Immunomodulatory composition

Abstract

To provide a component with immunomodulatory action, particularly one that can be easily taken orally.SOLUTION: The novel strain, Bifidobacterium longum subsp. longum MCC10345 (NITE BP-03751) is provided. Exopolysaccharide (EPS) produced by the strain is used as an active ingredient of an immunomodulatory composition. The composition is suitable for immunomodulatory applications in epithelial cells of the upper respiratory tract. The immunomodulation in the invention is preferably caused by inhibition of inflammatory cytokine production, promotion of antiviral cytokine production, and / or promotion of antiviral protein production.SELECTED DRAWING: Figure 1

Description

[Technical field]

[0001] The present invention relates to immunomodulatory compositions. [Background technology]

[0002] There is a great deal of interest in protecting the body from pathogenic viruses, such as the influenza virus, which has been around for a long time, and novel viruses, which are new threats. In response to pathogenic viruses, the innate immune system is important for preventing the invasion and proliferation of viruses into the body, and it is known that various cytokines and antiviral proteins are involved in this. Therefore, components that can control the production of cytokines and proteins involved in immunity are being searched for (for example, Patent Document 1).

[0003] In recent years, it has been reported that extracellular polymeric substances (hereinafter, also referred to as "EPS") produced by enterobacteria have physiological activities useful for humans, such as immunomodulatory effects (Non-Patent Document 1). EPS is a polysaccharide composed of various sugar residues, but the constituent sugars, structure, size, and charge differ depending on the bacterial species and strain, and it is presumed that each has a different physiological activity. For example, it has been reported that EPS produced by certain lactic acid bacteria controls the production of inflammatory cytokines and promotes the production of antiviral cytokines (Patent Document 2). It has also been reported that EPS produced by certain Bifidobacterium bacteria has immunostimulatory and antiallergic effects (Patent Documents 3 to 5). [Prior art documents] [Patent documents]

[0004] [Patent Document 1] Patent No. 5852558 [Patent Document 2] Patent No. 7017864 [Patent Document 3] WO 07 / 007562 [Patent Document 4] Japanese Patent Application Publication No. 58-203913 [Patent Document 5] JP 2011-201781 A [Non-patent literature]

[0005] [Non-Patent Document 1] Kenji Fukuda, Applied Glycoscience, Vol. 5, No. 1, 31-35 (2015) Summary of the Invention [Problem to be solved by the invention]

[0006] An object of the present invention is to provide a component capable of adjusting immune activation and suppression in a well-balanced manner, particularly a component that can be easily taken orally. [Means for solving the problem]

[0007] Bifidobacterium bacteria are known to produce EPS, but the effects of EPS produced by specific strains remain largely unknown. As a result of intensive research aimed at solving the above problems, the present inventors discovered that EPS produced by a novel strain of Bifidobacterium has an excellent immunomodulatory effect, thereby completing the present invention.

[0008] That is, in a first aspect, the present invention relates to a novel Bifidobacterium bacterium, Bifidobacterium longum subsp. longum. ubsp. longum) MCC10345 (NITE BP-03751).

[0009] A second aspect of the present invention is EPS produced by Bifidobacterium longum subsp. longum MCC10345 (NITE BP-03751).

[0010] A third aspect of the present invention relates to Bifidobacterium longum subsp. longum MCC10345 (NITE BP-03751) and / or a compound produced by said bacterium. An immunomodulatory composition containing EPS. Here, the composition is preferably for immunomodulation in epithelial cells of the upper respiratory tract. Furthermore, the immune regulation is preferably caused by suppressing the production of inflammatory cytokines, promoting the production of antiviral cytokines, and / or promoting the production of antiviral proteins. Moreover, the inflammatory cytokine is preferably IL-8 and / or IL-1β. Moreover, the antiviral cytokine is preferably IFNβ and / or IFNλ2. In addition, the antiviral protein is preferably Mx2. In a third aspect of the present invention, the EPS is preferably contained in one or more selected from the group consisting of a bacterial cell of Bifidobacterium longum subsp. longum MCC10345, a culture of the bacterium, and a treated product of the bacterial cell. The composition according to the third aspect of the present invention is preferably a food or drink, and more preferably a food or drink used for maintaining, improving, or strengthening immune function. The composition according to the third aspect of the present invention is preferably a pharmaceutical product, more preferably a pharmaceutical product used to treat or prevent a viral infectious disease. Effect of the Invention

[0011] According to the present invention, the production of IFNβ, IFNλ2, and Mx2 can be promoted. This can also lead to immunostimulation. According to the present invention, the production of IL-8 and IL-1β can be suppressed. This can also lead to immunosuppression. Since the composition of the present invention can be orally ingested, it can be easily used in the form of food, beverage, or pharmaceutical products. [Brief description of the drawings]

[0012] [Figure 1] Graph showing the relative expression level of IFNβ gene in lung epithelial cell A549 with addition of EPS sample (n=4). **: p<0.01, Student t-test. [Diagram 2] Graph showing the relative expression level of the Mx2 gene in lung epithelial cell A549 to which EPS sample was added (n=4). *: p<0.05, Student t-test. [Diagram 3] Graph showing the relative expression level of IL-8 gene in lung epithelial cell A549 with addition of EPS sample (n=4). **: p<0.01, Student t-test. [Figure 4] Graph showing the relative expression level of IL-1β gene in lung epithelial cell A549 with EPS sample added (n=4). *: p<0.05, Student t-test. [Diagram 5] Graph showing the relative expression level of the IFNβ gene in intestinal epithelial cells HT29 to which EPS samples were added (n=4). *: p<0.05, ***: p<0.001, Student t-test. [Figure 6] Graph showing the relative expression level of the IFNλ2 gene in intestinal epithelial cells HT29 to which an EPS sample was added (n=4). ***: p<0.001, Student t-test. [Figure 7] Graph showing the relative expression level of the IFNβ gene in intestinal epithelial cells HT29 to which EPS samples were added (n=4). *: p<0.05, ***: p<0.001, Student t-test. [Figure 8] Graph showing the relative expression level of IFNβ gene in intestinal epithelial cells HT29 to which EPS samples were added (n=4). ***: p<0.001, Student t-test. [Figure 9] Graph showing the relative expression level of the IFNλ2 gene in intestinal epithelial cells HT29 to which heated bacteria had been added (n=4). *: p<0.05, **: p<0.01, ***: p<0.001, Student t-test. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0013] The present invention will now be described in detail. However, the present invention is not limited to the following embodiments and can be freely modified within the scope of the present invention.

