Composition for promoting interferon-λ production and method for producing the same

Abstract

To provide a composition containing lactic acid bacteria, showing high interferon-λ production promoting effect on cells other than dendritic cells.SOLUTION: A composition containing cultured or processed Enterococcus bacteria as an active ingredient is administered to a subject, promoting the production of interferon-λ in the subject.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to a composition containing lactic acid bacteria as an active ingredient and promoting the production of interferon-λ, and a method for producing the same. [Background technology]

[0002] The immune system is a self-defense system that constantly monitors and repels cancer cells that develop inside the body, as well as bacteria and viruses that invade from outside. Research into compositions and foods that boost immunity has been ongoing for a long time.

[0003] Interferon-λ (IFN-λ) is a physiologically active substance (cytokine) belonging to the type III interferon family. In humans, the IFN-λ family is composed of four proteins: IFN-λ1 (IL-29), IFN-λ2 (IL-28A), IFN-λ3 (IL-28B), and IFN-λ4. Its production has been confirmed by dendritic cells, hepatocytes, intestinal epithelial cells, lung epithelial cells, and other cells. IFN-λ has the ability to activate innate immunity, including antiviral activity, and can exhibit antiviral activity, immunostimulatory activity, inhibitory activity against viral infections (e.g., hepatitis B virus, hepatitis C virus), antitumor growth activity, antitumor activity, and anticancer activity. Therefore, it is expected to be used in foods and beverages, therapeutic agents, preventive agents, ameliorative agents, and palliatives that possess these activities (Non-Patent Document 1).

[0004] It is expected that the physiological activity of IFN-λ can be induced in vivo not only by directly administering IFN-λ into the body, but also by increasing the expression or production level of IFN-λ in the body, i.e., by administering a substance that enhances IFN-λ production into the body. Patent Document 1 reports that Lactococcus lactis JCM20101 and JCM5805 strains enhance IFN-λ production in plasmacytoid dendritic cells. Patent Document 2 reports that Tetragenococcus halophilus KK221 strain enhances IFN-λ production in BDCA3DC dendritic cells. Patent Document 3 reports that in intestinal epithelial cells (HT-29 cells) mock-infected with poly:IC, a synthetic double-stranded RNA, Bifidobacterium breve MCC1274 strain, M-16V strain, Bifidobacterium longum subsp. longum BB536 strain, and Bifidobacterium longum subsp. infantis M-63 strain enhance IFN-λ production. [Prior art documents] [Patent documents]

[0005] [Patent Document 1] International Publication No. 2012 / 091081 [Patent Document 2] International Publication No. 2017 / 175774 [Patent Document 3] Patent Publication No. 2019-167327 [Non-patent literature]

[0006] [Non-Patent Document 1] Lazear HM et al., Shared and distinct functions of type I and type III interferons. Immunity 50 (4): 907-923 (2019) Summary of the Invention [Problem to be solved by the invention]

[0007] The lactic acid bacteria described in Patent Documents 1 and 2 have the effect of further enhancing the IFN-producing ability of dendritic cells, which already have a high IFN-producing ability. On the other hand, the lactic acid bacteria described in Patent Document 3 enhance the IFN-λ producing ability of intestinal epithelial cells, but this effect was confirmed in cells whose immune activity had been previously enhanced. Therefore, there is a need for lactic acid bacteria that significantly promote IFN-λ production in cells other than dendritic cells, without requiring any other immune activation treatment.

[0008] An object of the present invention is to provide a composition containing lactic acid bacteria that exhibits a high effect of promoting IFN-λ production in cells other than dendritic cells, and a method for producing a composition containing lactic acid bacteria that exhibits a high effect of promoting IFN-λ production in cells other than dendritic cells. [Means for solving the problem]

[0009] In order to achieve the above-mentioned object, the present inventors conducted extensive research and found that Enterococcus bacteria increase the amount of IFN-λ produced by colonic epithelial cells without the need for other immunostimulatory treatments, thereby completing the present invention.

[0010] The present invention encompasses the following. (1) A composition for enhancing interferon-λ production in a subject, comprising a culture or a processed product of Enterococcus bacteria as an active ingredient. (2) The composition described in (1), wherein the Enterococcus bacterium is Enterococcus cathelliflavus KB1733 strain (accession number NITE AP-03535). (3) The composition according to (1) or (2), wherein the culture or treated product of the Enterococcus bacterium is sterilized. (4) The composition according to any one of (1) to (3), which increases the expression level of a target antiviral protein gene. (5) The composition described in (4), wherein the antiviral protein gene is at least one gene selected from the group consisting of the Mx dynamin-like GTPase 1 (Mx1) gene, the 2'-5'-oligoadenylate synthetase 1 (OAS1) gene, and the interferon-stimulated gene 15 (ISG15) gene. (6) A pharmaceutical composition comprising the composition according to any one of (1) to (5). (7) A food composition comprising the composition according to any one of (1) to (5). (8) A composition for external use, comprising the composition according to any one of (1) to (5). (9) A method for producing a composition for enhancing interferon-λ production in a subject, the method comprising the steps of culturing Enterococcus bacteria and obtaining a culture or treated product of the Enterococcus bacteria. (10) The method according to (9), wherein the Enterococcus bacterium is Enterococcus casselliflavus KB1733 strain (accession number NITE AP-03535). [Effects of the Invention]

