Novel lactic acid bacteria, and composition, food, drink and pharmaceutical product containing lactic acid bacteria

Abstract

Provided are: a lactic acid bacterium which, by enhancing the production of IL-12p70 and IFN-Lambda, can improve resistance against infection by infectious microorganisms or viruses, alleviate stress, and suppress excessive inflammatory reactions that cause deterioration of symptoms; also provided are: a composition for immunoactivation, a composition for relieving stress, and a composition for suppressing inflammation, each of which contains the lactic acid bacteria; and a food, drink, or pharmaceutical product containing the compositions. The lactobacillus sake MG-LAB279 strain (the preservation number is NITE BP-03645) is adopted as a raw material, and the lactobacillus sake MG-LAB279 strain (the preservation number is NITE BP-03645) is adopted as an auxiliary material; a composition for immunoactivation, a composition for relieving stress, and a composition for inhibiting inflammation, each of which contains the strain or a cell component thereof as an active ingredient; and foods, drinks and pharmaceuticals containing these compositions.

Description

Technical Field

[0001] The present invention relates to a novel lactic acid bacterium, namely, Lactobacillus sakei MG-LAB279 strain (accession number: NITE BP-03645), and a composition, food and drink, and medicine containing the lactic acid bacterium. Background Art

[0002] Interferon is a type of cytokine family, and has been identified as a factor that inhibits viral infection. It is now clear that it has immunomodulatory effects including antiviral effects, and also exhibits various activities such as anti-tumor or anti-cancer, and cell proliferation inhibition. In humans, interferon is divided into type I interferon, type II interferon, and type III interferon. IFN-λ is a cytokine discovered in 2003 and belongs to type III interferon. Its subtypes are known to include IFN-λ1 (also known as IL-29), IFN-λ2 (also known as IL-28A), and IFN-λ3 (also known as IL-28B). Regarding IFN-λ, it has been confirmed that it is produced by dendritic cells, hepatocytes, intestinal epithelial cells, lung epithelial cells, etc., and like type I interferon, it has the function of activating natural immunity (the natural immunity is a system that protects the organism from infection by bacteria or viruses, etc.).

[0003] Like type I interferon, IFN-λ induces the expression of IFN-induced gene group (ISG) and inhibits viral infection. It is also known that IFN-λ can enhance the antiviral effect by acting synergistically with type I interferon. So far, it is believed that IFN-λ has functions that overlap with type I interferon. In recent years, it has attracted much attention to show antiviral activity by acting independently. Since the receptors of type I interferon are expressed in all cells, they cause inflammation throughout the body along with immune response, so excessive inflammatory reactions occur. Excessive inflammatory reactions accompanied by viral infection become the cause of serious symptoms such as multiple organ dysfunction, so it is important to suppress excessive inflammatory reactions throughout the body. On the other hand, the receptors of IFN-λ are only present in the above-mentioned hepatocytes, intestinal epithelial cells, lung epithelial cells, etc., so it can also be expected to impart selective physiological activity to the ingestor and suppress its side effects.

[0004] The immune response in the body is greatly affected by the balance of inflammatory and anti-inflammatory substances. For example, inflammatory cytokines such as interleukin-6 (IL-6) and tumor necrosis factor (TNFα) are involved in the initial response to infectious microorganisms and can amplify the inflammatory response. On the other hand, anti-inflammatory cytokines such as IL-10 have the effect of suppressing inflammatory responses. Normally, the immune response stops when the infection is eliminated, but if the regulatory function is lost, it will cause excessive production of immune cytokines, causing systemic damage to host cells (immune storm). Factors identified as the main causes of immune storms are known to include IL-6, TNFα, interferon, etc. In order to maintain the homeostasis of the immune response, it is necessary to regulate the production of inflammatory cytokines and anti-inflammatory cytokines.

[0005] Interleukin-12p70 (IL-12p70) has the effects of inducing differentiation from helper T precursor cells (Th0) to helper T cell type 1 (Th1) or activating natural killer cells (NK cells). Th1 cells increase the activity of phagocytes such as T cells or monocytes by producing interleukin-2 (IL-2) and interferon-γ (IFN-γ), thereby improving resistance to infection caused by viruses or bacteria. In addition, NK cells have the ability to specifically kill cancer cells or virus-infected cells. Therefore, by enhancing the production of IL-12p70, resistance to viral infection can be improved, and cancer prevention can be expected. Improving resistance to viral infection means improving, alleviating or treating the symptoms of future viral diseases, or preventing, suppressing or delaying the state of suffering from viral diseases.

[0006] Resistance to viral infection also affects stress status. Stress is generated by responding to various harmful stimuli (stressors) of various organisms. Modern people are subjected to various stresses such as changes in living or working environments, complex interpersonal relationships, physical and mental fatigue, etc. It is known that if an organism responds to stress, the activity of the sympathetic nerve-adrenal medulla system or the hypothalamus-pituitary-adrenal cortex system increases. Symptoms such as increased blood pressure, sweating, salivation, and awakening occur due to the increase in the sympathetic nerve-adrenal medulla system. It is known that due to the increase in the hypothalamus-pituitary-adrenal cortex system, for example, the secretion of adrenocortical stimulating hormone (ACTH) from the anterior pituitary gland is promoted, and the concentration of glucocorticoids such as cortisol, corticosterone, and cortisone in the blood increases. These glucocorticoids are generally called stress hormones. The effects of glucocorticoids are mostly related to the body's response to stress (activation of energy metabolism, promotion of cardiac function), and play an important role in maintaining homeostasis. On the other hand, however, it is known that glucocorticoids have adverse effects on the body by reducing the proliferation ability and function of immune cells, etc. Therefore, it is considered important to relieve stress in order to maintain immune function.

[0007] When improving the immune function for preventing viral infection, an immunostimulating composition having an immunostimulating effect is used. As an immunostimulating composition, a compound derived from nature is desirable. In particular, from the perspective of being able to be taken in daily life and having little concern about side effects, a food is desirable. As a food having an immunostimulating effect, polysaccharides of basidiomycetes represented by shiitake mushrooms or Agaricus bisporus, or edible microorganisms such as lactic acid bacteria, Aspergillus or yeast, or their cell wall components, etc. are known.

[0008] Patent document 1 states that the Lactococcus lactis subsp. Lactis JCM5805 strain promotes IFN-λ production by dendritic cells pDC. In addition, Patent document 2 states that the halophilic Tetragenococcus KK221 strain promotes IFN-λ production by dendritic cells BDCA3DC. Patent document 3 states that the IFN-λ production promoting effect of Bifidobacterium bacteria as an active ingredient is described. Patent document 4 states that the Lactobacillus paracasei MCC1375 strain has the effect of preventing influenza virus infection by promoting IL-12 production and activating NK cells.

