunostimulatory strains, and a mixture of 4 immunosuppressant strains) was diluted in the culture medium and treated for 24 hours. L. rhamnosus GG was treated as a positive control group. Promega's Griess Reagent system was used to measure the amount of NO produced. 50 μl of cultured medium was transferred to a new 96-well plate. At this time, a NO standard solution was used to dilute it to 1.56–100 μM for the NO standard curve and added. 50 μl of sulfanilamide solution was added to each well, and the reaction was allowed to proceed at room temperature for 5 minutes in the dark. After adding another 50 μl of NED solution to each well and allowing the reaction to proceed 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(a)).

[0088] The experimental results confirmed that the mixture of the seven bacterial strains described in this application produced a higher amount of NO compared to the mixture of four immunosuppressant bacterial strains and L. rhamnosus GG.

[0089] (2) Confirmation of anti-inflammatory activity For culturing Raw264.7 cells, a mouse-derived macrophagous cell line, a DMEM culture medium (Welgene, Korea) containing 10% fetal bovine serum (Welgene, Korea) and penicillin-streptomycin (Welgene, Korea) was used. Raw264.7 cells were placed in 3x10⁶ well plates in a 96-well plate. 4After dispensing at the cells / well concentration, the cells were cultured overnight in a 37°C CO2 incubator. After removing the culture medium from each well, a mixture of strains (a mixture of 7 strains, a mixture of 3 immunostimulatory strains, and a mixture of 4 immunosuppressant strains) was diluted in the culture medium and treated for 2 hours. To induce inflammation in Raw264.7 cells, LPS was treated in each well, and then the cells were cultured for another 24 hours in a 37°C CO2 incubator. The amount of NO produced was measured using the Promega Griess Reagent system. 50 μl of cultured medium was transferred to a new 96-well plate. At this time, a standard NO solution was used to dilute it to 1.56–100 μM for the NO standard curve and added. 50 μl of sulfanilamide solution was added to each well, and the reaction was allowed to proceed at room temperature for 5 minutes in the dark. After adding 50 μl of NED solution to each well and allowing it to react at room temperature for 5 minutes while blocking out light, 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] The experimental results confirmed that the mixture of seven bacterial strains described in this application suppressed excessive NO production induced by inflammatory substances to a greater extent compared to the mixture of three immunostimulant strains and the LPS-only treatment group.

[0091] Overall, it can be seen that the mixture of the seven bacterial strains of this application can regulate immune function by increasing NO production (immune-enhancing ability) or by suppressing excessive NO production induced by inflammatory substances (anti-inflammatory ability).

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

[0093] The scope of this application is defined by the claims, which are set forth below rather than by the detailed description above, and all modified or altered forms derived from the meaning and scope of the claims, as well as the concept of equivalents thereof, should be interpreted as being included within the scope of this application. [Accession Number]

[0094] Depository name: Korea Center for Microbial Conservation (KCCM) Accession number: KCCM42959 Date of acceptance: 20101112 Depository name: Korea Center for Microbial Conservation (KCCM) Accession number: KCCM43246 Date of acceptance: 20161028 Depository name: Korea Center for Microbial Conservation (KCCM) Accession number: KCCM80146 Date of acceptance: 20170516 Depository name: Korea Center for Microbial Conservation (KCCM) Accession number: KFCC11771P Date of acceptance: 20180530 Depository name: Korea Center for Microbial Conservation (KCCM) Accession number: KCCM12629P Date of acceptance: 20191114 Depository name: Korea Center for Microbial Conservation (KCCM) Accession number: KCCM12650P Date of acceptance: 20191231 Depository name: Korea Center for Microbial Conservation (KCCM) Accession number: KCCM12919P Date of acceptance: 20201223 JPEG0007857437000001.jpg247170JPEG0007857437000002.jpg248170JPEG000 7857437000003.jpg247170JPEG0007857437000004.jpg247170JPEG00078574370 00005.jpg247170JPEG0007857437000006.jpg247170JPEG0007857437000007.j pg247170JPEG0007857437000008.jpg247170JPEG0007857437000009.jpg248170

Claims

1. A composition for maintaining immune homeostasis by increasing or suppressing NO production, comprising a mixed strain containing Lactobacillus reuteri LM1071 strain (KCCM12650P) and Lactobacillus plantarum LM1001 strain (KCCM42959) as an active ingredient, The aforementioned mixed strains consist of Lactobacillus reuteri LM1071 strain (KCCM12650P) and Lactobacillus plantarum LM1001 strain (KCCM42959) in a ratio of 8:1 to 1:

4. A composition for maintaining immune homeostasis by increasing or suppressing NO production.

2. A food composition for maintaining immune homeostasis, comprising the composition for maintaining immune homeostasis described in claim 1 as an active ingredient, through increasing or suppressing NO production.

3. A health functional food composition for maintaining immune homeostasis, comprising the composition for maintaining immune homeostasis described in claim 1 as an active ingredient, through increasing or suppressing NO production.

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