[0011] The present invention provides a composition comprising lactic acid bacteria that exhibits a high IFN-λ production-promoting effect in cells other than dendritic cells, and a method for producing a composition comprising lactic acid bacteria that exhibits a high IFN-λ production-promoting effect in cells other than dendritic cells. [Brief explanation of the drawings]

[0012] [Figure 1] This graph shows the relationship between the amount of Enterococcus cathelliflavus KB1733 culture added and the amount of IFN-λ1 produced by WiDr cells. The error bars in the graph indicate standard error. The "*" in the graph indicates that a significant difference was found compared to the control (p<0.05) by Steel's test (n=6). [Figure 2]This graph shows the relationship between the amount of Enterococcus cathelliflavus KB1733 culture added and the amount of IFN-λ3 produced by WiDr cells. The error bars in the graph indicate standard error. An asterisk (*) in the graph indicates a significant difference (p<0.05) from the control by Steel's test (n=6). [Figure 3] This graph shows the relationship between the amount of Mx1 gene expression in WiDr cells and the treatment time with a culture of Enterococcus cathelliflavus KB1733 strain. The error bars in the graph indicate standard error. The "*" in the graph indicates a significant difference (p<0.05) from the value at time 0 in the Steel test (n=6). [Figure 4] This graph shows the relationship between the amount of OAS1 gene expression in WiDr cells and the treatment time with a culture of Enterococcus cathelliflavus KB1733 strain. The error bars in the graph indicate standard error. The "*" in the graph indicates a significant difference (p<0.05) from the 0-hour value in the Steel test (n=6). [Figure 5] This graph shows the relationship between the amount of ISG15 gene expression in WiDr cells and the time of treatment with a culture of Enterococcus cathelliflavus KB1733 strain. The error bars in the graph indicate standard error. An asterisk (*) in the graph indicates a significant difference (p<0.05) from the value at time 0 in the Steel test (n=6). DETAILED DESCRIPTION OF THE INVENTION

[0013] 1. Composition The composition of the present invention is a composition for enhancing interferon-λ (IFN-λ) production in a subject, which comprises a culture or processed product of Enterococcus as an active ingredient. The composition of the present invention is characterized in that it can promote IFN-λ production in a subject by administering it to the subject by means such as oral ingestion.

[0014] The present inventors have found that Enterococcus, a type of lactic acid bacteria, promotes IFN-λ production in WiDr cells, a colon cancer cell line. WiDr cells are derived from the colon epithelium and are not naturally high IFN-producing cells like dendritic cells, but are closer to normal somatic cells. Colon epithelial cells often come into contact with pharmaceutical and food ingredients when orally administered, and promotion of IFN-λ production in these cells is thought to significantly contribute to improving the body's immune function.

[0015] The present inventors further found that Enterococcus bacteria promote the expression of various IFN-inducible antiviral protein genes in WiDr cells, confirming that Enterococcus bacteria not only promote IFN-λ production in colonic epithelial cells but also actually induce immune defense functions in the cells.

[0016] As used herein, the term "subject" refers to a human or non-human animal, or a cell derived from a human or non-human animal. Non-human animals are not particularly limited, and examples thereof include mammals, birds, reptiles, amphibians, and fish, and preferably include chickens, cows, horses, pigs, dogs, and cats. The composition of the present invention is characterized in that, when administered to a subject, it can increase the amount of IFN-λ produced by the subject.

[0017] The IFN-λ whose production amount is increased by the composition of the present invention may be any of IFN-λ1, IFN-λ2, IFN-λ3 and IFN-λ4, but IFN-λ1 and / or IFN-λ3 are particularly preferred.

[0018] As used herein, "IFN-λ1" refers to a protein having an amino acid sequence that shares 60% or more, 65% or more, 70% or more, 80% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity with the amino acid sequence of SEQ ID NO: 1. The amino acid sequence of SEQ ID NO: 1 is the sequence of Accession No. AAN86125.

[0019] As used herein, "IFN-λ2" refers to a protein having an amino acid sequence that shares 60% or more, 65% or more, 70% or more, 80% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity with the amino acid sequence of SEQ ID NO: 2. The amino acid sequence of SEQ ID NO: 2 is the sequence of Accession No. AAN86126.