[0009] Prior art literature

[0010] Patent Literature

[0011] Patent Document 1: Japanese Patent Application Publication No. 2017-201984

[0012] Patent Document 2: International Publication No. 2017 / 175774

[0013] Patent Document 3: Japanese Patent Application Publication No. 2019-167327

[0014] Patent Document 4: International Publication No. 2012 / 133827 Summary of the invention

[0015] Technical problem to be solved by the invention

[0016] However, although preparations or medicines for regulating immune function that use lactic acid bacteria such as Lactobacillus or Lactococcus as active ingredients have been disclosed, on the other hand, there are no disclosed strains that use lactic acid bacteria or compositions derived from lactic acid bacteria as main ingredients and have the effect of enhancing the production of IL-12p70 and IFN-λ to sufficiently improve resistance to viral infections, and food and beverages with a high effect of preventing viral infections have not yet been developed.

[0017] The technical problem to be solved by the present invention is to provide an immune activation composition, a stress relief composition, an inflammation suppression composition, etc., which can effectively improve, alleviate or treat the symptoms of viral diseases, or prevent, inhibit or delay the state of suffering from viral diseases by enhancing the production of IL-12p70 and IFN-λ and activating natural immunity.

[0018] Technical solutions for solving technical problems

[0019] In order to solve the above technical problems, the inventors of the present invention have repeatedly conducted intensive studies and found that the sake Lactobacillus MG-LAB279 strain can enhance the production of IL-12p70 and IFN-λ and has excellent viral infection prevention effects, thereby completing the present invention.

[0020] In order to achieve the above object, the present invention provides the following lactic acid bacteria, and a composition, food, drink, and medicine containing the same.

[0021] [1] A Lactobacillus sakei MG-LAB279 strain, with the accession number being NITE BP-03645.

[0022] [2] A composition comprising the strain and / or its bacterial components described in [1] above.

[0023] [3] An immunostimulating composition comprising the strain described in [1] above and / or its bacterial components as an active ingredient.

[0024] [4] The immunostimulating composition according to [3] above, which does not cause inflammation.

[0025] [5] A composition for preventing or treating viral infection, comprising the strain and / or its bacterial component described in [1] above as an active ingredient.

[0026] [6] A composition for activating NK cells, comprising the strain described in [1] above and / or its bacterial components as an active ingredient.

[0027] [7] A composition for promoting interferon λ production, comprising the strain and / or its bacterial component described in [1] above as an active ingredient.

[0028] [8] A composition for promoting interleukin-12p70 production, comprising the strain described in [1] above and / or its bacterial components as an active ingredient.

[0029] [9] A composition for alleviating stress, comprising the strain described in [1] above and / or its bacterial components as an active ingredient.

[0030]

[10] A composition for suppressing inflammation, comprising the strain and / or its bacterial component described in [1] above as an active ingredient.

[0031]

[11] A skin-improving composition comprising the strain and / or its bacterial component described in [1] above as an active ingredient.

[0032]

[12] A food or beverage comprising the MG-LAB279 strain described in [1] above or the composition described in any one of [2] to

[11] above.

[0033]

[13] A pharmaceutical product comprising the MG-LAB279 strain described in [1] above or the composition described in any one of [2] to

[11] above.

[0034] Effects of the Invention

[0035] The present invention can provide a lactic acid bacterium and an immunostimulating composition, a stress relieving composition, an inflammation suppressing composition and the like containing the lactic acid bacterium, as well as a food, beverage and a medicine containing these compositions, which can improve resistance to infection caused by infectious microorganisms or viruses, relieve stress and suppress excessive inflammatory reactions that cause worsening of symptoms by enhancing the production of IL-12p70 and IFN-λ. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] Figure 1 This is a diagram showing the measurement results (IFN-λ production amount) of Test Examples 2 and 3 in a graph.

[0037] Figure 2A This is a diagram showing the measurement and calculation results (MX1 gene expression level) of Test Example 2 in a graphical form.

[0038] Figure 2B This is a diagram graphically showing the measurement and calculation results (OAS1 gene expression level) of Test Example 2.

[0039] Figure 3 This is a diagram showing the measurement results of Test Example 4 (Result 1: serum corticosterone concentration).

[0040] Figure 4 This is a diagram showing the measurement and calculation results of Test Example 4 (Result 2: IFN-λ gene expression level) in a graph.

[0041] Figure 5 This is a diagram showing the measurement results of Test Example 4 (Result 3: TNFα concentration in serum).

[0042] Figure 6 This is a diagram showing the observation results of Test Example 5 (Result 1: General condition score after influenza virus infection) in a graph.

[0043] Figure 7 This is a diagram showing the observation results of Test Example 5 (Result 2: survival rate after influenza virus infection) in a graph.

[0044] Figure 8 This is a diagram showing the measurement results of Test Example 6 (Result 3: number of viruses in the lungs) in a graph.

[0045] Fig. 9 This is a diagram showing the measurement results of Test Example 6 (Result 4: NK activity) in a graph.

[0046] Fig.10 This is a diagram showing the observation results of Test Example 7 (Result 1: General condition score after influenza virus infection) in a graph.

[0047] Fig.11 This is a diagram showing the observation results of Test Example 7 (Result 2: survival rate after influenza virus infection) in a graph.

[0048] Fig.12 This is a diagram showing the measurement results of Test Example 8 (Result 3: number of viruses in the lungs) in a graph.

[0049] Fig.13 This is a diagram showing the measurement results of Test Example 8 (Result 4: NK activity) in a graph.

[0050] Fig.14A This is a diagram showing the observation results of Test Example 8 (Result 5: lung lesions in the bronchus) in a graph.

[0051] Fig. 14BThis is a diagram showing the observation results of Test Example 8 (Result 5: lung lesions in alveoli) in a graph.

[0052] Fig.15 This is a diagram showing the measurement results (IFN-λ production amount) of Test Example 9 in a graph.

[0053] Fig.16 This is a diagram showing the measurement results (IL-10 concentration) of Test Example 10 in a graph.

[0054] Fig.17 This is a diagram showing the measurement results of Test Example 11 (Result 1: Body weight change rate after induction of colitis) in a graph.

[0055] Fig.18 This is a diagram showing the measurement results of Test Example 11 (Result 2: large intestine length after induction of colitis).

[0056] Fig.19 This is a diagram showing the observation results of Test Example 11 (Result 2: colitis symptom score after colitis induction) in a graph. DETAILED DESCRIPTION

[0057] Hereinafter, a specific embodiment of the present invention (hereinafter referred to as “the present embodiment”) will be described in detail, but the present invention is not limited thereto and various modifications can be made without departing from the gist of the present invention.