[0014] Bifidobacterium longum subsp. longum MCC10345 (NITE BP-03751) of the present invention (hereinafter also referred to as "Bifidobacterium longum subsp. longum MCC10345") is a novel Bifidobacterium bacterium isolated from human feces as an isolation source. In order to investigate the genetic properties of this bacterium, the 16SrRNA gene base sequence was identified by a conventional method. Furthermore, a homology search of the 16SrRNA gene base sequence of each Bifidobacterium bacterium was performed by BLAST analysis in the database of the National Center for Biotechnology Information (NCBI). As a result, it was confirmed that Bifidobacterium longum subsp. longum MCC10345 has 99% homology in the base sequence with Bifidobacterium longum subsp. longum JCM1217, which is the type strain of Bifidobacterium longum subsp. longum, and is a Bifidobacterium bacterium belonging to Bifidobacterium longum subsp. longum. Bifidobacterium longum subsp. longum MCC10345 was deposited at the Patent Microorganism Deposit Center of the National Institute of Technology and Evaluation (Postal Code: 292-0818, Address: Room 122, 2-5-8 Kazusa Kamatari, Kisarazu City, Chiba Prefecture) on September 14, 2022 under the Budapest Convention. The international deposit has been made in accordance with the provisions of the present application and has been assigned the accession number NITE BP-03751.

[0015] In this specification, Bifidobacterium longum subsp. longum MCC10345 is not limited to the strain deposited or registered under the name of the bacterium in a specified institution (hereinafter, for convenience of explanation, also referred to as the "deposited strain"), but also includes strains substantially equivalent thereto (also referred to as "derived strains" or "derived strains"). Regarding bacteria, a "strain substantially equivalent" to the deposited strain refers to a strain belonging to the same species as the deposited strain, and further having a genome sequence similarity (average nucleotide identity value) of preferably 99.0% or more with respect to the deposited strain, and more preferably 99.0% or more with respect to the deposited strain. It refers to a strain that has an identity of at least 99.5%, more preferably 100%, and preferably has the same bacteriological properties as the deposited strain. For bacteria, a strain that is substantially equivalent to the deposited strain may be, for example, a derived strain with the deposited strain as a parent strain. Derived strains include strains bred from the deposited strain and strains that have naturally arisen from the deposited strain. Breeding methods include modification by genetic engineering techniques and modification by mutation treatment. Mutation treatments include irradiation with X-rays, irradiation with ultraviolet rays, and N-methyl-N'-nitro-N-nitrosoguaia. Examples of mutations that may be present include treatment with mutagens such as nidin, ethyl methanesulfonate, and methyl methanesulfonate. Strains that have arisen naturally from the deposited strain include strains that have arisen naturally during use of the deposited strain. Such strains include mutant strains that have arisen naturally upon culturing (e.g., subculturing) of the deposited strain. Derivative strains may be constructed by one type of modification, or by two or more types of modifications.

[0016] As the Bifidobacterium longum subsp. longum MCC10345 of the present invention, a bacterial cell obtained by culturing the deposited strain or a derived strain (derived strain) can be used. The culture method is not particularly limited as long as Bifidobacterium longum subsp. longum MCC10345 can be grown. For example, the culture method may be a method commonly used for culturing Bifidobacterium longum subsp. longum. The method can be used as is or with appropriate modifications. The culture temperature may be, for example, 25 to 50°C, and is preferably 35 to 42°C. The culture can be preferably carried out under anaerobic conditions, for example, while aerating anaerobic gas such as carbon dioxide gas. The culture can also be carried out under microaerobic conditions such as liquid static culture. The culture can be carried out, for example, until the bacteria grow to a desired extent.

[0017] The medium used for the culture is Bifidobacterium longum subsp. longum. As long as MCC10345 can grow, the medium is not particularly limited. For example, a medium that is usually used for culturing the bacteria can be used as it is or after appropriate modification. That is, as the carbon source, for example, sugars such as galactose, glucose, fructose, mannose, cellobiose, maltose, lactose, sucrose, trehalose, starch, starch hydrolysate, and blackstrap molasses can be used depending on the assimilation. As the nitrogen source, for example, ammonium salts such as ammonia, ammonium sulfate, ammonium chloride, and ammonium nitrate, and nitrates can be used. In addition, as the inorganic salt, for example, sodium chloride, potassium chloride, potassium phosphate, magnesium sulfate, calcium chloride, calcium nitrate, manganese chloride, and ferrous sulfate can be used. In addition, organic components such as peptone, soybean flour, defatted soybean meal, meat extract, and yeast extract can be used. Specific examples of media commonly used for culturing the bacteria include Reinforced Clostridial medium, de Man, Rogosa, and Sharpe medium (MRS medium), modified MRS medium (mMRS medium), TOS propionate medium (TOSP medium), TOS propionate mupirocin medium (TOSP Mup medium), Gifu Anaerobic Medium (GAM) medium, and Yeast Extract-casein Hydrolysate Acid (YCFA) medium.