[0020] As used herein, "IFN-λ3" refers to a protein having an amino acid sequence that shares 60% or more, 65% or more, 70% or more, 80% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity with the amino acid sequence of SEQ ID NO: 3. The amino acid sequence of SEQ ID NO: 3 is the sequence of Accession No. AAN86127.

[0021] As used herein, "IFN-λ4" refers to a protein having an amino acid sequence that shares 60% or more, 65% or more, 70% or more, 80% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity with the amino acid sequence of SEQ ID NO: 4. The amino acid sequence of SEQ ID NO: 4 is the sequence of Accession No. NP_001263183.

[0022] As used herein, "sequence identity" refers to the percentage of identical amino acids or bases in the entire overlapping amino acid sequence (including the amino acid that serves as the translation initiation point) or base sequence (including the initiation codon) in optimal alignment of two amino acid or base sequences, with or without gaps introduced, and is calculated using formula (1). Sequence identity can be easily determined using BLAST (Basic Local Alignment Search Tool), an algorithm commonly used in this field. For example, BLAST is available to anyone from websites such as NCBI (National Center for Biotechnology Information) and KEGG (Kyoto Encyclopedia of Genes and Genomes), and sequence identity can be easily determined using default parameters. Sequence identity (%) = number of matches (ignoring gaps) / length of shorter sequence (length excluding gaps) × 100... Equation (1)

[0023] The composition of the present invention comprises a culture or a processed product of Enterococcus bacteria. As used herein, a "culture" of bacteria refers to live cells, killed cells, disrupted live or killed cells, freeze-dried live or killed cells, disrupted freeze-dried products, culture fluid, culture fluid extract, etc. The culture may contain a portion of the cells or a processed product of the cells, and may also contain DNA, RNA, etc. extracted from the cells. A "processed product" of bacteria refers to cells that have been subjected to, for example, enzyme treatment, heat treatment, extraction treatment, salting-out treatment, ethanol precipitation treatment, drying treatment, etc.

[0024] As used herein, "Enterococcus" refers to lactic acid bacteria belonging to the phylum Firmicutes, also known as Enterococcus. These are Gram-positive cocci that primarily exist in the intestinal tract of animals as diplococci or short streptococci.

[0025] The Enterococcus bacterium contained in the composition of the present invention is preferably Enterococcus casseliflavus. In particular, the Enterococcus casseliflavus KB1733 strain is preferred. The Enterococcus casseliflavus KB1733 strain was received for international deposit under the Budapest Treaty at the Patent Microorganisms Depositary of the National Institute of Technology and Evaluation (Room 122, 2-5-8 Kazusa Kamatari, Kisarazu City, Chiba Prefecture) on September 10, 2021, under the accession number NITE AP-03535. The Enterococcus casseliflavus KB1733 strain was isolated from pickles, a traditional fermented food, and has been used as a food. It can be said to be a highly safe bacterium, especially when taken orally.

[0026] In the composition of the present invention, the culture or processed product of Enterococcus bacteria may contain viable bacteria, but it is preferably sterilized, i.e., the bacteria are contained in the composition in a state in which they cannot grow or multiply. It has been confirmed that the culture or processed product of the active ingredient, Enterococcus casselliflavus KB1733 strain, in the composition of the present invention, is effective even without containing viable bacteria. The sterilized composition of the present invention has the advantage that, compared to a composition containing viable bacteria, it is easier to manage the temperature, time, etc. during transportation and storage when distributing it on the market as a pharmaceutical, food, etc.

[0027] Interferons (IFNs) are classified into three types: type I, type II, and type III, and each type induces a specific immune response. IFN-mediated signaling promotes the upregulation of MHC class I and II molecules, activating numerous downstream signaling cascades and resulting in the production of antiviral mechanisms, particularly various antiviral proteins. More than 120 IFN-inducible antiviral proteins are known, including Mx dynamin-like GTPase 1 (Mx1), 2'-5'-oligoadenylate synthetase 1 (OAS1), interferon-stimulated gene 15 (ISG15), viperin, protein kinase R (PKR), and interferon regulatory factor 7 (IRF7).

[0028] As used herein, "Mx dynamin-like GTPase 1 (Mx1) gene" refers to a gene having a nucleotide sequence that shares 60% or more, 65% or more, 70% or more, 80% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity with the nucleotide sequence set forth in SEQ ID NO: 5. Mx1 is known as a protein that exhibits intracellular antiviral activity against a wide range of viruses, including influenza, parainfluenza, measles, coxsackievirus, and hepatitis B. The nucleotide sequence set forth in SEQ ID NO: 5 is the sequence of Accession No. NG_027788.