[0058] The MG-LAB279 strain belonging to Lactobacillus sakei according to the present embodiment (hereinafter also referred to as “bacteria”) is a lactic acid bacterium deposited in the NPMD-National Institute for Technology Evaluation Patent Microorganism Depository on May 2, 2022 with the accession number: NITE BP-03645.

[0059] The MG-LAB279 strain is characterized in that it promotes the production of interferon-λ (IFN-λ) and interleukin-12p70 (IL-12p70).

[0060] MG-LAB279 strain can be provided in the form of a composition containing it. As a composition, there can be listed compositions for immune activation (for example, compositions for preventing or treating viral infections, compositions for NK cell activation, compositions for promoting interferon λ production, compositions for promoting interleukin-12p70 production), compositions for stress relief, compositions for suppressing inflammation, and compositions for improving skin, but they are not limited to these. These can be used in food, beverages, medicines, feeds, etc. The following is a detailed description.

[0061] The composition for immune activation, stress relief, and inflammation suppression of the present embodiment contains Lactobacillus sakei MG-LAB279 strain as an active ingredient. The Lactobacillus sakei MG-LAB279 strain used in the composition of the present embodiment may be a live cell, a dead cell, or both, preferably a dead cell.

[0062] The inventors of the present invention selected strains with excellent flavor and physical properties when applied to food from a variety of lactic acid bacteria in plants or fermented foods and humans, and thus selected the sake Lactobacillus MG-LAB279 strain. After research, the inventors of the present invention found that the production of IL-12p70 and IFN-λ was improved by taking the sake Lactobacillus MG-LAB279 strain. Therefore, by taking the sake Lactobacillus MG-LAB279 strain, it can be expected that natural immunity will be activated and resistance to diseases caused by viruses and the like will be improved.

[0063] Furthermore, the compositions such as the immunoactivating composition, stress relieving composition, and inflammation suppressing composition of the present embodiment use compositions derived from the natural lactic acid bacteria Lactobacillus sakei MG-LAB279 strain that exists in nature, and therefore have a very high safety effect.

[0064] As a medium for culturing Lactobacillus sakei MG-LAB279 strain, various mediums can be used, such as milk medium, medium containing milk components, semi-synthetic medium not containing these, etc. As such a medium, a reduced skim milk medium obtained by reducing skim milk and sterilizing it by heating can be exemplified.

[0065] The culture method of these sake Lactobacillus MG-LAB279 strains is carried out by static culture or neutralization culture with constant pH control, and there is no particular limitation on the culture method as long as the conditions are such that the bacteria grow well. Regarding the bacterial bodies, the bacterial bodies obtained by culturing according to the conventional method for culturing lactic acid bacteria and separating from the obtained culture by a collection method such as centrifugation can be directly used.

[0066] In addition, dead cells can be generally obtained by heating the cells. The heating conditions are not particularly limited as long as the cells die, but generally, sufficient effects can be obtained by heating at 90°C for about 30 minutes.

[0067] For the sake Lactobacillus MG-LAB279 strain contained in the compositions such as the immune activation composition, stress relief composition and inflammation suppression composition of the present embodiment, as a bacterial body, it is not limited to the bacterial body obtained by purification and separation, but may also be its culture or suspension, or other bacterial body-containing substances. It may also be bacterial components such as cytoplasmic components such as nucleic acids or cell wall parts obtained by treating the bacterial body with enzymes or physical methods. These can be used alone or in combination. Therefore, as the dead bacterial body of sake Lactobacillus MG-LAB279 strain, it may also be a culture or the bacterial body contained therein itself. In this case, the culture or the bacterial body contained therein itself may also be formulated in the food and drink products and medicines containing the compositions such as the immune activation composition, stress relief composition and inflammation suppression composition of the present embodiment.

[0068] In addition, in the composition for immune activation, stress relief, and inflammation suppression of the present embodiment, the bacteria can also be mixed with dextrin for easy handling. In this case, the composition for immune activation, stress relief, and inflammation suppression of the present embodiment can be used in the form of a composition containing 0.1 to 99% by mass of dead bacteria of Lactobacillus sakei MG-LAB279 strain and 1 to 99.9% by mass of dextrin.

[0069] The live bacteria and / or dead bacteria of Lactobacillus sakei MG-LAB279 strain can be formulated in any food and beverage in the form of a composition such as an immune activation composition, a stress relief composition, and an inflammation suppression composition, or can be added to raw materials in the manufacturing process of food and beverage. Examples of food and beverage include milk drinks, fermented milk, fruit juice drinks, soft drinks, jelly, candy, chocolate, dairy products, egg processed products such as mayonnaise, desserts such as cream cakes, bread, etc.

[0070] In addition, Lactobacillus sakei MG-LAB279 strain and / or its dead cells or cultured products can be formulated into any feed in the form of a composition such as an immunoactivating composition, a stress-relieving composition, and an inflammation-suppressing composition contained in livestock feed, or can be added to raw materials during the feed production process.

[0071] For the compositions such as the immune activation composition, stress relief composition and inflammation suppression composition of this embodiment, there are no special restrictions when formulating them as long as the sake Lactobacillus MG-LAB279 strain (dead bacteria, etc.) is used as the active ingredient. Excipients, stabilizers, flavoring agents, etc. allowed in the formulation can be appropriately mixed and can be formulated according to conventional methods.

[0072] In addition, for compositions such as the immune activation composition, stress relief composition, and inflammation suppression composition of the present embodiment, excipients, binders, disintegrants, lubricants, flavoring agents, suspending agents, coating agents, and any other drugs can be mixed to prepare formulations within the range that does not hinder the action of bacteria and cultures.

[0073] The compositions such as the immunoactivating composition, stress relieving composition and inflammation suppressing composition of the present embodiment can be used in a wide range of applications such as pharmaceuticals, quasi-pharmaceuticals, cosmetics, and food and beverages. These compositions can be tablets, pills, capsules, granules, powders, syrups, etc., and it is desirable to administer them orally.

[0074] In order to exert the immune activation effect, stress relief effect, inflammation suppression effect, etc., in the case of adults, the dosage can be adjusted so that the dead cells of Lactobacillus sakei MG-LAB279 strain are taken in an amount of 0.1 to 10 g / day, preferably 0.5 to 5 g / day. There is no particular limitation on the content ratio of lactic acid bacteria, and it can be appropriately adjusted according to its use, ease of production, or preferred daily dosage.