[0018] The bacterial cells may be live bacterial cells, killed bacterial cells, or a mixture of live bacterial cells and killed bacterial cells. The killed bacterial cells may be sterilized by any method, but for example, heat-treated bacterial cells (heat-sterilized bacterial cells) are preferable. The heat-sterilized bacterial cells may be obtained by treating the bacteria at 70 to 120°C for 10 to 40 minutes, at 80 to 110°C for 10 to 40 minutes, at 90 to 100°C for 10 to 40 minutes, or at 90 to 150°C for 5 to 30 seconds. Under these heat-sterilization conditions, it is possible to obtain a heat-sterilized bacterial cell having a high immunomodulatory effect, which will be described later. During the heat treatment, pressure may be applied. The temperature does not necessarily have to be constant, as long as it is within the above temperature range for a predetermined period of time. As the culture of the bacterial cells, for example, the culture obtained by culturing may be used as it is, the culture may be diluted or concentrated, or the bacterial cells recovered from the culture may be used. In addition, the culture may be a culture supernatant or a culture fraction. When the culture supernatant is used, for example, the supernatant of the culture solution obtained by culturing in MRS medium at 37°C for 16 hours can be preferably used. As the processed product of the bacterial cells, the bacterial cells or culture product can be heated, crushed, heat-dried, freeze-dried, or spray-dried, and dilutions or fractions thereof can be used.

[0019] The present invention relates to an EPS produced by Bifidobacterium longum subsp. longum MCC10345 (NITE BP-03751). Bifidobacterium longum subsp. longum MCC10345 can produce EPS by the general culture method described above. The bacterium usually secretes the EPS produced inside the cell outside the cell. Therefore, EPS is secreted from the above-mentioned Bifidobacterium longum subsp. longum MCC10345 (NITE BP- 03751) can be obtained by the culture method.

[0020] That is, in another aspect of the present invention, Bifidobacterium longum subsp. longum MCC10345 (NITE BP-03751) is cultured to obtain a culture. and recovering EPS from the culture. 1) may be a method for producing EPS produced by the A process for culturing Bifidobacterium longum subsp. longum MCC10345 (NITE BP-03751) to obtain a culture, and extracting EPS from the culture. The recovery process is carried out by the above-mentioned Bifidobacterium longum subsp. longum This can be done appropriately depending on the culture conditions of MCC10345 (NITE BP-03751). Cut. The step of recovering EPS from the culture may be a step of separating and purifying EPS from the culture, or a step of recovering EPS from the culture in a form contained in one or more selected from MCC10345 bacterial cells, a culture of the bacterial cells, and a treated product of the bacterial cells. The step of separating and purifying EPS from the culture is not particularly limited, and can be carried out by known methods such as centrifugation, membrane separation, etc. Membrane separation can be carried out using a dialysis membrane that removes EPS with a molecular weight of 100,000 or less. The process for recovering EPS from the culture in a form contained in one or more selected from MCC10345 bacterial cells, a culture of the bacterial cells, and a processed product of the bacterial cells is not particularly limited, and can be carried out by appropriately combining known methods such as centrifugation, dilution, concentration, crushing, heating, heat drying, freeze drying, and spray drying.

[0021] The composition of the present invention is Bifidobacterium longum subsp. longum MCC10345 (NITE BP-03751) and / or EPS produced by said bacteria Contains as an active ingredient. Bifidobacterium longum subsp. longum MCC10345 The bacterial cells described above can be used. When the EPS is contained in the composition of the present invention, it may be contained in a form contained in one or more selected from the group consisting of Bifidobacterium longum subsp. longum MCC10345 bacteria, a culture of the bacteria, and a processed product of the bacteria.

[0022] The content of EPS produced by Bifidobacterium longum subsp. longum MCC10345 in the composition of the present invention is not particularly limited and is appropriately set depending on the form of the composition, but for example, in the form when ingested, it is preferably 0.1 to 1000 μg / g or 0.1 to 1000 μg / mL, more preferably 1 to 1000 μg / g or 1 to 1000 μg / mL, and even more preferably 10 to 100 μg / g or 10 to 100 μg / mL. The structure of EPS is not known in detail, but the molecular weight is 100,000 or less. In addition, when the EPS produced by Bifidobacterium longum subsp. longum MCC10345 is contained in the form of a bacterial cell, a culture of the bacterial cell, and / or a processed product of the bacterial cell, the amount of the EPS produced by the Bifidobacterium longum subsp. longum MCC10345 is not particularly limited and is appropriately determined depending on the form of the composition. For example, when ingested, the amount of the bacterial cell is 1×10 4 ~1×10 13 cfu / g or 1×10 4 ~1×10 13 cfu / mL is preferred, and 1×10 5 ~1×10 12 cfu / g or 1×10 5 ~1×10 12 More preferably, it is 1×10 cfu / mL. 6 ~1×10 11 cfu / g or 1×10 6 ~1×10 11It is more preferable to use cfu / mL. In this specification, "cfu" stands for colony forming unit. When killed cells are used, , cfu / g or cfu / mL may be read as individual cells / g or individual cells / mL. When a culture supernatant is used as the bacterial culture, the content is preferably 0.1 to 100% by mass, more preferably 1 to 90% by mass, and even more preferably 10 to 80% by mass of the entire composition.

[0023] In addition, when the EPS produced by Bifidobacterium longum subsp. longum MCC10345 is contained in the form of a bacterial cell, a culture of the bacterial cell, and / or a processed product of the bacterial cell, the content of the EPS relative to the entire composition is preferably 0.001 mass % or more and less than 100 mass%, more preferably from 0.005 to 95 mass%, even more preferably from 0.01 to 85 mass%, even more preferably from 0.1 to 80 mass%, and still more preferably from 1 to 75 mass%. These contents may be within the range generally used when distributed as oral compositions.

[0024] The composition of the present invention has an immunomodulatory effect. Here, "immunomodulation" refers to achieving a balance so that the immune system functions appropriately, and includes "immunoactivation" and "immunosuppression." In this specification, "immunostimulation" refers to an action in the direction of enhancing immune response, specifically including an action of improving a state where immune response is reduced, and an action of enhancing immune response from a normal or good state. In addition, the action in the direction of enhancing immune response may include an action of protecting against viral infection and suppressing viral proliferation (collectively referred to as antiviral action) caused by immunostimulation, and an action of treating or preventing infectious diseases and an action of improving or alleviating symptoms of the disease. Here, "prevention" refers to the prevention or delay of the onset of a disease or symptom in the subject of application, or the reduction in the risk of a disease or symptom in the subject of application. In addition, examples of infectious diseases include diseases caused by influenza virus, norovirus, RS virus, etc., and COVID-19, etc. As used herein, the term "immunosuppression" includes an effect of suppressing inflammation (anti-inflammatory effect) so as to prevent an excessive immune response.