[0029] As used herein, the term "2'-5'-oligoadenylate synthetase 1 (OAS1) gene" refers to a gene having a nucleotide sequence that shares 60% or more, 65% or more, 70% or more, 80% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity with the nucleotide sequence set forth in SEQ ID NO: 6. OAS1 binds to and activates inactive RNase L, degrading mRNA and rRNA and suppressing the growth of viruses and bacteria. The nucleotide sequence set forth in SEQ ID NO: 6 is the sequence of Accession No. NG_011530.

[0030] As used herein, the term "interferon-stimulated gene 15 (ISG15) gene" refers to a gene having a nucleotide sequence that shares 60% or more, 65% or more, 70% or more, 80% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity with the nucleotide sequence set forth in SEQ ID NO: 7. The nucleotide sequence of SEQ ID NO: 7 is the sequence of Accession No. NG_033033.

[0031] The composition of the present invention preferably has the effect of increasing the expression level of an antiviral protein gene in a subject. The antiviral protein gene is not particularly limited as long as it is a gene for a protein derived from a subject and has antiviral activity, but is preferably at least one gene selected from the group consisting of the Mx1 gene, the OAS1 gene, and the ISG15 gene, which are particularly useful indicators of IFN activity.

[0032] The form of the composition of the present invention is not particularly limited, and may be any form such as an aqueous solution, dry powder, freeze-dried product, or frozen product, and the form can be appropriately selected depending on the use of the composition described below.

[0033] The compositions of the present invention are compositions for administration to a subject. The administration form is not particularly limited as long as it allows the active ingredient to be taken up by the subject, and may be oral or parenteral administration, although the administration form may be appropriately selected depending on the intended use of the composition, as described below. The compositions of the present invention are highly safe even when administered to a subject by oral ingestion, etc. Regardless of the administration form, they have the effect of promoting IFN-λ production in a subject, and, in relation to IFN, may have antiviral, immunostimulatory, anti-infectious, anti-hepatitis B, anti-hepatitis C, anti-tumor growth, anti-tumor, and anti-cancer effects in a subject.

[0034] 2. Use of the composition The use of the composition of the present invention is not particularly limited, and it may be incorporated, for example, into pharmaceuticals, quasi-drugs, cosmetics, food and beverages, feed, etc. Here, the pharmaceuticals, quasi-drugs, and cosmetics refer to those defined in the "Act on Ensuring Quality, Efficacy, and Safety of Pharmaceuticals, Medical Devices, etc. (PMD Act)." Hereinafter, the compositions of the present invention will be described separately as "pharmaceutical compositions," "edible compositions," and "external compositions" according to their uses, but these are not completely separate and may overlap with each other.

[0035] 2-1 Pharmaceutical Composition The pharmaceutical composition of the present invention is characterized by containing the composition of the present invention described in the section "1. Composition." As used herein, the term "pharmaceutical composition" refers to a composition for treating and / or preventing a disease in humans or non-human animals, and primarily includes pharmaceuticals and some quasi-drugs as defined by the Pharmaceutical and Medical Device Act.

[0036] The pharmaceutical composition of the present invention contains a culture or processed product of Enterococcus bacteria as an active ingredient. In addition to the active ingredient, the pharmaceutical composition of the present invention may contain a pharmaceutically acceptable carrier, if necessary. The term "pharmaceutically acceptable carrier" as used herein refers to additives commonly used in the pharmaceutical formulation field. Examples include excipients, binders, disintegrants, fillers, emulsifiers, flow additives, lubricants, etc.

[0037] Examples of excipients include sugars such as monosaccharides, disaccharides, cyclodextrins, and polysaccharides (more specifically, but not limited to, glucose, sucrose, lactose, raffinose, mannitol, sorbitol, inositol, dextrin, maltodextrin, starch, and cellulose), metal salts (e.g., sodium chloride, sodium or calcium phosphate, calcium sulfate, magnesium sulfate, calcium carbonate), citric acid, tartaric acid, glycine, low-, medium-, and high-molecular-weight polyethylene glycols (PEGs), pluronics, kaolin, silicic acid, or combinations thereof.

[0038] Examples of binders include starch paste using corn, wheat, rice, or potato starch, simple syrup, glucose solution, gelatin, tragacanth, methylcellulose, hydroxypropylmethylcellulose, sodium carboxymethylcellulose, shellac, and / or polyvinylpyrrolidone.

[0039] Examples of disintegrants include the above-mentioned starches, lactose, carboxymethyl starch, cross-linked polyvinylpyrrolidone, agar, laminaran powder, sodium hydrogen carbonate, calcium carbonate, alginic acid or sodium alginate, polyoxyethylene sorbitan fatty acid esters, sodium lauryl sulfate, stearic acid monoglyceride, or salts thereof.

[0040] Examples of fillers include the above-mentioned sugars and / or calcium phosphate (for example, tricalcium phosphate or calcium hydrogen phosphate).