[0075] The immunoactivation composition of the present embodiment can achieve immunoactivation without causing inflammation, and in this respect, it subverts the technical common sense that inflammation is induced during immunoactivation, and is more useful than the existing immunoactivation composition accompanied by inflammation. Among them, "not causing inflammation" mentioned in the present embodiment includes the following situation: the inflammation caused in the human or animal who ingested the immunoactivation composition does not reach the level where the side effects accompanied by inflammation can be observed. Regarding "not causing inflammation", for example, it can be confirmed as follows: as in Test Example 8 described later, in the test using inflammation parameters represented by pathological histological examination, no increase in the score indicating the degree of inflammation is observed compared with the control group (control group) treated with viral infection (no significant difference).

[0076] Example

[0077] Hereinafter, the present invention will be described in more detail with reference to Examples and Test Examples. However, these are merely illustrative and the present invention is not limited thereto.

[0078] 〔Identification of strains〕

[0079] The identification of the strain can be performed by analyzing the 16S rRNA gene according to the method described in "The Science of Lactic Acid Bacteria and Bifidobacteria" edited by the Japanese Society of Lactic Acid Bacteria (Kyoto University Academic Press) (analysis can also be performed on a commission basis).

[0080] The 16S rRNA gene of MG-LAB279 strain was deciphered by the known method described above. The deciphered 16S rRNA gene of MG-LAB279 strain had 99.9% homology with that of Lactobacillus sakei subsp. sakei DSM20017 strain, indicating that this strain belongs to Lactobacillus sakei strain.

[0081] [Test Example 1]

[0082] [Study on the promotion of IL-12p70 production in mouse spleen cells]

[0083] Spleen cells collected from the spleen of 6-week-old BALB / c male mice were adjusted to 5.0 × 10 cells / mL using RPMI1640 (GIBCO) medium containing 10% fetal bovine serum (FBS, BIOWEST), antibiotics (penicillin / streptomycin) 100 units / mL (GIBCO), and 50 μM mercaptoethanol (hereinafter referred to as RPMI1640 medium). 4 Cells / well were seeded in a 96-well plate (IWAKI). Various 21 unknown lactic acid strains including Lactobacillus sakei MG-LAB279 were added to 20 μg / mL and cultured separately (no lactic acid strains were added to the control group). Culture was carried out at 37°C and 5% CO2.

[0084] (ELISA test)

[0085] 100 μL of supernatant from mouse spleen cells cultured for 48 hours was collected from each 96-well culture plate and analyzed by ELISA. Washing in ELISA detection was performed using PBS buffer containing 0.05% Tween20 and plate washing solution (BIO-RAD, Immuno Wash1575). 100 μL of 0.8 μg / mL anti-mouse IL-12B monoclonal antibody (SIN Sino Biological) was added to the 96-well immunoplate (Thermo) and allowed to stand overnight at 4°C to solidify. After washing, 350 μL of 5% skim milk (Wako) was added and allowed to stand overnight at 4°C for blocking. After washing, 50 μL of culture supernatant was added, reacted at 37°C for 2 hours, washed, and then 100 μL of 0.8 μg / mL biotin-labeled anti-mouse IL-12 (p35) monoclonal antibody (MABMabtechAB) was added and labeled at 37°C for 2 hours. After washing, add 100 μL of 0.8 μg / mL HRP-streptavidin complex (Streptavidin complex, manufactured by Amersham) and react at room temperature for 1 hour. After washing, add 100 μL of TMB substrate (Sera Care), perform enzyme reaction, and then add 100 μL of 0.3 M sulfuric acid to stop the reaction. IL-12p70 was detected based on the absorption at 450 nm using a microplate reader (Perkin Elmer, ARVO X3). In addition, the amount of IL-12 produced was calculated based on a calibration curve prepared using an IL-12p70 standard (Pepro Tech). The measurement results are shown in Table 1 below (only the results of three lactic acid strains including the MG-LAB279 strain with the highest IL-12p70 production are shown. The results of the SHIELD lactic acid bacteria of Test Example 3 are also shown). Among them, the remaining two lactic acid bacteria other than the MG-LAB279 strain shown in Table 1 are the MG-LAB302 strain (deposit number: NITE BP-03646) and the MG-LAB533 strain (deposit number: NITE BP-03647) deposited in the NPMD-National Institute for Technology Evaluation Patent Microorganism Depository on May 2, 2022.

[0086] [Table 1]

[0087]

[0088] The results showed that the Lactobacillus sakei MG-LAB279 strain most strongly induced the production of IL-12p70 among the 21 lactic acid strains evaluated.

[0089] [Test Example 2]

[0090] [Study on the promotion of IFN-λ production in human epithelial cells]

[0091] (Preparation of samples for extraction of lactic acid bacteria nucleic acid)

[0092] Each lactic acid strain (the same 21 species as in Test Example 1) that was frozen and stored was inoculated into 5 mL of MRS medium (BDDifco) and cultured at 30°C overnight. After collecting the bacteria from these lactic acid bacteria culture solutions by centrifugation, water was added for re-suspending and centrifugation was performed to obtain bacterial particles. The nucleic acid components of these bacterial particles were extracted using a commercially available nucleic acid extraction kit (NucleoBond AXG column manufactured by Takara). Specifically, the above-mentioned bacterial particles were suspended in a buffer, and at least one lysozyme selected from proteinase K (proteinase K), lysozyme (Lysozyme, Wako Pure Chemical Industries, Ltd.), and Labiase (Ozeki Shuzo) was added and treated at 37°C overnight. Lysis buffer was added to these enzyme-treated solutions, treated at 50°C for 30 minutes, and the resulting treated solution was supplied to the column to obtain a nucleic acid eluate. An equal amount of isopropanol was added to these nucleic acid eluates, mixed by inversion, and the resulting mixed solution was centrifuged. The supernatant was discarded, 70% ethanol was added to the obtained precipitate, and the mixture was centrifuged. The precipitate after the supernatant was removed was dissolved in Nuclease Free Water (QIAGEN) to obtain nucleic acid extraction samples of each lactic acid strain.

[0093] (IFN-λ production test)

[0094] HT-29 cell line obtained from ECACC (European Collection of Cell Cultures) was cultured in a 10 cm culture dish using 10 mL of McCoy 5A (HyClone) medium (hereinafter referred to as McCoy medium) containing 10% fetal bovine serum (FBS, BIOWEST) and 100 units / mL of penicillin-streptomycin (Gibco). The culture was carried out at 37°C and 5% CO2. HT-29 cells cultured to 70-80% confluence were plated at 4.0×10 4Cells were seeded at a concentration of cells / well in a 96-well tissue culture flat-bottom plate (IWAKI) and cultured overnight. A total of 150 μL of McCoy's medium, 50 μL of McCoy's medium containing 1% lipofectamin 2000 (Lipofectamin 2000, invitrogen), and 50 μL of McCoy's medium containing the above-mentioned nucleic acid extraction test object adjusted to a nucleic acid concentration of 15 μg / mL were added to each well and cultured for 20 hours. Only 150 μL of McCoy's medium containing 0.33% lipofectamin 2000 was added to the sample without the addition of the test object, and it was used as a control group. 50 μL of McCoy's medium containing 0.6 μg / mL of Poly(:IC) (R&D Systems Inc.) was added instead of the nucleic acid from lactic acid bacteria, and it was used as a positive control group. The experiment was carried out three times in a row.

