IFN-α production inducer activator, IL-12 production inducer activator, antiviral activator, and immunostimulant

Abstract

The present invention clarifies novel properties of lactic acid bacteria of the genus Weissella, particularly the Weissella cibaria HRL3 strain deposited under accession number NITE P-04107, and provides new uses based on these properties. [Solution] An IFN-α production inducer activator that activates the induction of interferon-α (IFN-α) production in plasmacytoid dendritic cells (pDCs), and an IL-12 production inducer activator that activates the induction of interleukin-12 (IL-12) production in myeloid dendritic cells (mDCs), each containing at least one of Weissella lactic acid bacteria cells or a processed lactic acid bacteria cell product as an active ingredient. Also, an antiviral activator and an immunostimulator based on the above-mentioned effects.

Description

[Technical Field]

[0001] The present invention relates to an IFN-α production inducer activator, an IL-12 production inducer activator, an antiviral activator, and an immunostimulant containing at least one of the cells or a processed cell product of a lactic acid bacterium belonging to the genus Weissella as an active ingredient. [Background technology]

[0002] It has been widely known that lactic acid bacteria have various physiological functions (bioactivities). For example, sleep-promoting agents containing lactic acid bacteria belonging to the genus Lactobacillus, Lactococcus, Leuconostoc, Pediococcus, or Weissella, or cultures thereof, as active ingredients, are known (see Patent Document 1).

[0003] Lactic acid bacteria of the genus Weissella are commonly found in nature and are sometimes used in the production of fermented foods. Furthermore, known products that utilize the physiological functions of Weissella lactic acid bacteria include, for example, an anti-inflammatory agent containing vesicles derived from Weissella lactic acid bacteria (see Patent Document 2) and a cancer preventive or therapeutic agent containing the bacteria or a culture thereof as an active ingredient (see Patent Document 3). [Prior art documents] [Patent documents]

[0004] [Patent Document 1] Patent Publication No. 2021-123562 [Patent Document 2] Special Publication No. 2022-511913 [Patent Document 3] Special Publication No. 2023-513097 Summary of the Invention [Problem to be solved by the invention]

[0005] However, there is still room for further research on Weissella lactic acid bacteria, and their physiological functions have not yet been fully elucidated.

[0006] The present invention has been made in view of the above circumstances, and aims to clarify the novel physiological functions of lactic acid bacteria of the genus Weissella and to provide new uses based on these functions. [Means for solving the problem]

[0007] Through intensive research by the inventors of the present invention, it has been found that lactic acid bacteria of the genus Weissella activate the induction of interferon-α (IFN-α) production in plasmacytoid dendritic cells (pDCs). It has also been found that lactic acid bacteria of the genus Weissella activate the induction of interleukin-12 (IL-12) production in myeloid dendritic cells (mDCs). Based on these findings, the inventors of the present invention have concluded that at least one of Weissella lactic acid bacteria cells and a processed product of lactic acid bacteria cells can activate the antiviral properties of the body and can also activate immunity, thereby completing the present invention. The present invention includes the following embodiments.

[0008] (1) An IFN-α production inducer containing at least one of the cells of a lactic acid bacterium belonging to the genus Weissella and a processed cell product of the lactic acid bacterium as an active ingredient, which activates the induction of interferon-α (IFN-α) production in plasmacytoid dendritic cells (pDCs). (2) The IFN-α production inducer activator according to (1) above, wherein the lactic acid bacterium is classified as Weissella cibaria. (3) The IFN-α production inducer activator according to (2) above, wherein the lactic acid bacterium is Weissella cibaria HRL3 strain isolated from Hakutsuru Reishi and deposited under accession number NITE P-04107. (4) The IFN-α production inducer activator according to (1) above, wherein the bacterial cells and the treated bacterial cells contain nucleic acid derived from the lactic acid bacteria. (5) The IFN-α production inducer activator according to any one of (1) to (4) above, which is in the form of a food or drink, a pharmaceutical product, a feed, or an active ingredient composition to be incorporated therein. (6) An IL-12 production inducer activator containing at least one of the cells of a lactic acid bacterium belonging to the genus Weissella and a processed cell product of the lactic acid bacterium as an active ingredient, which activates the induction of interleukin-12 (IL-12) production in myeloid dendritic cells (mDCs). (7) The IL-12 production inducer activator according to (6) above, wherein the bacterial cells and the treated bacterial cells contain ribonucleic acid (RNA) derived from the lactic acid bacteria. (8) The IL-12 production inducer activator according to (6) or (7) above, which is in the form of a food or drink, a pharmaceutical product, a feed, or an active ingredient composition to be incorporated therein. (9) An antiviral activator containing at least one of the cells of a lactic acid bacterium belonging to the genus Weissella and a processed cell product of the lactic acid bacterium as an active ingredient, which activates the antiviral properties of the body by activating the induction of interferon-α (IFN-α) production in plasmacytoid dendritic cells (pDCs), or activates the induction of interleukin-12 (IL-12) production in myeloid dendritic cells (mDCs). (10) The antiviral activator according to (9) above, wherein the bacterial cells and the treated bacterial cells contain nucleic acid derived from the lactic acid bacteria. (11) The antiviral activator according to (9) or (10) above, which is in the form of a food or drink, a medicine, a feed, or an active ingredient composition to be incorporated therein. (12) An immunostimulant containing at least one of the cells of a lactic acid bacterium belonging to the genus Weissella and a processed cell product of the lactic acid bacterium as an active ingredient, which activates the immune system of the living body by activating the induction of interferon-α (IFN-α) production in plasmacytoid dendritic cells (pDCs), or activates the induction of interleukin-12 (IL-12) production in myeloid dendritic cells (mDCs). (13) The immunostimulating agent according to (12), wherein the bacterial cells and the treated bacterial cells contain nucleic acid derived from the lactic acid bacteria. (14) The immunostimulant according to (12) or (13) above, which is in the form of a food or drink, a pharmaceutical product, a feed, or an active ingredient composition to be incorporated therein. [Effects of the Invention]