[0041] Examples of emulsifiers include sorbitan fatty acid esters, glycerin fatty acid esters, sucrose fatty acid esters, and propylene glycol fatty acid esters.

[0042] Examples of flow regulators and lubricants include silicates, talc, stearates or polyethylene glycol.

[0043] Such carriers are primarily used to facilitate the formation of the dosage form and to maintain the dosage form and pharmacological effect, and may be used appropriately as needed. In addition to the above-mentioned additives, flavoring agents, solubilizing agents, suspending agents, diluents, surfactants, stabilizers, absorption enhancers, bulking agents, wetting agents, humectants, adsorbents, disintegration inhibitors, coating agents, coloring agents, preservatives, antioxidants, perfumes, flavoring agents, sweeteners, buffers, etc. may also be included as needed.

[0044] The pharmaceutical composition of the present invention may also contain other drugs, for example, other antibiotics in a predetermined amount, as long as the effect of promoting IFN-λ production is not lost.

[0045] The dosage form of the pharmaceutical composition of the present invention is not particularly limited as long as it does not inactivate the culture or processed product of Enterococcus bacteria, which is the active ingredient, and other additional active ingredients. For example, it may be any liquid, solid, or semisolid. Specific dosage forms include, for oral administration, tablets such as sugar-coated tablets, buccal tablets, coated tablets, and chewable tablets, lozenges, pills, powders, capsules including soft capsules, granules, suspensions, emulsions, syrups including dry syrups, and liquids such as elixirs. For parenteral administration, injectables such as intravenous injection, subcutaneous injection, intraperitoneal injection, and intramuscular injection, transdermal absorption tapes, aerosols, and suppositories for transdermal, nasal, pulmonary, enteral, buccal, and transmucosal administration are preferred from the viewpoints of convenience and versatility.

[0046] The pharmaceutical composition of the present invention preferably contains a culture or processed product of Enterococcus bacteria in an amount that is effective in promoting IFN production and is highly unlikely to cause serious side effects. The dose of the culture or processed product of Enterococcus bacteria to be administered is not particularly limited, but may be, for example, 1 x 10 converted into the number of bacterial cells. 8 ~1×10 12 Cells / individual / day, specifically, 1 x 10 9 ~1×10 11 The number of cells per individual per day can be adjusted.

[0047] The number of times the pharmaceutical composition of the present invention is administered is not particularly limited as long as the IFN production-promoting effect is sufficient and no serious side effects occur, but is preferably administered, for example, once every three days to five times a day, and particularly once to three times a day. The administration period of the pharmaceutical composition of this embodiment is not particularly limited, but can be, for example, 1 to 24 weeks, and particularly 8 to 12 weeks.

[0048] 2-2 Edible composition The edible composition of the present invention is characterized by containing the composition of the present invention described in the section "1. Composition." As used herein, "edible composition" refers to a composition intended for oral intake as nutrition by humans or non-human animals. An edible composition suitable for human consumption is also referred to as a food or drink, and an edible composition suitable for non-human animal consumption is also referred to as a feed.

[0049] The form of the edible composition (food, drink, feed) of the present invention is not particularly limited, and examples include processed foods, health foods (nutritional supplements, nutritionally functional foods, foods for the sick, foods for specified health uses, foods with functional claims, etc.), supplements, foods for the sick (hospital food, food for sick people, nursing care food, etc.), confectionery, oils and fats, dairy products, retort foods, microwave foods, frozen foods, seasonings, health supplements, beverages, and nutritional drinks.

[0050] The shape and properties of the edible composition (food, drink, feed) of the present invention are not particularly limited, and examples thereof include solid, semi-solid, gel, liquid, powder, etc. In particular, when used as a health food or supplement, it is preferable to make it in the form of granules, capsules, tablets, chewable preparations, drink powders, energy drinks, smoothies, jellies, gummies, candies, gums, etc., to enable continuous and convenient intake.

[0051] The amount of culture or processed product of Enterococcus bacteria contained in the edible composition of the present invention is not particularly limited as long as it is an amount that is unlikely to cause health damage. For example, 8 ~1×10 12 Cells / individual / day, specifically, 1 x 10 9 ~1×10 11 The number of cells per individual per day can be adjusted.

[0052] 2-3 External composition The topical composition of the present invention is characterized by containing the composition of the present invention described in "1. Composition." As used herein, "topical composition" refers to a composition used to treat and / or cure diseases of the body surface, such as the skin, hair, or nails of humans or non-human animals, or to improve and / or maintain the health or beauty of the body surface, and is intended to be applied, rubbed, sprayed, or contacted with the body surface by a similar method. This primarily includes some drugs and quasi-drugs under the Pharmaceutical and Medical Device Act, as well as cosmetics.