[0095] (ELISA test)

[0096] 100 μL of the supernatant of HT-29 cells cultured for 20 hours was recovered from each 96-well culture plate, and the production of each test body was quantified using an ELISA detection kit (DuoSet ELISA Development System Human IL-29 / IFN-λ1, R&D Systems Inc.). Specifically, after preparing the test body according to the attached protocol, the absorbance of each test body was measured at a wavelength of 450 nm, and the IFN-λ production was quantified using a calibration curve prepared using the standard sample attached to the detection kit. The measurement results are shown in Figure 1 (Only the results of the top three lactic acid strains producing the most IFN-λ are shown. The results of the SHIELD lactic acid bacteria of Test Example 3 are also shown.) The measured values ​​are shown as the average of three consecutive measurements, and the error bars are shown as standard errors.

[0097] like Figure 1 As shown, it was found that the Lactobacillus sakei MG-LAB279 strain most strongly induced IFN-λ production among the 21 lactic acid strains evaluated.

[0098] [Confirmation of viral infection prevention effects]

[0099] (Analysis of expression levels of MX1 and OAS1)

[0100] The test used nucleic acid extraction test subjects adjusted with Lactobacillus sakei MG-LAB279, Pediococcus pentosaceus MG-LAB533, and Lactobacillus plantarum MG-LAB302. Poly(:IC) (R&D Systems Inc.) was used as the positive control group. The culture was carried out in the same manner as the IFN-λ production test, and after removing the supernatant, the total RNA (total RNA) test subjects adjusted with the CellAmp Direct RNA Prep Kit for RT-PCR (CellAmp Direct RNA Prep Kit for RT-PCR, TAKARA) were used in the analysis. Expression level analysis by quantitative PCR used the one-step method PrimeScript TM RT-PCR Kit (One Step PrimeScript TM RT-PCR Kit, TAKARA) and quantitative PCR apparatus (TAKARA, TP-600). Primers of sequence 1 and sequence 2 in Table 2 were used for the determination of MX1, sequence 3 and sequence 4 were used for the determination of OAS1, and sequence 5 and sequence 6 were used for the determination of GAPDH. The gene expression levels of MX1 and OAS1 and GAPDH as a reference gene were measured, and the expression levels were evaluated by dividing the above gene expression levels by the reference gene expression levels to obtain relative values. The measurement results are shown in Figure 2A and Figure 2B The measured values ​​are shown as the average of four consecutive measurements, and the error bars are shown as standard errors.

[0101] [Table 2]

[0102] Serial Number Base sequence 5'→3' 1 Positive ACA GGA CCA TCG GAA TCT TG 2 Reverse CCC TTC TTC AGG TGG AAC AC 3 Positive TGT CCA AGG TGG TAA AGG GTG 4 Reverse CCG GCG ATT TAA CTG ATC CTG 5 Positive GAA GGT GAA GGT CGG AGT C 6 Reverse GAA GAT GGT GAT GGG ATT TC

[0103] like Figure 2A and Figure 2B As shown, it can be seen that the sake Lactobacillus MG-LAB279 strain strongly promotes the expression of MX1 and OAS1. MX1 and OAS1 are known to be proteins with interferon induction, and have the effect of decomposing RNA from viruses or inhibiting viral RNA replication. Therefore, it can be considered that the sake Lactobacillus MG-LAB279 strain has a viral infection prevention effect.

[0104] [Test Example 3]

[0105] [Comparison with SHIELD lactic acid bacteria (registered trademark)]

[0106] In Test Examples 1 and 2, SHIELD lactic acid bacteria (registered trademark), which has a large market share, was used as a comparative example and the test was conducted in the same manner. The measurement results are shown in Tables 1, 3 and Figure 1 middle.

[0107] [Table 3]

[0108]

[0109] According to Table 1, Table 3 and Figure 1 It is known that the lactic acid bacteria Lactobacillus sakei MG-LAB279 strain strongly induces the production of IL-12p70 and IFN-λ, and its activity is higher than that of SHIELD lactic acid bacteria (registered trademark) (Lactobacillus paracasei MCC1849 strain), which has a large market share. Therefore, it is known that the dead cells of the lactic acid bacteria Lactobacillus sakei MG-LAB279 strain can be used as a cellular immune activator.

[0110] [Test Example 4]

[0111] [Analysis of IFN-λ expression in intestinal lymph nodes using restraint stress mice]

[0112] (Raising mice)

[0113] The test used 8-week-old female C57BL / 6J mice (Charles River, Japan) after 1 week of pre-feeding. Animals were kept in a breeding room maintained at a management temperature of 20-26°C, a management humidity of 40-70% RH, and a light and dark cycle of 12 hours each (lighting: 7:00-19:00). During the pre-feeding period, sterilized polycarbonate flat-bottomed cages (213mm×324mm×131mm) with floor mats (Pal Soft, Oriental Yeast Industry) were used. In addition, considering the breeding environment, the floor mats were replaced twice a week.

[0114] (Grouping of mice)

[0115] The mice were divided into three groups, namely, a group given a normal diet (hereinafter referred to as the "control group"), a group given a normal diet and subjected to restraint stress (hereinafter referred to as the "stress group"), and a group administered with the lactic acid bacteria Lactobacillus sakei MG-LAB279 strain and subjected to restraint stress (hereinafter referred to as the "279 strain administration group"), with 3, 5, and 4 mice in each group.

[0116] (Test food and administration method)

[0117] The test food for the control group and the stress group was powdered feed (MF, Oriental Yeast Industry Co., Ltd.). The test food for the strain-administered group was the above-mentioned powdered feed mixed with 0.5% dead bacteria of the lactic acid bacteria Lactobacillus sakei MG-LAB279 strain. After grouping, the powdered feed (MF) or the feed mixed with the test substance was placed in a feeding machine (MF-4S, SHIN FACTORY Co., Ltd.) and allowed the mice to ingest it freely. For the feed, the feed was supplemented or replaced with new feed twice a week. The administration of the test food continued until the end of the experiment.