[0025] Furthermore, the site where the immunomodulatory effect of the composition of the present invention is exerted is not particularly limited, but is usually epithelial cells, such as epithelial cells of the upper respiratory tract and epithelial cells of the intestinal tract, with upper respiratory tract epithelial cells being more preferred. Therefore, the composition of the present invention is suitable for use in the prevention or treatment of viruses that can infect the upper respiratory tract and cause disease.

[0026] As shown in the examples below, EPS produced by Bifidobacterium longum subsp. longum MCC10345 suppresses the production of inflammatory cytokines (IL-8, IL-1β, etc.), promotes the production of antiviral cytokines (IFNβ, IFNλ2, etc.), and promotes the production of antiviral proteins (Mx2, etc.). Here, "promotion of production" refers to an increase in the amount or rate of production after application of the composition of the present invention compared to before application, or when the composition of the present invention is applied compared to when it is not applied. "Suppression of production" refers to a decrease in the amount or rate of production after application of the composition of the present invention compared to before application, or when the composition of the present invention is applied compared to when it is not applied. The promotion or suppression of production can be confirmed by, but is not limited to, measuring the blood concentrations of these cytokines or proteins or measuring the mRNA expression levels of these genes.

[0027] Activation of the innate immune system against viral infection is mediated by Toll-like receptors (TLR3, 7, 9, etc.) or RIG-like receptors (RIG-I, MDA-5, etc.) are detected by cells It works by inducing the gene expression of antiviral cytokines (IFNs) and inflammatory cytokines upon viral sensitization, which activates downstream signal transduction pathways. The virus-infected cells themselves are induced to enter an antiviral state by their own IFNs, while surrounding cells are induced to enter an antiviral state by the binding of secreted IFNs to the IFN receptor. Type I and type III IFNs activate the transcription factor ISG consisting of STAT1, STAT2, and IRF9 via the JAK-STAT pathway. They form an F3 complex and activate the expression of IFN-stimulated genes (ISGs). The actual antiviral effector molecules are the MxA protein, protein kinase R (PKR), and 2′5′-oligoadenylate synthetase (OAS), which belong to the ISGs. On the other hand, the induction of inflammatory cytokine expression via TLRs etc. due to viral infection promotes the local induction and activation of inflammatory immune cells in order to eliminate virus-infected cells, but their excessive activation leads to the spread of inflammation and is a factor in the decline of the body's defense function. In this regard, IFNβ preparations used to treat multiple sclerosis (MS) This can be understood from the fact that it has been reported that it enhances the production of anti-inflammatory cytokines and suppresses excessive T cell activation (Iran, J. Neurol., 2013;12:149-56.). Therefore, the composition of the present invention suppresses the production of inflammatory cytokines (IL-8, IL-1β, etc.), promotes the production of antiviral cytokines (IFNβ, IFNλ2, etc.), and promotes the production of antiviral proteins (Mx2, etc.), thereby providing the effect of suppressing excessive immune activation and inflammation while activating the immune system. In other words, the composition of the present invention exerts a biological defense function via the innate immune system by adjusting the immune activation and inhibition in an appropriate balance. The immunomodulatory effect of the composition of the present invention may be mediated through mechanisms other than the suppression of inflammatory cytokine production, the promotion of antiviral cytokine production, and the promotion of antiviral protein production.

[0028] The subject to which the composition of the present invention is administered (recipient) and the subject to which the composition is ingested (recipient) are not particularly limited as long as they are animals, but are usually humans, and preferably healthy humans (i.e., people who are not suffering from any disease or illness). They may also be adults, children, infants, newborns, etc. Furthermore, there are no particular limitations on the gender.

[0029] In this specification, "administering the bacterial cell or EPS to a subject" may be synonymous with "allowing the subject to ingest the bacterial cell or EPS." Ingestion is usually voluntary (free intake), but may be forced (forced intake). That is, the administration step may specifically be, for example, a step of blending Bifidobacterium longum subsp. longum MCC10345 and / or EPS produced by the bacteria into food, drink, or feed and supplying the same to the subject, thereby allowing the subject to ingest it freely.

[0030] The timing of intake (administration) of the composition of the present invention is not particularly limited, and can be appropriately selected depending on the condition of the subject to be taken (administered).

[0031] The intake (administration) amount of the composition of the present invention is appropriately selected depending on the age, sex, condition, other conditions, etc. of the subject to be ingested (administered). The intake (administration) amount of the composition of the present invention, as the intake (administration) amount of EPS produced by Bifidobacterium longum subsp. longum MCC10345 according to the present invention, is, for example, preferably 0.01 to 100 mg / day, more preferably 0.1 to 100 mg / day, and even more preferably 0.1 to 10 mg / day for adults. Alternatively, when the EPS produced by Bifidobacterium longum subsp. longum MCC10345 is contained in the form of a bacterial cell, a culture of the bacterial cell, and / or a processed product of the bacterial cell, the amount of the bacterial cell is 1×10 7 ~1×10 12 cfu / mL / day range is preferred, with 1×10 8 ~1×10 11 A range of 1×10 cfu / mL / day is more preferred. 9 ~1×10 11 cfu / mL / day is more preferred. Regardless of the amount or period of ingestion (administration), the composition of the present invention can be ingested (administered) once a day or in multiple divided doses.

[0032] The intake (administration) period of the composition of the present invention is not particularly limited. In addition, there is no particular upper limit to the intake (administration) period, and continuous, long-term intake (administration) is possible.

[0033] The composition of the present invention may be administered orally or parenterally, preferably orally. Parenteral administration includes transdermal, intravenous, rectal, and inhalation.