[0009] According to the present invention, novel uses of Weissella lactic acid bacteria can be provided, including IFN-α production inducer activators, IL-12 production inducer activators, antiviral activators, and immunostimulants, each of which contains at least one of Weissella lactic acid bacteria cells and treated lactic acid bacteria cells as an active ingredient. [Brief explanation of the drawings]

[0010] [Figure 1] 1 is a table showing the sugar metabolism pattern of the Weissella cibaria HRL3 strain. [Figure 2] 1 is a graph showing the growth curve of Weissella cibaria HRL3 strain. [Figure 3] 1 is a bar graph showing the results of evaluating the IFN-α production induction and activation activity and IL-12 production induction and activation activity of the Weissella cibaria HRL3 strain. [Figure 4] 1 is a bar graph showing the results of evaluating the effect of nuclease treatment on the IFN-α production induction activation activity and IL-12 production induction activation activity of the Weissella cibaria HRL3 strain. DETAILED DESCRIPTION OF THE INVENTION

[0011] Next, an embodiment of the present invention will be described. Note that the embodiment described below does not limit the invention according to the claims, and not all of the elements and combinations thereof described in the embodiment are necessarily essential to the solution of the present invention.

[0012] (I) Lactic acid bacteria of the genus Weissella, bacterial cells, and processed bacterial cells In one embodiment of the present invention, the active ingredient is a Weissella genus lactic acid bacterium cell or a processed lactic acid bacterium cell product. Lactic acid bacteria of the Weissella genus are lactic acid bacteria belonging to the Lactobacillus order, Lactobacillaceae family (Lactobacillaceae family). Preferably, the lactic acid bacterium is classified as Weissella cibaria. More preferably, the lactic acid bacterium is a strain isolated from White Crane Reishi or a mutant thereof. Even more preferably, the lactic acid bacterium is Weissella cibaria HRL3 strain or a mutant thereof.

[0013] As used herein, "bacterial cells" includes both live and dead bacterial cells. The bacterial cells may be frozen or dried. Furthermore, as used herein, "treated bacterial cells" refers to a substance containing components derived from bacterial cells, obtained by a process of disrupting the bacterial cells or a process of extracting components from the bacterial cells. Treated bacterial cells include not only those containing part or all of the bacterial cells, such as cytoplasm or cell wall fractions obtained by treating the bacterial cells by physical means, but also those that do not contain the bacterial cells themselves, such as extracts from the bacterial cells.

[0014] The term "mutant strain" as used herein means a strain that has been mutated by a person skilled in the art using a method well known to those skilled in the art to the extent that the mutation does not affect the main properties of the strain, or a strain that a person skilled in the art can confirm to be equivalent to such a mutation.

[0015] Furthermore, the bacterial cells and treated bacterial cells according to one embodiment of the present invention preferably contain nucleic acid derived from lactic acid bacteria of the genus Weissella. As used herein, "nucleic acid" includes both deoxyribonucleic acid (DNA) and ribonucleic acid (RNA).

[0016] As shown in the Examples below, DNA derived from Weissella lactic acid bacteria is thought to be strongly involved in activating the induction of interferon-α (IFN-α) production in plasmacytoid dendritic cells (pDCs). Furthermore, RNA derived from Weissella lactic acid bacteria is thought to be strongly involved in activating the induction of interleukin-12 (IL-12) production in myeloid dendritic cells (mDCs). Furthermore, the above RNA is thought to be involved in activating the induction of interferon-α (IFN-α) production in plasmacytoid dendritic cells (pDCs).

[0017] (II) Weissella cibaria HRL3 strain Here, we will describe the Weissella civaria HRL3 strain, a recently discovered novel strain. The Weissella civaria HRL3 strain was deposited on April 25, 2024, at the Patent Microorganisms Depositary, National Institute of Technology and Evaluation (Room 122, 2-5-8 Kazusa Kamatari, Kisarazu City, Chiba Prefecture, 292-0818, Japan). The accession number is NITE P-04107. Hereinafter, this strain may be simply referred to as the "HRL3 strain."

[0018] The HRL3 strain is a lactic acid bacterium extracted from Rhinacanthus nasutus (L.) Kurz, also known as white crane reishi grass, which is used as an ingredient in traditional Chinese medicine and food. While typical lactic acid bacteria utilize glucose to produce lactic acid, the HRL3 strain is unique in that it uses fructose as a sugar source for lactic acid fermentation.