[0118] (Methods of coping with stress)

[0119] The 8-week-old C57BL / 6J mice of each group were allowed to eat the above feed freely and were raised for 12 days. The stress group and the 279-strain administration group were placed in a 50 mL conical tube on the 13th day of feeding, and were restrained with absorbent cotton and silicon plugs to prevent them from moving. In addition, considering the feeding environment, a stomata and an excretion port for removing feces and urine were provided on the conical tube for restraint in a manner that does not hinder breathing. The restraint was performed for 18 hours, during which they were raised in a water-free and food-free state. The control group was not restrained, and other than that, they were raised in the same way as other groups.

[0120] (Corticosterone determination in serum)

[0121] After the restraint was completed, autopsy was performed under isoflurane anesthesia, and whole blood was collected through the posterior vena cava. The obtained blood was injected into CAPIJECT II (TERUMO) and inverted 8 to 10 times for mixing. After standing at room temperature for 30 minutes, the blood was centrifuged at 3000g for 10 minutes to collect the serum. The corticosterone concentration in the obtained serum was measured using a corticosterone ELISA Kit (Corticosterone ELISA Kit, Cayman Chemical Company). The test was performed by comparing the two groups between the stress group and the 279-strain administration group (without corresponding t-test). The significance level was 5% (hazard rate). Figure 3 )

[0122] (Expression analysis using intestinal lymph nodes)

[0123] After the restraint was completed, autopsy was performed under isoflurane anesthesia to collect the mesenteric lymph nodes. The mesenteric lymph nodes were placed in a solution containing 1 mL of TRIzol TMThe cells were disrupted using a bead cell disruptor (TOMY) in a 2 mL volumetric tube containing reagent (Invitrogen) and zirconium oxide beads (TOMY). 0.2 mL of chloroform was added to the resulting cell solution and stirred. After the water layer was recovered, RNA from the intestinal membranous lymph nodes was obtained using the isopropanol precipitation method. Expression analysis using quantitative PCR was performed using the one-step PrimeScript TM RT-PCR Kit (One Step PrimeScript TM RT-PCR Kit, TAKARA) and quantitative PCR apparatus (TAKARA, TP-600). Primers of sequence 7 and sequence 8 in Table 4 were used for the determination of IFN-λ, and primers of sequence 9 and sequence 10 were used for the determination of RPL19. The gene expression levels of IFN-λ and RPL19 as a reference gene were measured, and the expression levels were evaluated by dividing the former by the latter to obtain a relative value.

[0124] [Table 4]

[0125] Serial Number Base sequence 5'→3' 7 Positive ACC CTG AAG GTC TGG GAG AAA 8 Reverse CTG TGG CCT GAA GCT GTG TA 9 Positive ATG AGT ATG CTC AGG CTA CAG A 10 Reverse GCA TTG GCG ATT TCA TTG GTC

[0126] (Determination of inflammatory cytokine TNFα in serum)

[0127] After the restraint was completed, autopsy was performed under isoflurane anesthesia, and the inflammatory cytokine TNFα in the serum prepared by whole blood collected from the posterior vena cava was measured using a Mouse TNFα ELISA Kit (Proteintech).

[0128] (Result 1: Effect of test food on serum corticosterone concentration)

[0129] like Figure 3 As shown, in the control group, the serum corticosterone concentration increased due to the restraint stress treatment compared with the untreated group. On the other hand, in the 279-strain administration group, although an increase in serum corticosterone concentration was also seen, it was significantly improved compared with the control group. Therefore, it can be seen that the lactic acid bacteria Lactobacillus sakei MG-LAB279 strain has the effect of alleviating (reducing) stress caused by physical or mental fatigue and causing immune dysfunction.

[0130] (Result 2: Effect of test food on IFN-λ expression in intestinal membranous lymph nodes)

[0131] The measurement results are shown in Figure 4. It was confirmed that the expression level of IFN-λ gene in the intestinal membranous lymph nodes was higher in the 279-strain intake group than in the control group and the stress group. That is, it was shown that even in a state where there is a concern about low immune function due to stress, oral intake of Lactobacillus sakei MG-LAB279 strain bacteria can enhance the expression of interferon-λ in the intestinal membranous lymph nodes.

[0132] (Result 3: Effect of test food on TNFα in serum)

[0133] The results of the measurement of inflammatory cytokine TNFα in serum are shown in Figure 5 .

[0134] like Figure 5 As shown, in the control group, the serum TNFα concentration increased due to the restraint stress treatment compared to the untreated group. On the other hand, an increase in serum corticosterone concentration was also seen in the 279-strain administration group, but the increase was suppressed compared to the control group. Therefore, it can be seen that the lactic acid bacteria Lactobacillus sakei MG-LAB279 strain not only has the effect of alleviating (reducing) stress that is the cause of immunodeficiency, but can also be expected to inhibit systemic inflammation associated with stress. In addition, since stress relief and inflammation suppression can be achieved, it can be expected that the improvement effect on skin problems such as skin cracking, dryness, and rough skin caused by stress or inflammation can be expected.

[0135] [Test Example 5]

[0136] [Evaluation of status scores and survival rates of influenza-infected mice (Part 1)]

[0137] (Raising mice)

[0138] The animals used in the experiment were 5-week-old female BALB / c strain (BALB / c Cr Slc) (Japan SLC Co., Ltd.) after 1 week of pre-feeding. The animals were kept in a breeding room maintained at a management temperature of 18-28°C, a management humidity of 30-80% RH, and a light and dark cycle of 12 hours each (lighting: 6:00-18:00). During the pre-feeding period, sterilized plastic cages (W: 175mm×D: 245mm×H: 125mm) with floor mats were used. In addition, considering the breeding environment, each cage was enriched with the environment (nesting sheets, ANIMEC Co., Ltd.).

[0139] (Grouping of mice)

[0140] The mice were divided into five groups, namely, an influenza virus infection untreated group (hereinafter referred to as the "untreated group"), a control group treated with influenza virus infection (hereinafter referred to as the "control group"), a group administered with lactic acid bacteria Lactobacillus sakei MG-LAB 279 strain before influenza virus infection treatment (hereinafter referred to as the "279 strain administration group"), a group administered with Pediococcus pentosaceus MG-LAB 533 strain before influenza virus infection treatment (hereinafter referred to as the "533 strain administration group"), and a group administered with Lactobacillus plantarum MG-LAB 302 strain before influenza virus infection treatment (hereinafter referred to as the "302 strain administration group"), with 8 mice in each group.