[0053] The form of the topical composition of the present invention is not particularly limited as long as it can be applied to the body surface, and may be any form such as a transdermal absorption tape, aerosol, patch, lotion, gel, cream, spray, or pack.

[0054] The topical composition of the present invention may contain other components as needed in addition to a culture or processed product of Enterococcus bacteria. Examples of other components include excipients, binders, disintegrants, fillers, emulsifiers, flow regulators, lubricants, etc. Details of each component are as described in the section "2-1 Pharmaceutical Compositions."

[0055] The amount of culture or processed product of Enterococcus bacteria contained in the topical composition of the present invention is not particularly limited as long as it is an amount that is unlikely to cause health hazards. For example, the amount may be 1 x 10 8 ~1×10 12 Cells / individual / day, specifically, 1 x 10 9 ~1×10 11 The number of cells per individual per day can be adjusted.

[0056] 3. Method for producing the composition The production method of the present invention is a method for producing a composition for enhancing IFN-λ production in a subject, and is characterized by comprising the steps of culturing Enterococcus bacteria and obtaining a culture or treated product of the Enterococcus bacteria.

[0057] In the production method of the present invention, the culture conditions for Enterococcus bacteria, the collection method of the culture or treated product as an active ingredient, and the treatment method are not particularly limited, as long as a culture or treated product of Enterococcus bacteria that exhibits the effect of enhancing IFN-λ production and antiviral protein gene expression can be obtained.

[0058] In the production method of the present invention, the Enterococcus bacterium is preferably Enterococcus casseliflavus, and particularly preferably the Enterococcus casseliflavus KB1733 strain.

[0059] 3-1 Cultivation process The production method of the present invention includes a step of culturing Enterococcus bacteria. One example of the culturing step is to inoculate Enterococcus bacteria into an MRS medium and culture them for 20 to 24 hours at 30 to 37°C, which is the optimum temperature for the bacteria, to obtain a culture solution.

[0060] 3-2 Processing process The production method of the present invention includes a step of obtaining a culture or a processed product of Enterococcus bacteria. More specifically, it includes a processing step of processing the culture solution obtained in the culturing step to obtain a culture or a processed product that can be used as a composition.

[0061] As an example of the processing step, the following method can be used. The bacterial cells are collected from the culture solution obtained in the culture step and washed with sterilized water or physiological saline. The obtained bacterial cells are subjected to a drying process after heat sterilization or in an untreated state to obtain a dried sterilized powder or a dry powder as the active ingredient. Note that the active ingredient is not limited to a powder, and may also be in the form of an aqueous solution or a frozen product.

[0062] The drying method used in the processing step is not particularly limited, but may include natural drying, air drying, freeze drying, etc. The sterilization method is also not particularly limited, but may include, for example, heat sterilization, pressure sterilization, osmotic sterilization, etc.

[0063] The composition obtained by the production method of the present invention can promote IFN-λ production in a subject by administering it to the subject by oral ingestion or the like. Furthermore, in relation to IFN, the composition may have antiviral effects, immunostimulatory effects, anti-infectious disease effects, anti-hepatitis B effects, anti-hepatitis C effects, anti-tumor growth effects, anti-tumor effects, and anti-cancer effects in the subject. [Example]

[0064] The present invention will be described in more detail below with reference to examples, but the technical scope of the present invention is not limited to the following examples.

[0065] Example 1: Evaluation test of the promotion of IFN-λ production in WiDr cells by Enterococcus lactic acid bacteria [1-1] Preparation of test samples A frozen Enterococcus genus bacterial suspension (Enterococcus cathelliflavus KB1733 strain) was added to MRS liquid medium (Thermo Fisher Scientific) and cultured at 30°C for 24 hours.

[0066] After 24 hours of incubation, the culture medium was added to MRS liquid medium and further incubated at 30°C for 24 hours. The culture was then inoculated into 40 mL of MRS liquid medium in a 50 mL centrifuge tube and incubated statically at 30°C for 20-24 hours. The mixture was then centrifuged at 2,590 × g and 4°C for 10 minutes, and the supernatant was removed. Physiological saline was added to the bacterial cells, and the cells were suspended by vortexing. The mixture was then centrifuged under the same conditions, and the supernatant was removed. 3 mL of ultrapure water was added, and the bacterial cells were suspended by vortexing. The entire suspension was transferred to a 15 mL centrifuge tube, sterilized by heating at 100°C for 30 minutes, cooled under running water, and frozen overnight in a -80°C deep freezer. The 15 mL centrifuge tube containing the frozen suspension was capped and covered with a Bemcot® filter sterilized at 121°C for 15 minutes. The tube was then dried for 6 days using a vacuum freeze dryer (AGC TECHNO GLASS, FREEZE-DRYER, SRG-40M). After drying, the Bemcot® filter was removed from the 15 mL centrifuge tube containing the sterilized bacteria. The dried sample was finely ground with a microspatula, and 10 mg of bacterial powder was transferred to a 1.5 mL tube. One mL of DMEM (high glucose) medium (Thermo Fisher Scientific) supplemented with 10% fetal bovine serum (FBS) and 100 μg / mL penicillin and streptomycin (hereafter referred to as "complete medium") was added and suspended by vortexing to obtain a 10 mg / mL sterilized bacteria suspension. The sterilized bacteria suspension was serially diluted with complete medium to prepare sterilized bacteria suspensions with concentrations of 1, 3, 10, and 30 μg / mL, which served as test samples.