[0034] When the composition of the present invention is intended to be orally ingested (administered), it is preferably in the form of a food or drink. Another aspect of the present invention is the use of Bifidobacterium longum subsp. longum MCC10345 and / or EPS produced by said bacterium in the manufacture of an immunomodulatory composition. Another aspect of the present invention is the use of Bifidobacterium longum subsp. longum MCC10345 and / or EPS produced by said bacterium in immunomodulation. Another aspect of the present invention is a method for producing a bacterial strain of Bifidobacterium longum subsp. longum. A method for regulating immunity, comprising administering to a subject MCC10345 and / or EPS produced by the bacterium. The form and properties of the food and beverage products are not particularly limited as long as they do not impair the effects of the present invention and can be orally ingested (administered), and they can be produced by conventional methods using raw materials that are usually used for food and beverage products, except that they contain the EPS produced by Bifidobacterium longum subsp. longum MCC10345 itself, cells of the bacterium, a culture of the bacterium, and / or a processed product of the cells.

[0035] Food and drink products include, regardless of their form, liquid, paste, gel, solid, powder, etc., such as tablet confectionery; liquid food (nutritional food for tube feeding); wheat flour products such as bread, macaroni, spaghetti, noodles, cake mix, fried chicken powder, breadcrumbs, etc.; instant noodles, cup noodles, retort / prepared foods, pre-cooked canned foods, microwave foods, instant soups / stews, instant miso soup / cleansing soups, canned soups, freeze-dried foods, other instant foods, etc.; canned agricultural products, canned fruit, jams / mackerels, etc. - Agricultural processed products such as malts, pickles, boiled beans, dried agricultural goods, cereals (processed grain products); processed marine products such as canned seafood, fish ham and sausage, seafood paste products, seafood delicacies, tsukudani (fried fish paste), etc.; processed livestock products such as canned livestock paste, livestock ham and sausage, etc.; dairy products such as processed milk, milk drinks, yogurt, lactic acid bacteria drinks, cheese, ice cream, modified milk powder, cream, other dairy products, etc.; fats and oils such as butter, margarines, vegetable oils, etc.; soy sauce, miso, sauces, Basic seasonings such as processed tomato seasonings, mirin, vinegar, etc.; complex seasonings and foods such as cooking mixes, curry bases, sauces, dressings, noodle soups, spices, and other complex seasonings; frozen foods such as frozen ingredient foods, semi-cooked frozen foods, and cooked frozen foods; confectioneries such as caramel, candy, chewing gum, chocolate, cookies, biscuits, cakes, pies, snacks, crackers, Japanese sweets, rice snacks, bean snacks, dessert snacks, jellies, and other confectioneries; These include carbonated drinks, natural fruit juice, fruit juice drinks, soft drinks with fruit juice, fruit pulp drinks, fruit drinks with fruit particles, vegetable drinks, soy milk, soy milk drinks, coffee drinks, tea drinks, powdered drinks, concentrated drinks, sports drinks, nutritional drinks, alcoholic drinks, and other beverages, as well as other commercially available foods such as baby food, furikake, and ochazuke nori; infant formula; enteral nutritional food; health functional foods (foods for specified health uses, foods with nutritional functions, foods with functional claims), and nutritional supplements such as supplements.

[0036] When the food or drink is in the form of a supplement, it can be formulated into solid preparations such as powders, granules, tablets, capsules, etc., which may be enteric-coated, etc.; liquid preparations such as solutions, syrups, suspensions, emulsions, etc. When formulating such preparations, the ingredients, carriers, and methods for formulating pharmaceuticals described below can be followed.

[0037] In addition, one aspect of the food and drink may be feed, such as pet food, livestock feed, and fish feed. The form of the feed is not particularly limited, and may be EPS itself produced by Bifidobacterium longum subsp. longum MCC10345, the bacterial cells of the bacteria, a culture of the bacteria and / or a processed product of the bacterial cells, as well as, for example, grains such as corn, wheat, barley, rye, milo, etc.; vegetable oil cakes such as soybean oil cake, rapeseed oil cake, palm oil cake, linseed oil cake, etc.; bran such as wheat bran, rice bran, defatted rice bran, etc.; manufacturing residues such as corn gluten meal, corn jam meal, etc.; animal feeds such as fish meal, skim milk powder, whey, yellow grease, tallow, etc.; The feed may contain yeasts such as lure yeast and brewer's yeast; mineral feeds such as calcium phosphate tribasic and calcium carbonate; fats and oils; simple amino acids; sugars, etc.

[0038] When the composition of the present invention is in the form of a food or drink (including feed), it can be provided and sold as a food or drink labeled for its immunomodulatory use. Furthermore, the EPS itself produced by Bifidobacterium longum subsp. longum MCC10345 according to the present specification, the bacterial cells, a culture of the bacterial cells, and / or a processed product of the bacterial cells can be used for the production of such food or drink.

[0039] Such "indication" acts include all acts for informing consumers of the above-mentioned uses, and any expression that can recall or infer the above-mentioned uses falls under the category of "indication" acts in this invention, regardless of the purpose of the indication, the content of the indication, the object or medium on which it is displayed, etc. Furthermore, it is preferable that the "labeling" be done in an expression that allows consumers to directly recognize the above-mentioned uses.Specific examples include the act of transferring, delivering, displaying for the purpose of transferring or delivering, or importing food and beverage products or product packaging on which the above-mentioned uses are written, displaying or distributing advertisements, price lists, or transaction documents related to the products and writing the above-mentioned uses in information containing the above-mentioned uses and providing them by electromagnetic means (such as the Internet), etc.

[0040] On the other hand, the content of the labeling is preferably a labeling approved by the government etc. (for example, a labeling approved based on various systems established by the government and made in a manner based on such approval, etc.) In addition, it is preferable that such a labeling content is affixed to the packaging, containers, catalogs, pamphlets, POP and other sales site promotional materials, other documents, etc.