[0019] The HRL3 strain is a short bacillus classified as Weissella cibaria, and the observed colony formation conditions were aerobic, with a growth pH of 6.8 and a growth temperature of 30°C. The HRL3 strain was discovered in lactic acid bacterial colonies obtained by aerobic culture of bacteria living in the aboveground parts (leaves, stems, and flowers) of Hakutsuru Reishi in a fructose-yeast extract-polypeptone (FYP) medium. A specific method for collecting the HRL3 strain will be described in Example 1 below.

[0020] The inventors of the present invention evaluated the sugar utilization ability of the HRL3 strain using the API 50 CH bacterial identification test kit (bioMérieux, France). The evaluation method was as follows. First, the HRL3 strain was cultured in 12 mL of FYP liquid medium (described below) at 29°C under anaerobic conditions for 24 hours. The culture was then centrifuged, the supernatant was removed, and the resulting pellet was suspended in 2 mL of suspension medium (included in the kit) to prepare a suspension. Next, the above suspension was added to 5 mL of suspension medium (included in the kit) and suspended, and the McFarland turbidity was adjusted to 2. Furthermore, a volume of the suspension (bacterial solution) twice the amount of the above suspension (1.0 mL) was mixed with 10 mL of API 50 CHL medium (included in the kit) and poured into the microtube of an API 50 CH plate (included in the kit). Two drops of mineral oil were then added to the microtube, and the culture was then cultured at 29°C for 48 hours. The presence or absence of sugar utilization (fermentation) was determined by the color tone of the indicator.

[0021] The results (carbohydrate metabolism patterns) obtained by the above method are shown in Figure 1. For comparison, Figure 1 also shows the carbohydrate metabolism patterns of the Weissella civaria II-I-59 strain (reference strain), Weissella civaria CHJ3 strain, Weissella civaria CCUG33604 strain, Weissella civaria CCUG34912 strain, and Weissella civaria CCUG38043 strain obtained from the BacDive database.

[0022] As a result, it was confirmed that the HRL3 strain is capable of assimilating both glucose (GLU) and fructose (FRU), similar to the reference strain, Weissella civaria II-I-59, but differs from it in that it can also assimilate galactose (GAL) and other sugars.

[0023] The inventors of the present invention also obtained growth curves for the HRL3 strain on glucose and fructose as follows: First, the HRL3 strain was added to FYP liquid medium or glucose-yeast extract-polypeptone (GYP) liquid medium and cultured at 30°C for 24 hours under aerobic conditions (shaking culture) or anaerobic conditions (sealed static culture). The bacterial solution was sampled at 0, 6, 12, 18, and 24 hours of culture, and the absorbance at 660 nm was measured using a microplate reader (Thermo Fisher Scientific, USA).

[0024] The FYP liquid medium (pH = 6.8) used in this specification has the following composition: The following composition is for one lot. (FYP liquid medium composition) D-Fructose (Fujifilm Wako Pure Chemical Industries, Ltd.): 100g Yeast extract (Nacalai Tesque Inc.): 10g Bacto Peptone (Becton Dickinson, USA): 5g Sodium acetate (Fujifilm Wako Pure Chemical Industries, Ltd.): 2g Tween® 80 (Sigma-Aldrich, USA): 0.5g Magnesium sulfate heptahydrate (Fujifilm Wako Pure Chemical Industries, Ltd.): 0.2g Manganese sulfate pentahydrate (Fujifilm Wako Pure Chemical Industries, Ltd.): 0.01g Ferrous sulfate heptahydrate (Fujifilm Wako Pure Chemical Industries, Ltd.): 0.01g Sodium chloride (Fujifilm Wako Pure Chemical Industries, Ltd.): 0.01g Cycloheximide (Fujifilm Wako Pure Chemical Industries, Ltd.): 0.05g Sodium azide (Fujifilm Wako Pure Chemical Industries, Ltd.): 0.05g Distilled water: 1L

[0025] Furthermore, the GYP liquid medium (pH = 6.8) in this specification has the same composition as the above FYP liquid medium, except that it contains the same amount (100 g per lot) of D-glucose (Fujifilm Wako Pure Chemical Industries, Ltd.) instead of D-fructose.

[0026] The growth curve obtained by the above method is shown in Figure 2. It was confirmed that the HRL3 strain can assimilate both glucose and fructose, and that its growth is promoted under aerobic conditions.

[0027] The inventors of the present invention also commissioned Azenta Inc. to perform 16S rRNA sequence analysis of the HRL3 strain. The HRL3 strain was frozen and stored in the presence of glycerol before being sent. The bacterial DNA was extracted by alkaline extraction, amplified by PCR, and then used for Sanger sequencing. The resulting sequence data was then used to perform a homology search using BLAST.

[0028] The strain closest to HRL3 was Weissella civaria II-I-59 (type strain), with 99% identity in the 16S rRNA gene sequence, confirming that HRL3 belongs to the Weissella civaria family.