[0067] [1-2] Cell culture test WiDr cells (JCRB Cell Bank, National Institutes of Biomedical Innovation, Health and Nutrition, No. JCRB0224) were maintained in a 100 mm diameter dish (AGC TECHNO GLASS) at 37°C and 5% CO2. The medium was changed every 2-3 days. Subculture was performed twice a week. The medium was removed, the cells were washed with 10 mL of PBS, and then 2 mL of 0.25% (w / v) trypsin-1 mM EDTA solution was added. The cells were incubated at 37°C and 5% CO2 for 3 minutes to detach the cells from the bottom of the dish. 8 mL of complete medium was added to stop the reaction, and the detached cells were collected. After counting the number of cells using trypan blue staining, the viable cell count in complete medium was 1.1 x 10 6 After confirming that the passaged WiDr cells were 80-90% confluent, the WiDr cells were cultured in a 6-well plate (AGC TECHNO GLASS) at a concentration of 1 × 10 6 Cells were seeded at 1000 x g per well and cultured at 37°C in a 5% CO2 environment. After 48 hours of culture, the medium was removed, and 0.5 mL of PBS was added to wash the cells. After removing the PBS, 0.5 mL of PBS was added and washed again. After removing the PBS, 1,000 μL of each concentration of sterilized cell suspension was added to each well and incubated for 48 hours. As a control, complete medium without sterilized cell suspension was added and incubated in the same manner. After incubation, the cell supernatant was collected in a 1.5 mL tube and centrifuged (1,500 x g, 4°C, 5 minutes). After centrifugation, the supernatant was collected in a 1.5 mL tube and stored at -80°C. The concentrations of IFN-λ1 and IFN-λ3 in the cell supernatant were measured. IFN-λ1 concentrations were measured using an IL-29 Human ELISA Kit (Thermo Fisher Scientific) according to the kit's protocol. The IFN-λ3 concentration was measured by chemiluminescent enzyme immunoassay (CLEIA) according to the method described in M. Sugiyama, et al., Hepatology Research, 42(11) pp. 1089-1099 (2012).

[0068] [1-3]Result The concentrations of IFN-λ1 and IFN-λ3 in the WiDr cell culture medium when each killer cell suspension was added are shown in Figures 1 and 2, respectively. It was confirmed that the addition of killer cells significantly increased the production of IFN-λ1 and IFN-λ3 by WiDr cells.

[0069] Example 2: Evaluation test of the amount of antiviral protein gene expression increased by Enterococcus lactic acid bacteria [2-1] Preparation of test samples A 10 μg / mL suspension of the fungicide was prepared under the same conditions as in Example 1 and used as a test sample.

[0070] [2-2] Cell culture test WiDr cells were maintained in a 100 mm diameter dish (AGC TECHNO GLASS) at 37°C in a 5% CO2 environment. The medium was changed every 2-3 days, and subculture was performed twice a week. The medium was removed, the cells were washed with 10 mL of PBS, and then 2 mL of 0.25% (w / v) trypsin-1 mM EDTA solution was added. The cells were incubated at 37°C and 5% CO2 for 3 minutes to detach the cells from the bottom of the dish. 8 mL of complete medium was added to stop the reaction, and the detached cells were collected. After counting the number of cells using trypan blue staining, the viable cell count in complete medium was 1.1 x 10 6 After confirming that the passaged WiDr cells were 80-90% confluent, the WiDr cells were transferred to a 35 mm dish (AGC TECHNO GLASS) at a density of 1 × 10 6 The cells were seeded at 1 cell per dish and cultured at 37°C in a 5% CO2 environment. 48 hours after the start of culture, the medium was removed, and 0.5 mL of PBS was added to wash the cells. After removing the PBS, 0.5 mL of PBS was added again, and the cells were washed again. After removing the PBS, 1,000 μL of a 10 μg / mL sterilized cell suspension was added to each well and incubated for 0, 4, 8, 24, and 48 hours. After each incubation, the medium was removed, and 0.5 mL of PBS was added to wash the cells. After removing the PBS, 0.5 mL of PBS was added again, and the cells were washed again. The 35 mm dishes from which the supernatant had been removed were stored at -80°C.