[0041] In addition, "labeling" also includes labeling as health food, functional food, enteral nutritional food, special purpose food, health functional food (specified health food, nutrient functional food, functional food), dietary supplement, medical drug, etc. Among these, in particular, labeling approved by the Consumer Affairs Agency, for example, labeling approved under the system related to specified health food, nutrient functional food, or functional food, or a similar system, can be mentioned. Specifically, labeling as specified health food, labeling as conditional specified health food, labeling that affects the structure or function of the body, labeling for reducing disease risk, labeling of functionality based on scientific evidence, etc. can be mentioned. More specifically, labeling as specified health food (especially labeling of health use) and similar labeling as specified in the Cabinet Office Ordinance on the Permission of Special Use Labeling Provided in the Health Promotion Act (Cabinet Office Ordinance No. 57 of August 31, 2009) are typical examples. Examples of such claims include "activates immune function," "helps regulate immune function in healthy individuals," "supports immune function," "supports regulation of immune function in healthy individuals," "combats viruses," "prevents viral infections," "increases resistance to infectious diseases," "increases the body's defense mechanisms," and the like.

[0042] The composition of the present invention may also be in the form of a pharmaceutical product. Another aspect of the present invention is the use of Bifidobacterium longum subsp. longum MCC10345 and / or EPS produced by said bacterium in the manufacture of an immunomodulatory composition. Another aspect of the present invention is Bifidobacterium longum subsp. longum MCC10345 and / or EPS produced by said bacterium for use in immunomodulation. Another aspect of the present invention is a method for producing a bacterial strain of Bifidobacterium longum subsp. longum. A method for regulating immunity, comprising administering to a subject MCC10345 and / or EPS produced by the bacterium.

[0043] The route of administration of pharmaceuticals may be either oral or parenteral, with oral being preferred. Parenteral intake (administration) includes transdermal, intravenous, rectal, inhalation, etc. The pharmaceutical form can be formulated into a desired dosage form depending on the administration method. For example, in the case of oral administration, it can be formulated into solid preparations such as powders, granules, tablets, and capsules; liquid preparations such as solutions, syrups, suspensions, and emulsions. In addition, in the case of parenteral administration, it can be formulated into suppositories, ointments, injections, and the like. In the formulation, in addition to the EPS produced by Bifidobacterium longum subsp. longum MCC10345 itself, the bacterial cells, the culture of the bacterial cells, and / or a processed product of the bacterial cells, components such as excipients, pH adjusters, colorants, and flavoring agents that are usually used in formulations can be used. It is also possible to use other medicinal ingredients, known or future components with immunomodulatory effects, and promoters / suppressors of immune system cytokines and immune system proteins in combination. In addition, formulation can be carried out by a known method according to the dosage form. When formulating, a carrier usually used in formulation may be appropriately mixed. Such carriers include excipients, binders, disintegrants, lubricants, stabilizers, flavoring agents, etc.

[0044] Examples of excipients include sugar derivatives such as lactose, sucrose, glucose, mannitol, and sorbitol; starch derivatives such as corn starch, potato starch, α-starch, dextrin, and carboxymethyl starch; cellulose derivatives such as crystalline cellulose, hydroxypropyl cellulose, hydroxypropylmethyl cellulose, carboxymethyl cellulose, and calcium carboxymethyl cellulose; gum arabic; dextran; pullulan; silicate derivatives such as light anhydrous silicic acid, synthetic aluminum silicate, and magnesium aluminometasilicate; phosphate derivatives such as calcium phosphate; carbonate derivatives such as calcium carbonate; and sulfate derivatives such as calcium sulfate.

[0045] Examples of binders include gelatin, polyvinylpyrrolidone, macrogol, and the like, in addition to the above-mentioned excipients.

[0046] Examples of disintegrants include the above-mentioned excipients, as well as chemically modified starch or cellulose derivatives such as croscarmellose sodium, sodium carboxymethyl starch, and crosslinked polyvinylpyrrolidone.

[0047] Examples of lubricants include talc; stearic acid; metal stearates such as calcium stearate and magnesium stearate; colloidal silica; waxes such as pea gum and geranium stearate; boric acid; glycol; carboxylic acids such as fumaric acid and adipic acid; sodium carboxylates such as sodium benzoate; sulfates such as sodium sulfate; leucine; lauryl sulfates such as sodium lauryl sulfate and magnesium lauryl sulfate; silicic acids such as silicic anhydride and silicic acid hydrate; starch derivatives, and the like.

[0048] Examples of the stabilizer include paraoxybenzoic acid esters such as methylparaben and propylparaben; alcohols such as chlorobutanol, benzyl alcohol, and phenylethyl alcohol; benzalkonium chloride; acetic anhydride; and sorbic acid.

[0049] Examples of flavoring agents include sweeteners, acidulants, and fragrances. In the case of a liquid preparation for oral administration, examples of the carrier used include solvents such as water.

[0050] The timing of taking the pharmaceutical agent of the present invention is not particularly limited, and may be before or after a meal, between meals, or before going to bed. EXAMPLES

[0051] The present invention will be described in more detail below using examples, but the present invention is not limited to these examples.

[0052] <Test Example 1> (1) Purpose Test Example 1 was carried out to clarify the mycological properties of the MCC10345 strain. (2) Acquisition of MCC10345 stock MCC10345 strain was isolated from a human fecal sample. MCC10345 strain was cultured anaerobically at 37°C for 48 hours on BL agar medium (Eiken Chemical Co., Ltd., E-MG16, 5% sterile defibrated horse blood added). The bacteriological properties are shown in Table 1.

[0053] [Table 1]

[0054] (3) Analysis of the 16S rRNA gene sequence The 16S rRNA gene base sequence of the MCC10345 strain was analyzed. A sample DNA solution for PCR was prepared from the MCC10345 strain cultured in (2), and the 16S rRNA gene was amplified by PCR using this sample DNA solution to obtain a DNA amplification product from which the 16S rRNA gene sequence of the MCC10345 strain was determined. Based on this base sequence information, a homology search was performed on the 16S ribosomal RNA (SrRNA) gene sequence precursors in the international sequence database (Genbank) of the National Center for Biotechnology Information (NCBI) using the Basic Local Alignment Search Tool (BLAST). The results showed 99% homology with Bifidobacterium longum subsp. longum JCM1217. Ta.

[0055] <Test Example 2> (1) Purpose Test Example 2 is a study on the EPS produced by Bifidobacterium longum subsp. longum MCC10345 (NITE BP-03751) using human lung epithelial cells. The purpose of this study was to confirm the immunomodulatory effect of . The relative expression levels of the IFNβ, Mx2, IL-8, and IL-1β genes were used as indicators of the immunomodulatory effect.