[0029] (III) IFN-α production inducer activator, IL-12 production inducer activator, antiviral activator, and immunostimulant As used herein, the term "IFN-α production inducer activator" refers to a substance that activates the induction of interferon-α (IFN-α; hereinafter, also referred to as "IFN-α") production in plasmacytoid dendritic cells (pDC; hereinafter, also referred to as "pDC").

[0030] Dendritic cells are antigen-presenting cells present in the body, and pDCs in particular play a key role in activating immune function, primarily against viruses, by producing type I interferon. IFN-α is a type of type I interferon produced by pDCs and is primarily involved in innate immunity against viruses.

[0031] As used herein, the term "IL-12 production inducer / activator" refers to a substance that activates the induction of interleukin-12 (IL-12; hereinafter, also referred to as "IL-12") production in myeloid dendritic cells (mDCs; hereinafter, also referred to as "mDCs").

[0032] mDCs are dendritic cells different from the pDCs described above, and are known to primarily have the function of antigen presentation, but also produce cytokines. IL-12 is a type of interleukin produced by mDCs and plays an important role in the immune system, acting as a T cell stimulator and NK cell stimulator, for example. In this specification, "IL-12" refers to "IL-12p70."

[0033] As used herein, the term "antiviral activator" refers to a substance that activates the antiviral properties of the body by activating the induction of IFN-α production in pDCs, and a substance that activates the antiviral properties of the body by activating the induction of IL-12 production in mDCs. Furthermore, as used herein, the term "immunostimulatory agent" refers to a substance that activates the immune system of the body by activating the induction of IFN-α production in pDCs, and a substance that activates the immune system of the body by activating the induction of IL-12 production in mDCs.

[0034] (IV) Foods, beverages, medicines, feeds, or compositions containing active ingredients to be incorporated therein (Active ingredient composition) The IFN-α production-inducing activator, IL-12 production-inducing activator, antiviral activator, and immunopotentiator of this embodiment can be used in the form of an active ingredient composition to be incorporated into foods, beverages, pharmaceuticals, or feed. When used as an active ingredient composition, at least one of the active ingredient, the Weissella lactic acid bacteria cells or a treated cell product, can be used as is. Alternatively, a culture or fermentation broth (including culture supernatant) of Weissella lactic acid bacteria, or a crude or purified product thereof, can also be used. Furthermore, when the active ingredient is a treated cell product, a composition containing a substance used in treating the cells (e.g., extraction solvent) can also be used as the active ingredient composition.

[0035] Furthermore, the bacterial cells may not only be live bacterial cells, but also those sterilized by heat sterilization or the like (killed bacterial cells). When Weissella lactic acid bacteria are killed by heat treatment, the conditions are preferably 65 to 85°C for 1 minute or more, for example, about 10 minutes, 30 minutes, or 60 minutes, and more preferably 70 to 75°C for 1 minute or more, for example, about 5 minutes, 10 minutes, or 30 minutes. As will be explained in Example 2 below, even heat-treated bacterial cells can be expected to have the effect of inducing and activating IFN-α production and IL-12 production. Furthermore, live bacteria may undergo morphological changes during delivery or display after product production, so killed bacterial cells, which do not undergo further morphological changes, are preferably used.

[0036] The active ingredient composition of this embodiment may further contain, as needed, an appropriate amount of nutritional components suitable for the maintenance and growth of Weissella lactic acid bacteria. Specific examples of such nutritional components include carbon sources used in culture media for culturing microorganisms, such as fructose, glucose, sorbose, ribose, lyxose, xylose, arabinose, lactulose, and sucrose; nitrogen sources, such as yeast extract and peptone; vitamins; minerals; trace metal elements; and other nutritional components. Examples of vitamins include vitamin B, vitamin D, vitamin C, vitamin E, and vitamin K. Examples of trace metal elements include zinc and selenium. Examples of other nutritional components include various oligosaccharides, such as lactoferrin oligosaccharides, soybean oligosaccharides, lactitol, fructooligosaccharides, and galactooligosaccharides. The amount of these oligosaccharides is not particularly limited, but is preferably selected from a range that generally provides a concentration of approximately 1 to 30% by weight of the composition of this embodiment.

[0037] The amount of Weissella lactic acid bacteria in the active ingredient composition of this embodiment is generally such that the number of bacteria is 10 8 ~10 13 The amount can be appropriately selected from the range of amounts that will give approximately 100,000 live bacteria (not necessarily the number of viable bacteria). The active ingredient composition of this embodiment is preferably prepared in the form of a food or drink, a pharmaceutical product, etc., as described below, after being appropriately blended with an appropriate edible carrier (food material), a pharmaceutically acceptable carrier, etc.

[0038] Furthermore, the above-mentioned bacterial cells and treated bacterial cells preferably contain nucleic acids derived from lactic acid bacteria of the genus Weissella, because, as shown in Example 3 below, the IFN-α production-inducing and IL-12 production-inducing and activating effects (and thus antiviral activation effects) of lactic acid bacteria of the genus Weissella are thought to be mainly attributable to the nucleic acids of the lactic acid bacteria.