[0071] [2-3] Total RNA extraction Cells from each well were detached using 900 μL of TRIzol™ (Thermo Fisher Scientific) and pipetted to collect the cell extract in a 1.5 mL ringlock tube. The sample was placed on ice, 180 μL of chloroform was added, vortexed, and then allowed to stand at room temperature for at least 3 minutes. Centrifuged at 12,000 × g and 4°C for 15 minutes, and 250 μL of the supernatant was transferred to a 1.5 mL tube. 200 μL of isopropanol was added and vortexed. After allowing to stand at room temperature for 10 minutes, the tube was centrifuged at 12,000 × g and 4°C for 10 minutes. The supernatant was removed, and the tube was air-dried for approximately 10 minutes. 75% ethanol was added to the precipitate, and the mixture was centrifuged at 12,000 × g and 4°C for 5 minutes. The supernatant was removed. This procedure was repeated twice. The RNA was dried at room temperature and then dissolved in 30-50 μL of RNase-free distilled water to prepare an RNA extraction solution. A 0.1-fold volume of 3M sodium acetate solution and 2.5-fold volume of ethanol were added to the RNA extraction solution, mixed gently, and then left to stand overnight at -80°C. The mixture was centrifuged at 12,000 × g, 4°C, and 10 minutes. The supernatant was removed, and the tube was air-dried for approximately 10 minutes. 75% ethanol was added to the precipitate, and the mixture was centrifuged at 12,000 × g, 4°C, and 5 minutes. The supernatant was removed, and the RNA was dried at room temperature and then dissolved in 30 μL of RNase-free distilled water to prepare an RNA solution.

[0072] [2-4]cDNA synthesis Reverse transcription from RNA to cDNA was performed using the Prime Script RT reagent Kit (Takara Bio) according to the manufacturer's instructions. Specifically, the enzyme mix, extracted RNA solution, and water were placed in a microtube and heated at 37°C for 15 minutes and then at 85°C for 5 seconds using a thermal cycler. The prepared cDNA solution was stored at -80°C.

[0073] [2-5] Quantitative PCR The gene abundances of Mx1, OAS1, ISG15, and GAPDH in each cDNA solution (equivalent to mRNA abundance) were measured by real-time PCR using SYBR Premix ExTaq II (Takara Bio) on a 7500 Fast Real-time PCR system (Applied Biosystems). SYBR Premix ExTaq II, PCR primers (forward primer (F) and reverse primer (R)), each cDNA sample, and RNase-free distilled water were added to a 96-well PCR plate and placed in a PCR machine. PCR was performed using an initial denaturation at 95°C for 30 seconds followed by 40 cycles of temperature changes from 95°C for 5 seconds to 60°C for 30 seconds. The sequences of the primers used are listed in Table 1 as SEQ ID NOS: 8–15. The expression levels of each gene were normalized by the expression level of the internal control gene, glyceraldehyde 3-phosphate dehydrogenase (GAPDH), and analyzed as a relative value, with the gene expression level at time 0 under the same conditions set to 1.

[0074] [Table 1]

[0075] [2-6]Result The gene expression levels of Mx1, OAS1, and ISG15 in WiDr cells upon addition of the killer cell suspension are shown in Figures 3, 4, and 5, respectively. It was confirmed that addition of the killer cell suspension promoted the expression of various IFN-inducible antiviral protein genes.

Claims

1. A composition for enhancing interferon-λ production in a subject, comprising a bactericidal form of Enterococcus cathelliflavus as an active ingredient.

2. The composition according to claim 1, wherein the Enterococcus casselliflavus is the Enterococcus casselliflavus KB1733 strain (accession number NITE AP-03535).

3. The composition according to claim 1 or 2, which increases the expression level of an antiviral protein gene in a subject.

4. The composition of claim 3, wherein the antiviral protein gene is at least one gene selected from the group consisting of the Mx dynamin-like GTPase 1 (Mx1) gene, the 2'-5'-oligoadenylate synthetase 1 (OAS1) gene, and the interferon-stimulated gene 15 (ISG15) gene.

5. A pharmaceutical composition for enhancing interferon-λ production in a subject, comprising the composition according to any one of claims 1 to 4.

6. An edible composition for enhancing interferon-λ production in a subject, comprising the composition according to any one of claims 1 to 4.

7. A composition for external use for enhancing interferon-λ production in a subject, comprising the composition according to any one of claims 1 to 4.

8. A method for producing a composition for enhancing interferon-λ production in a subject, comprising the steps of culturing Enterococcus cathelliflavus and obtaining a killed form of Enterococcus cathelliflavus.

9. The production method according to claim 8, wherein the Enterococcus casselliflavus is the Enterococcus casselliflavus KB1733 strain (accession number NITE AP-03535).

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