[0141] (Test food and administration method)

[0142] The test food for the untreated group and the control group was powdered feed (MF, Oriental Yeast Industry Co., Ltd.). The test food for the 279-strain administration group was powdered feed mixed with 0.5% dead bacteria of the lactic acid bacteria Lactobacillus sakei MG-LAB279 strain. The test food for the 533-strain administration group was powdered feed mixed with 0.5% dead bacteria of the lactic acid bacteria Pediococcus pentosaceus MG-LAB533 strain. The test food for the 302-strain administration group was powdered feed mixed with 0.5% dead bacteria of the lactic acid bacteria Lactobacillus plantarum MG-LAB302 strain. After grouping, the powdered feed (MF) or the test substance mixed feed was placed in the feeding machine, and the mice were allowed to ingest it freely. For the feed, it was replaced with new feed twice a week, and the leftover feed was discarded. The administration of the test food continued until the end of the experiment.

[0143] (How influenza viruses infect people)

[0144] Influenza virus PR8 [A / PR / 8 / 34 (H1N1)] was used to treat influenza virus infection in mice. The frozen inoculation virus stock solution was thawed before use and 2 × 10 3 PFU / mL, and used it as the inoculated virus solution. On the day of virus inoculation (15 days after the start of the experiment), 0.05 mL (1×10 2 PFU / mouse) was dripped into the nasal cavity. The virus solution was stirred before each inoculation.

[0145] (Weight measurement)

[0146] Body weight was measured once a week from the start of the test food intake to the day before virus inoculation (14th day from the start of the test), and body weight was measured every day using an electronic balance after the virus inoculation day (15th day from the start of the test). The body weight measured on the virus inoculation day was used as a reference. When a weight loss of more than 20% was observed, the animals were bled from the abdominal aorta under isoflurane anesthesia for euthanasia. Euthanized animals were excluded from the measurement objects.

[0147] (Determination of general status score)

[0148] The general condition was observed once every day after the day of virus ingestion. The general condition score was calculated as the total score of each site of discovery using the general condition score table in Table 5. In the case of two or more sites of discovery with a score of 3 in the general condition score, euthanasia was performed by bleeding from the abdominal aorta under isoflurane anesthesia. Euthanized animals were excluded from the measurement objects. Figure 6 In the table, the day of influenza virus infection treatment was taken as day 0, and the general status score was expressed over time until 7 days after infection. Each value was expressed as mean ± standard error, and in the comparison with the control group, when the P value was less than 0.05 when using the Wilcoxon Rank Sum Test, it was judged to be statistically significantly different and marked with "*", and when the P value was less than 0.01, it was marked with "**".

[0149] [Table 5]

[0150]

[0151] (Determination of survival rate)

[0152] When a body weight loss of 20% or more was confirmed or two or more sites with a score of 3 were found in the general condition score, the animals were euthanized by bleeding from the abdominal aorta under isoflurane anesthesia for humane reasons. The euthanized animals were treated as deaths on that day when calculating the survival rate. Figure 7 In the data, the day of influenza virus infection treatment was set as day 0, and the survival rate until 7 days after infection was expressed over time. Each value was expressed as mean ± standard error, and in the comparison with the control group, when the P value was less than 0.05 when using the log-rank test, it was judged to be statistically significantly different and marked with "*", and when the P value was less than 0.01, it was marked with "**".

[0153] (Result 1: Effect of test food on general condition score)

[0154] like Figure 6 As shown, in the control group, the general condition score increased from the third day after influenza virus infection. On the other hand, in the 279-strain administration group, although the increase in the general condition score due to influenza virus infection can be seen, it has improved compared with the control group. In addition, in the 302-strain administration group and the 533-strain administration group, there has been some improvement compared with the control group, but the degree of improvement is not as great as that of the 279-strain administration group.

[0155] (Result 2: Effect of test food on survival rate)

[0156] like Figure 7 As shown, in the control group, individuals who died began to be seen on the 6th day after influenza virus infection, and all individuals died on the 8th day after infection. On the other hand, no individuals died in the 279-strain administration group. In addition, the survival rate in the 533-strain administration group and the 302-strain administration group increased compared with the control group, but the degree of improvement was not as great as that in the 279-strain administration group.

[0157] [Test Example 6]

[0158] [Evaluation of viral counts and NK activity in the lungs of influenza-infected mice (Part 1)]

[0159] The feeding of mice, the test food, the method of administering the test food, and the method of infecting with influenza virus were carried out in the same manner as in Test Example 5. The mice were grouped in the same manner as in Test Example 5 except that 5 mice were used per group. Five days after the day of virus inoculation (the 15th day from the start of the test), the mice were bled from the abdominal aorta under isoflurane anesthesia for euthanasia, and the number of viruses in the lungs and the evaluation of NK activity were carried out.

[0160] (Determination of the number of viruses in the lungs)

[0161] The lungs removed from the mice were fragmented and homogenized with 2 mL of HBSS (Hank's Balanced Salt Solution, Life Technologies Corporation) using a blender (Physcotron (registered trademark), NS-51, Microtec Nition Co., Ltd.). The extract was filtered with a sterilized mesh (100 μm, cell strainer; FALCONTM) to remove cell clumps and impurities, and centrifuged (190 × g, 5 minutes) using a centrifuge (AX-310, Tomy Seiko Co., Ltd.), and the supernatant was used as tissue fluid, and about 1 mL was dispensed into a serum tube and frozen for storage.

[0162] The lung tissue fluid was thawed and an appropriate dilution was prepared using MEM (Minimum Essential Medium Eagle, SIGMA). 0.1 mL of the original solution and the diluted lung tissue fluid were added to each well of a 12-well plate seeded with MDCK cells, and the cells were allowed to adsorb the virus for 1 hour.

[0163] The medium A (10 mL of 10×MEM, 3 mL of 7.5% sodium bicarbonate, 2 mL of 200 mmol / L L-glutamine, 1 mL of 1% DEAE dextran, 1 mL of 15% glucose, 1 mL of 10% BSA, 40 μL of 2.5% trypsin, 1 mL of antibiotics, and 31 mL of sterile distilled water) and medium B (0.8 g of agarose, 50 mL of distilled water) used to measure the concentration of the virus stock solution were mixed in equal amounts, and multiple layers of 1.5 mL were stacked on the cells. After the agarose solution solidified, the cells were cultured for 2 days using a carbon dioxide culture device. After that, neutral red was added to the medium mixed with medium A and medium B, and the resulting medium was stacked in multiple layers. The number of virus plaques was measured on the second day. The number of viruses was calculated using the number of plaques calculated based on the maximum dilution factor at which the number of plaques could be measured. If all samples cannot be measured, prepare again.

[0164] Figure 8 This is a graph showing the number of viruses (PFU / lung) in lung tissue on the 5th day after influenza virus infection (the 20th day after the start of the test). Each value is expressed as mean ± standard error, and when the P value is less than 0.01 when using the Wilcoxon rank sum test in comparison with the control group, it is judged to be statistically significant and marked with "**".