[0039] (Pharmaceuticals) When the IFN-α production-inducing activator, IL-12 production-inducing activator, antiviral activator, and immunostimulant of the present embodiment are formulated into pharmaceutical forms, they are prepared and put into practical use by using at least one of the active ingredient, Weissella lactic acid bacteria cells or a treated cell product, together with a suitable pharmaceutically acceptable pharmaceutical carrier. Examples of such pharmaceutical carriers include diluents and excipients commonly used in this field, such as fillers, extenders, binders, wetting agents, disintegrants, surfactants, and lubricants.

[0040] The dosage unit form of a pharmaceutical product can be selected from a variety of forms, and preferred are oral dosage forms, such as tablets, pills, powders, liquids, suspensions, emulsions, granules, and capsules.

[0041] When forming into tablets, the pharmaceutical carrier may be, for example, lactose, sucrose, sodium chloride, glucose, urea, starch, calcium carbonate, kaolin, crystalline cellulose, silicic acid, potassium phosphate, or other excipient; water, ethanol, propanol, simple syrup, glucose solution, starch solution, gelatin solution, carboxymethylcellulose, hydroxypropylcellulose, methylcellulose, polyvinylpyrrolidone, or other binder; sodium carboxymethylcellulose, calcium carboxymethylcellulose, low-substituted hydroxypropylcellulose, dry starch, alginate, or the like. Disintegrants such as sodium phosphate, agar powder, laminaran powder, sodium bicarbonate, and calcium carbonate; surfactants such as polyoxyethylene sorbitan fatty acid esters, sodium lauryl sulfate, and stearic acid monoglyceride; disintegration inhibitors such as sucrose, stearin, cocoa butter, and hydrogenated oil; absorption promoters such as quaternary ammonium salts and sodium lauryl sulfate; humectants such as glycerin and starch; adsorbents such as starch, lactose, kaolin, bentonite, and colloidal silicic acid; and lubricants such as purified talc, stearates, boric acid powder, and polyethylene glycol. Tablets can be coated with a conventional coating, such as sugar-coated tablets, gelatin-coated tablets, enteric-coated tablets, or film-coated tablets, or can be double- or multi-layered tablets, if necessary.

[0042] When forming into pills, the following may be used as pharmaceutical carriers: excipients such as glucose, lactose, starch, cacao butter, hardened vegetable oil, kaolin, talc, etc.; binders such as powdered gum arabic, powdered tragacanth, gelatin, ethanol, etc.; disintegrants such as laminaran, agar, etc.

[0043] Furthermore, the pharmaceutical product may contain coloring agents, preservatives, perfumes, flavoring agents, sweeteners, and other pharmaceuticals, if necessary.

[0044] There are no particular limitations on the method of administration of the pharmaceutical of this embodiment, and it is determined depending on the dosage form, the patient's age, sex and other conditions, the severity of the disease, etc. The dosage is selected appropriately depending on the method of use, the patient's age, sex and other conditions, the severity of the disease, etc., but it is generally considered appropriate to administer the active ingredient composition at about 0.5 to 100 mg per kg of body weight per day. The pharmaceutical can be administered to humans in 1 to 4 divided doses per day.

[0045] (Food and beverages) As used herein, "food and beverage" includes all products that are used orally for consumption, including beverages. In this specification, even products in the form of tablets or the like are considered to be included in the term "food and beverage" as long as they are used exclusively for consumption. For example, health foods, health supplements, foods for the sick, nutritional supplements, and functional health foods (foods for specified health uses, foods with nutrient functions) as defined by the Ministry of Health, Labor and Welfare are also included in the term "food and beverage." Health foods are foods intended to promote health, maintain, or improve health in a more positive sense than regular foods.

[0046] When the IFN-α production inducer activator, IL-12 production inducer activator, antiviral activator and immunostimulant of this embodiment are made into food and beverage products, they are prepared and put into practical use using appropriate materials acceptable in the food and beverage field together with at least one of the active ingredients, namely, the Weissella lactic acid bacteria cells and processed cells.

[0047] Examples of food and beverage forms include fermented milk, lactic acid bacteria beverages, fermented vegetable beverages, fermented fruit beverages, and fermented soy milk beverages. "Fermented milk" refers to milk or dairy products fermented with lactic acid bacteria or yeast to form a paste or liquid. Therefore, fermented milk includes both beverage forms and yogurt forms. "Lactic acid bacteria beverages" refer to beverages made by diluting milk or dairy products fermented with lactic acid bacteria or yeast to form a paste or liquid as the main ingredient with water.

[0048] Other forms of food and drink include, for example, fermented foods such as pickles, miso, fermented tea, and bread; infant foods such as baby food, powdered milk, and baby food; effervescent preparations, confectioneries such as gum, gummy candies, and pudding; noodles; nutritional supplements such as capsules, granules, powders, and tablets; and dairy products other than fermented milk and lactic acid bacteria drinks.

[0049] The content of the active ingredient composition in the food or beverage of this embodiment is not particularly limited and can be determined as appropriate. From the viewpoint of achieving the effects of inducing and activating IFN-α production, inducing and activating IL-12 production, or antiviral activity, the content of the active ingredient composition is, for example, preferably 0.001% by mass or more, more preferably 0.01% by mass or more, and even more preferably 0.1% by mass or more, relative to the total mass of each food or beverage. Meanwhile, the upper limit of the content of the active ingredient composition in the food or beverage is not particularly limited and can usually be adjusted as appropriate depending on the form of the food or beverage.