[0056] (2) Preparation of EPS samples Bifidobacterium longum subsp. longum MCC10345( NITE BP-03751) to Lactobacillus MRS Broth (DI The mixture was cultured at 37°C for 16 hours in a 30-well plate (manufactured by Fco). The culture solution was centrifuged at 10,000 rpm for 10 minutes, and the supernatant was collected. It was mixed with cold ethanol in an amount three times the amount of the supernatant and allowed to stand at 4°C for 24 hours. The precipitate was collected after standing, dissolved in distilled water for injection, centrifuged at 10,000 rpm for 10 minutes to remove unnecessary substances, and then freeze-dried to obtain an extract. The obtained extract was dissolved again in distilled water for injection at 15 mg / mL, and then added to DNas e (043-26773 Fujifilm Wako Co., Ltd., 7 μg / mL), RNase (7 40505 MACHEREY-NAGEL, 7 μg / mL), and Pronase E ( 107433 MERCK, 50μg / mL) and then further treated at 10,000 rpm, Unnecessary substances were removed by centrifugation for 10 minutes. The obtained supernatant was dialyzed for 72 hours using a dialysis membrane that removes molecules with molecular weights of 100,000 or less, and the obtained solution was freeze-dried to prepare an EPS sample.

[0057] (3) Cell testing Human lung epithelial cells A549 (ECACC 86012804) were cultured in F12K medium (10% F S) to 1 × 10 5 1 well per well in a 24-well plate and incubated at 37°C, 5% CO2 The cells were grown to 70% confluence under the conditions described above. The EPS samples prepared in (1) were added to the medium at final concentrations of 10, 30, and 100 μg / mL, and incubated at 37°C under 5% CO2 conditions for 4 hours. LyoVec (InvivoGen) was then added at a final concentration of 200 ng / mL, and incubated at 37°C for 18 hours. LyoVec is a pseudovirus-like stimulant, and its addition stimulates the cells. After incubation, the cells were incubated with RNeasy plus universal tissue kit (Qiagen) for 18 hours. RNA was extracted using TaKaRaPrime Quantitative PCR was performed using the Script RT Reagent kit, ExTaq II, and a target gene-specific primer, and the relative expression levels of each gene, IFNβ, Mx2, IL-8, and IL-1β, were measured using GAPDH as a standard.

[0058] (4) Results The results are shown in Figures 1 to 4. "nt" in Figures 1 to 4 indicates cases where neither cell stimulation with LyoVec nor addition of EPS sample was performed. The relative expression level of the antiviral cytokine IFNβ gene increased depending on the concentration of the EPS sample added, and a significant increase was observed in the 100 μg / mL added group compared to the non-addition group. In parallel with this, the relative expression level of the antiviral protein Mx2 gene, whose production is induced by IFNβ, increased, and a significant increase was observed in the 100 μg / mL added group compared to the non-addition group. On the other hand, the relative expression levels of the inflammatory cytokines IL-8 and IL-1β decreased depending on the concentration of the EPS sample added, and a significant decrease was observed in the 30 and 100 μg / mL added groups for IL-8, and in the 100 μg / mL added group for IL-1β, compared to the non-addition group. The concentration-dependent suppression of inflammatory cytokine gene expression by the EPS sample is thought to reflect the anti-inflammatory effect of IFNβ and the anti-inflammatory response of the EPS sample, and it is speculated that this may suppress the excessive immune response associated with viral infection.

[0059] <Test Example 3> (1) Purpose Test Example 3 is a study to determine whether EP produced by Bifidobacterium longum subsp. longum MCC10345 (NITE BP-03751) is effective in preventing and treating chronic obstructive pulmonary disease using human intestinal epithelial cells. The purpose of this study was to confirm the immunomodulatory effect of S. The relative expression levels of the IFNβ and IFNλ2 genes were used as indicators of the immunomodulatory effect.

[0060] (2) Preparation of EPS samples Using the same procedure as in Test Example 2, an EPS sample produced by the MCC10345 strain was prepared.

[0061] (3) Cell testing The same procedure as in Test Example 2 was carried out using human intestinal epithelial cells HT29 (EC91072201-G0), except that the relative expression levels of IFNβ and IFNλ2 were measured as indicators of immunomodulatory activity. EPS samples were added to the medium to give final concentrations of 1, 10, and 100 μg / mL.

[0062] (4) Results The results are shown in Figures 5 and 6. "Control" in Figures 5 and 6 refers to an example in which cells were stimulated with LyoVec but no EPS sample was added. As shown in Figure 5, when the EPS sample was added at 1 μg / mL, there was no change in the relative expression level of the antiviral cytokine IFNβ gene compared to the control. When the EPS sample was added at 10 μg / mL or 100 μg / mL, the relative expression level of the IFNβ gene increased significantly compared to the control, and the change was particularly remarkable at 100 μg / mL. As shown in Figure 6, there was no change in the relative expression level of the antiviral cytokine IFNλ2 gene when the EPS sample was added at 1 or 10 μg / mL compared to the control. When 100 μg / mL was added, the relative expression level of the IFNβ gene was significantly increased compared to the control.

[0063] <Test Example 4> (1) Purpose Test Example 4 is a comparison of the lactic acid bacteria produced by MCC10345 (NITE BP-03751) with other strains belonging to Bifidobacterium longum subsp. longum. The relative expression levels of the IFNβ gene in EPS were compared.

[0064] (2) Preparation of EPS samples An EPS sample was prepared by the same procedure as in Test Example 2. Furthermore, EPS samples were prepared by the same procedure for each of Bifidobacterium longum subsp. longum JCM1217T, JCM31944, and ATCC51870.

[0065] (3) Cell testing The same procedure as in Test Example 2 was carried out using human intestinal epithelial cells HT29 (EC91072201-G0), except that the relative expression level of IFNβ was measured as an index of immunomodulatory activity. EPS samples were added to the medium to give final concentrations of 10 and 100 μg / mL.