[0050] (feed) When the IFN-α production-inducing activator, IL-12 production-inducing activator, antiviral activator, and immunostimulant of this embodiment are in the form of feed, they can be in the form of, for example, a formulation for oral administration (aqueous solution, emulsion, granules, powder, capsules, tablets, etc.).

[0051] [Example] The present invention will be described in more detail below with reference to examples, but the present invention is not limited to these examples. In the following description, "%" in the numerical values ​​indicating the amount of each component added means mass percent concentration (mass %) unless otherwise specified.

[0052] [Example 1] Collection, cultivation, and killed cell preparation of Weissella cibaria HRL3 strain (Collection and isolation) First, the aboveground parts (leaves, stems, and flowers) of Hakutsuru Reishi (produced in Chiba Prefecture) were chopped into 1-2 cm pieces with scissors. The chopped Hakutsuru Reishi was added to FYP liquid medium and cultured with shaking at 29°C. After confirming that the culture solution had become cloudy and had become a suspension, the suspension was added to FYP liquid medium and cultured with shaking at 29°C for 48 hours. The suspension was then spread on FYP agar medium, which was prepared by adding calcium carbonate (Fujifilm Wako Pure Chemical Corporation) and agar (Fujifilm Wako Pure Chemical Corporation), and cultured aerobically at 29°C. After 48 hours of culture, acid production was determined by the dissolution of calcium carbonate around the colony, and lactic acid bacteria colonies that showed acid production were picked. The picked strains were then cultured statically in FYP liquid medium for 24 hours at 29°C. The isolated strain was stored in a deep freezer in 10% glycerol (Fujifilm Wako Pure Chemical Industries, Ltd.).

[0053] Using the above method, several fructophilic lactic acid bacteria strains were isolated, one of which was strain HRL3.

[0054] (Cultivation and preparation of killed cells) The frozen HRL3 strain was suspended in 5 mL of FYP liquid medium and subjected to static culture at 30°C under anaerobic conditions for 24 or 48 hours. The suspension was then centrifuged at 21,500 × g for 15 minutes, washed twice with Gibco PBS (Thermo Fisher Scientific, USA), and sterilized by heating at 65°C for 30 minutes to obtain killed cells. The killed HRL3 strain was freeze-dried using a freeze dryer (FDU-1200, Tokyo Rikakikai Co., Ltd.), weighed, and suspended in PBS to prepare a killed cell solution with a cell concentration of 1 mg / mL. In Example 2 described below, killed cells of the HRL3 strain were cultured in FYP liquid medium for 48 hours. In Example 3 described below, killed cells of the HRL3 strain were cultured in FYP liquid medium for 24 hours.

[0055] [Example 2] Evaluation of the IFN-α production-inducing and IL-12 production-inducing activity of the HRL3 strain (Spleen cell preparation) First, C57BL / 6 mice (CLEA Japan, Inc.) were euthanized by cervical dislocation and their spleens were removed. Spleen cells were then isolated from the spleens, and the cell suspension was passed through a 40 μm cell strainer (AS ONE Corporation). To remove red blood cells, the cells were treated with 0.17 M Tris-HCl buffer (pH = 7.65, FUJIFILM Wako Pure Chemical Corporation) containing 0.83% NH4Cl (FUJIFILM Wako Pure Chemical Corporation). For cell culture, RPMI-1640 medium (FUJIFILM Wako Pure Chemical Corporation) containing 10% fetal bovine serum (FBS) (SERANA, Germany) and penicillin-streptomycin solution (Sigma-Aldrich, USA) was used.

[0056] Spleen cells are rich in immune cells such as dendritic cells (pDCs and mDCs), macrophages, and T cells, and are relatively easy to obtain, making them suitable for early-stage experiments investigating the effects of certain substances on immune cells.

[0057] (Evaluation of IFN-α production induction and activation effects and IL-12 production induction and activation effects) 5 × 10 spleen cells were plated in a 96-well flat-bottom plate (Nunc, Denmark). 5Cells were seeded at 1000 cells / well, and CpG-ODN1668 (final concentration 100 ng / mL, Hokkaido System Science Co., Ltd.), LPS (final concentration 1 μg / mL, Sigma-Aldrich, USA), killed Lactobacillus plantarum JCM1149 cells (final concentration 100 μg / mL), and killed Lactobacillus plantarum HRL3 cells (final concentration 100 μg / mL) were added to separate wells. Cultures were then incubated for 24 hours at 37°C in the presence of 5% CO2. After incubation, the production of IFN-α and IL-12p70 in the culture supernatant was measured by ELISA. The IFN-α assay kit used was ELISA Flex:Mouse IFN-α (HRP) (MABTECH, USA). The IL-12p70 assay kit used was Mouse IL-12p70 ELISA Ready-SET-Go! (eBioscience, USA). All measurements were carried out according to the protocol recommended by the kit.

[0058] CpG-ODN1668 is a ligand of TLR9, and LPS (lipopolysaccharide) is a component of the outer membrane of the cell wall of Gram-negative bacteria. In this test, CpG-ODN1668 and LPS were used as controls (positive controls). In addition, Lactobacillus plantarum JCM1149 strain is a reference strain of Lactobacillus plantarum, and was used to compare its action with that of HRL3 strain. In addition, the killed cells of Lactobacillus plantarum JCM1149 strain were obtained by the same method as that of the killed cells of HRL3 strain (see the section "Cultivation and killed cell preparation" in Example 1).

[0059] (Evaluation results) Figure 3 is a bar graph showing the evaluation results of the IFN-α production induction activation activity and IL-12 production induction activation activity of the HRL3 strain. Figure 3(a) is a bar graph showing the IFN-α production induction activation activity, and Figure 3(b) is a bar graph showing the IL-12 production induction activation activity. In Figure 3, CpG-ODN1668 is described as "CpG-ODN", and Lactobacillus plantarum JCM1149 strain is described as "JCM1149". In Figure 3 and Figure 4 described below, significant differences (P<0.05) are indicated with an asterisk (*).

[0060] As shown in Figure 3, it was confirmed that the HRL3 strain has significant IFN-α production-inducing and IL-12 production-inducing activity. IFN-α is produced by plasmacytoid dendritic cells (pDCs), and IL-12 is produced by myeloid dendritic cells (mDCs). Therefore, it was found that killed cells of the HRL3 strain affect these dendritic cells. It was also confirmed that the HRL3 strain has significantly higher IFN-α production-inducing and IL-12 production-inducing activity than the common lactic acid bacterium Lactobacillus plantarum JCM1149 strain.

[0061] Therefore, the HRL3 strain, lactic acid bacteria classified as Weissella sibaria, which has high genetic homology with the HRL3 strain, and lactic acid bacteria of the Weissella genus are expected to be active ingredients in IFN-α production inducer activators, IL-12 production inducer activators, antiviral activators, and immunostimulants.

[0062] [Example 3] Evaluation of the effect of nuclease treatment on the IFN-α production-inducing and IL-12 production-inducing activity of the HRL3 strain Next, to investigate the basis of the IFN-α production-inducing and IL-12 production-inducing activity of the HRL3 strain, the effect of nuclease treatment was evaluated.

[0063] (Nuclease treatment and evaluation of IFN-α production induction and activation effects and IL-12 production induction and activation effects) Ribonuclease A (10 μg / mL, Nippon Gene Co., Ltd.), an RNase, or DNase I (20 U / mL, Nippon Gene Co., Ltd.), a DNase, was added to killed cells of the HRL3 strain and allowed to react for 2 hours at 37° C. to prepare samples. The samples were then used to measure the amounts of IFN-α and IL-12p70 produced by the same method as in Example 2 for evaluating the IFN-α production-inducing and IL-12 production-inducing activation activity.

[0064] For the RNase reaction, 10 mM Tris-HCl (pH = 8.0) (Fujifilm Wako Pure Chemical Corporation) was used as the buffer (RNase A buffer). For the DNase reaction, DNase reaction buffer (Nippon Gene Co., Ltd.) and RNase-free water (Sigma-Aldrich, USA) were used as the buffer (DNase I buffer). In this test, to examine the effect of the buffer, a sample using only the buffer (no RNase or DNase) was also prepared.

[0065] (Evaluation results) Figure 4 is a bar graph showing the results of evaluating the effect of nuclease treatment on the IFN-α production-inducing and IL-12 production-inducing activity of the HRL3 strain. Figure 4(a) is a bar graph showing the IFN-α production-inducing and IL-12 production-inducing activity, and Figure 4(b) is a bar graph showing the IL-12 production-inducing and activating activity. Note that "HRL3" at the bottom of each bar graph indicates the presence or absence of the HRL3 strain (+: present, -: absent; similarly for other items). "RNase A buffer" indicates the presence or absence of a buffer for the RNase reaction. "RNase A" indicates the presence or absence of an RNase. "DNase I buffer" indicates the presence or absence of a buffer for the DNase reaction. "DNase I" indicates the presence or absence of a DNase.

[0066] As shown in Figure 4(a), it was confirmed that the amount of IFN-α produced by the HRL3 strain was reduced both when RNA and DNA were degraded. Furthermore, it was confirmed that the amount of IFN-α produced was reduced more significantly when DNA was degraded than when RNA was degraded. In other words, it was confirmed that nucleic acids (especially DNA) are strongly involved in the IFN-α production-inducing activity of the HRL3 strain.

[0067] Furthermore, as shown in Figure 4(b), it was confirmed that the amount of IL-12 produced by the HRL3 cell line was reduced to almost zero when RNA was degraded, but was not significantly reduced when DNA was degraded. In other words, it was confirmed that RNA, among nucleic acids, is strongly involved in the IL-12 production-inducing activity of the HRL3 cell line.

[0068] From the above results, it can be said that when lactic acid bacteria classified as HRL3 strain, Weissella cibaria, and lactic acid bacteria of the genus Weissella are used as active ingredients of IFN-α production inducer activators, IL-12 production inducer activators, antiviral activators, and immunostimulants, it is preferable to contain nucleic acids derived from these lactic acid bacteria as the active ingredients.