[0066] (4) Results The results are shown in Figure 7. "Control" in Figure 7 refers to an example in which cells were stimulated with LyoVec but no EPS sample was added. The relative expression level of IFNβ in the MCC10345 strain was significantly increased compared to the control, regardless of whether 10 μg / mL or 100 μg / mL of EPS sample was added. This increase was more significant than that in JCM1217T, JCM31944, and ATCC51870.

[0067] <Test Example 5> (1) Purpose Test Example 5 was carried out to search for components contained in the EPS sample that contribute to the relative expression level of the IFN-β gene.

[0068] (2) Preparation of EPS samples The following samples were obtained by changing only the procedure of enzyme treatment of the culture supernatant extract of the EPS-producing bacteria in the procedure of Test Example 2. α-amylase+EPS: A sample in which the culture supernatant extract of EPS-producing bacteria was treated with α-amylase (A3176, SIGMA-ALDRICH). The treatment conditions were a final enzyme concentration of 0.25 mg / mL, 37°C, and 18 hours. This sample contains decomposed polysaccharides contained in the culture supernatant extract of EPS-producing bacteria. DNase+EPS: A sample in which the culture supernatant extract of EPS-producing bacteria was treated with DNase (043-26773 Fujifilm Wako Co., Ltd.). The treatment conditions were a final enzyme concentration of 7 μg / mL. The DNA contained in the culture supernatant extract of the EPS-producing bacteria was decomposed. This is a sample. Lipase+EPS: A sample in which the culture supernatant extract of EPS-producing bacteria was treated with Lipase (534781, SIGMA-ALDRICH). Treatment conditions were 0.1 mg / mL, 37°C, 18 hours. This is a sample in which lipids contained in the culture supernatant extract of EPS-producing bacteria have been decomposed. Proteinase K+EPS: The culture supernatant extract of EPS-producing bacteria was The sample was treated with ase K (29442-14, Nakarai Task). The treatment conditions were: The results were as follows: 50 μg / mL, 37°C, 18 hours. This is a sample in which protein has been decomposed. In addition, an EPS sample obtained by the same procedure as in Test Example 2 was used as a reference sample.

[0069] (3) Cell testing The same procedure as in Test Example 2 was carried out using human intestinal epithelial cells HT29 (EC91072201-G0), except that the relative expression level of IFNβ was measured as an index of immunomodulatory activity. The EPS sample was added to the cells to a final concentration of 100 μg / mL. In addition, α-amylase, DNase, Lipase, and Proteinase K were added in amounts equivalent to the amounts added during each enzyme treatment. Each amount was added to the cells alone.

[0070] (4) Results The results are shown in Figure 8. "Control" in Figure 8 is an example where cells were stimulated with LyoVec but no EPS sample or enzyme was added. As shown in Figure 8, the relative expression level of INFβ in α-amylase+EPS, i.e., the sample in which polysaccharides contained in the culture supernatant extract of EPS-producing bacteria were degraded, was significantly lower than that in the EPS sample. On the other hand, in the EPS sample treated with DNase, Lipase, or Proteinase, i.e., the sample in which DNA, lipids, or proteins contained in the culture supernatant extract of EPS-producing bacteria were degraded, the relative expression level of INFβ was comparable to that of the EPS sample in Test Example 2, or no significant decrease was observed. These results suggested that among the components contained in the culture supernatant extract of EPS-producing bacteria, the component that contributes to the increase in the relative expression level of INFβ is polysaccharides.

[0071] <Test Example 6> (1) Purpose Test Example 6 was carried out to examine the relationship between the heating temperature and the relative expression level of IFNλ2 in heated cells of the MCC10345 strain.

[0072] (2) Sample preparation Bifidobacterium longum subsp. longum MCC10345 (NITE BP-03751) was cultured in Lactobacillus MRS Broth (DI The culture was performed in a broth (manufactured by Fco) at 37° C. for 16 hours. The same MRS broth as in Test Example 2 was used. The cells were collected by centrifugation at 10,000 rpm for 10 minutes, and heated at room temperature (25°C), 70, 95, and 120°C to obtain heated cells.

[0073] (3) Cell testing The same procedure as in Test Example 2 was carried out using human intestinal epithelial cells HT29 (EC91072201-G0), adding heated bacterial cells instead of the EPS sample, and measuring the relative expression level of IFNλ2 as an index of immunomodulatory activity. The amount of bacterial cells added was 1x10 7 The CFU / well was adjusted.

[0074] (4) Results The results are shown in Figure 9. As the heat treatment temperature increased, the relative expression level of IFNλ2 increased, reaching a maximum when the heat treatment temperature was 95°C.

Claims

1. Bifidobacterium longum subsp. longum MCC10345 (NITE BP-03751).

2. Exopolysaccharide (EPS) produced by Bifidobacterium longum subsp. longum MCC10345 (NITE BP-03751).

3. NITE BP-03751 Bifidobacterium longum subsp. Gum MCC10345 (NITE BP-03751) and / or the bacteria produced An immunomodulatory composition comprising EPS.

4. The composition according to claim 3, for use in immunomodulation in epithelial cells of the upper respiratory tract.

5. The composition according to claim 3 or 4, wherein the immune modulation is caused by suppressing the production of inflammatory cytokines, promoting the production of antiviral cytokines, and / or promoting the production of antiviral proteins.

6. The composition according to claim 5, wherein the inflammatory cytokine is IL-8 and / or IL-1β.

7. The composition of claim 5 , wherein the antiviral cytokine is IFNβ and / or IFNλ2.

8. The composition of claim 5 , wherein the antiviral protein is Mx2.

9. The composition according to claim 3 or 4, wherein the EPS is contained in one or more selected from the group consisting of a bacterial cell of Bifidobacterium longum subsp. longum MCC10345, a culture of the bacterial cell, and a treated product of the bacterial cell.

10. The composition according to claim 3 or 4, which is a food or drink.

11. The composition of claim 10 for use in maintaining, improving or enhancing immune function.

12. The composition according to claim 3 or 4, which is a pharmaceutical product.

13. The composition of claim 12 for use in treating or preventing a viral infectious disease.

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