[0069] It has been known for some time that lactic acid bacteria and their nucleic acids can be used to promote IFN-α production or IL-12 production by acting on dendritic cells. For example, Japanese Patent Application Laid-Open No. 2016-73314 describes that lactic acid bacteria, which are cocci classified as Lactococcus Lactis subsp. Lactis, and their nucleic acids activate pDCs to promote IFN-α production.

[0070] However, it is not known that lactic acid bacteria of the genus Weissella, particularly those classified as Weissella sibaria, activate the induction of IFN-α production in pDCs and IL-12 production in mDCs, and thus have antiviral activation effects through these actions. Furthermore, the above-mentioned JP 2016-73314 A describes that the ability to induce IFN-α production in pDCs is not present in most strains and is not a universal activity.

[0071] Furthermore, JP 2016-73314 A suggests that the ability to induce IFN-α production in pDCs is a characteristic property of cocci. Since lactic acid bacteria of the genus Weissella are classified as short bacilli rather than cocci, the present invention is considered to have specificity from this perspective as well. Note that the tests in the above examples differ from the tests described in JP 2016-73314 A. Therefore, it should be noted that the values ​​described in the above examples cannot be simply compared with the values ​​described in JP 2016-73314 A.

[0072] (Prescription Example 1: Tablets) Tablets can be produced by mixing the following ingredients and compressing them according to a conventional method. The active ingredient composition used is killed cells of the HRL3 strain obtained in Example 1. (composition) Active ingredient composition (Example 1) 150 mg Cellulose 80mg Starch 20mg Sucrose fatty acid ester 2mg

[0073] (Formulation Example 2: Capsules) The following ingredients are mixed according to a conventional method and filled into a capsule base containing gelatin and glycerin to obtain soft capsules. The active ingredient composition uses killed cells of the HRL3 strain obtained in Example 1. composition Active ingredient composition (Example 1) 100 mg Beeswax 10mg Grape seed oil 110mg

Claims

1. An IFN-α production inducer activator that contains at least one of the cells of a lactic acid bacterium belonging to the genus Weissella and a processed cell product of the lactic acid bacterium as an active ingredient, and activates the induction of interferon-α (IFN-α) production in plasmacytoid dendritic cells (pDCs).

2. 2. The IFN-α production inducer activator according to claim 1, wherein the lactic acid bacterium is classified as Weissella cibaria.

3. 3. The IFN-α production inducer activator according to claim 2, wherein the lactic acid bacterium is Weissella cibaria HRL3 strain isolated from Hakutsuru Reishi and deposited under accession number NITE P-04107.

4. The IFN-α production inducer activator according to claim 1, wherein the bacterial cells and the treated bacterial cells contain nucleic acid derived from the lactic acid bacteria.

5. The IFN-α production inducer activator according to any one of claims 1 to 4, which is in the form of a food or drink, a pharmaceutical product, a feed, or an active ingredient composition to be incorporated therein.

6. An IL-12 production inducer activator that contains at least one of the cells of a lactic acid bacterium belonging to the genus Weissella and a processed cell product of the lactic acid bacterium as an active ingredient, and activates the induction of interleukin-12 (IL-12) production in myeloid dendritic cells (mDCs).

7. The IL-12 production inducer activator according to claim 6, wherein the bacterial cells and the treated bacterial cells contain ribonucleic acid (RNA) derived from the lactic acid bacteria.

8. The IL-12 production inducer activator according to claim 6 or 7, which is in the form of a food, drink, pharmaceutical, feed, or an active ingredient composition to be incorporated therein.

9. An antiviral activator comprising at least one of the cells of a lactic acid bacterium belonging to the genus Weissella and a processed cell product of the lactic acid bacterium as an active ingredient, which activates the antiviral properties of the living body by activating the induction of interferon-α (IFN-α) production in plasmacytoid dendritic cells (pDCs), or activates the induction of interleukin-12 (IL-12) production in myeloid dendritic cells (mDCs).

10. The antiviral activator according to claim 9 , wherein the bacterial cells and the treated bacterial cells contain nucleic acid derived from the lactic acid bacteria.

11. The antiviral activator according to claim 9 or 10, which is in the form of a food or drink, a pharmaceutical product, a feed, or an active ingredient composition to be incorporated therein.

12. An immunostimulant comprising at least one of the cells of a lactic acid bacterium belonging to the genus Weissella and a processed cell product of said lactic acid bacterium as an active ingredient, which activates the immune system of a living body by activating the induction of interferon-α (IFN-α) production in plasmacytoid dendritic cells (pDCs), or activates the induction of interleukin-12 (IL-12) production in myeloid dendritic cells (mDCs).

13. The immunostimulating agent according to claim 12 , wherein the bacterial cells and the treated bacterial cells contain nucleic acid derived from the lactic acid bacteria.

14. The immunostimulant according to claim 12 or 13, which is in the form of a food or drink, a pharmaceutical product, a feed, or an active ingredient composition to be incorporated therein.

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