[0165] (Evaluation of NK activity)

[0166] Add HBSS to the spleen removed from the mouse and cut it into small pieces. Use the rubber part of the inner barrel of a plastic syringe to gently crush it in a plastic dish to make 2 mL of a single cell suspension. After removing tissue debris through a cell strainer, add the cell suspension to a 50 mL centrifuge tube and centrifuge (1000 rpm, 190 × g, 5 minutes). After removing the supernatant, add 2 mL of red blood cell lysate (water for injection: Japanese Pharmacopoeia, Otsuka distilled water, Otsuka Pharmaceutical Co., Ltd.) and mix thoroughly for 3 seconds, and add an equal amount of 2 × PBS. Obtain the supernatant without taking the precipitate, centrifuge (1000 rpm, 190 × g, 5 minutes), wash the cells with culture medium, and resuspend to 1 mL using RPMI1640 with 10% FBS. Count the living cells using trypan blue solution to prepare an appropriate cell number (effector cells).

[0167] The cell suspension of YAC-1 cells was transferred to a 15 mL centrifuge tube and centrifuged (1000 rpm, 190×g, 5 minutes). After removing the supernatant, 1 mL of RPMI1640 was added and mixed thoroughly, and the appropriate cell count (target cells) was prepared using a hemocytometer.

[0168] Using the prepared effector cells and target cells, the cell damage rate measured using an LDH cytotoxicity Detection Kit (Takara Bio Co., Ltd.) was evaluated as NK activity.

[0169] Fig. 9 It is a graph showing the NK activity of mouse spleen cells on the 5th day after influenza virus infection (the 20th day after the start of the test). The horizontal axis represents the ratio of the number of effector cells to the number of target cells. That is, mouse spleen cells and YAC-1 cells were mixed in a ratio of 10:1, 20:1, and 40:1, and the cell damage rate was measured, and the measurement results were shown as NK activity. Among them, each value is represented by the mean value ± standard error. In the comparison with the control group, when the P value is less than 0.05 when using the Wilcoxon rank sum test, it is judged to be statistically significantly different and marked with "*".

[0170] (Result 3: Effect of test food on the number of viruses in the lungs)

[0171] like Figure 8 As shown, no virus was detected in the untreated group, while the number of viruses in the control group increased. On the other hand, in the 279-strain administration group, although the number of viruses in the lungs increased due to influenza virus infection, it was reduced compared to the control group. In addition, no reduction in the number of viruses in the lungs was observed in the 302-strain administration group and the 533-strain administration group. Therefore, it can be seen that the 279 strain has an antiviral effect that reduces the number of viruses in the body, thereby improving the survival rate.

[0172] (Result 4: Effect of test food on NK activity)

[0173] like Fig. 9 As shown, the NK activity of spleen cells in the 279-strain administration group increased compared with the control group. On the other hand, no increase in NK activity was observed in the 533-strain administration group and the 302-strain administration group relative to the control group. Therefore, it can be seen that the 279-strain has the effect of activating NK cells.

[0174] [Test Example 7]

[0175] [Evaluation of status scores and survival rates of influenza-infected mice (Part 2)]

[0176] (Raising mice)

[0177] The same method as in Experimental Example 5 was used.

[0178] (Grouping of mice)

[0179] The mice were divided into five groups, namely, an influenza virus infection-untreated group (hereinafter referred to as the "untreated group"), a control group treated with influenza virus infection (hereinafter referred to as the "control group"), a group administered with lactic acid bacteria Lactobacillus sakei MG-LAB279 strain before influenza virus infection treatment (hereinafter referred to as the "279 strain administration group"), a group administered with Lactococcus lactis subsp. lactis JCM5805 strain before influenza virus infection treatment (hereinafter referred to as the "JCM5805 strain administration group"), and a group administered with SHIELD lactic acid bacteria before influenza virus infection treatment (hereinafter referred to as the "SHIELD lactic acid bacteria administration group"), with 8 mice in each group.

[0180] (Test food and administration method)

[0181] The test food for the untreated group and the control group was a powdered feed (MF, Oriental Yeast Industry Co., Ltd.). The test food for the 279-strain administration group was a powdered feed mixed with 0.5% dead cells of the lactic acid bacteria Lactobacillus sakei MG-LAB279 strain. The test food for the JCM5805-strain administration group was a powdered feed mixed with 0.5% dead cells of the lactic acid bacteria Lactococcus lactis subspecies JCM5805 strain. The test food for the SHIELD lactic acid bacteria administration group was a powdered feed mixed with 3.3% (0.5% in terms of dead cells of lactic acid bacteria) of SHIELD lactic acid bacteria. The administration method was carried out in the same manner as in Test Example 5.

[0182] (How influenza viruses infect people)

[0183] The same procedure as in Test Example 5 was carried out for treating mice infected with influenza virus.

[0184] (Weight measurement)

[0185] The body weight was measured in the same manner as in Test Example 5.

[0186] (Determination of general status score)

[0187] The general condition score was measured in the same manner as in Test Example 5. The general condition score was calculated as the total score of each site of discovery using the general condition score table in Table 5. When two or more sites of discovery with a score of 3 were found in the general condition score, the animals were bled from the abdominal aorta under isoflurane anesthesia for euthanasia. The euthanized animals were excluded from the measurement. Fig.10 In the table, the day of influenza virus infection treatment was set as day 0, and the general condition score was expressed over time until 8 days after infection. Each value was expressed as mean ± standard error, and in the comparison with the control group, when the P value was less than 0.05 when using the Wilcoxon rank sum test, it was judged to be statistically significantly different and marked with "*", and when the P value was less than 0.01, it was marked with "**".

[0188] (Determination of survival rate)

[0189] The survival rate was measured in the same manner as in Test Example 5. Fig.11 In the table, the day of influenza virus infection treatment was set as day 0, and the survival rate was expressed over time until day 13 after infection. Each value was expressed as mean ± standard error, and when the P value was less than 0.05 in the comparison with the control group using the log-rank test, it was judged to be statistically significantly different and marked with "*", and when the P value was less than 0.01, it was marked with "**".

[0190] (Result 1: Effect of test food on general condition score)

[0191] like Fig.10 As shown, in the control group, the general condition score increased from the 4th day after influenza virus infection. On the other hand, in the 279-strain administration group, although the general condition score increased due to influenza virus infection, it was improved compared with the control group. In addition, in the JCM5805-strain administration group and the SHIELD lactic acid bacteria administration group, there was improvement compared with the control group, but the degree of improvement was not as great as that of the 279-strain administration group.

[0192] (Result 2: Effect of test food on survival rate)

[0193] like Fig.11 As shown, in the control group, individuals who died were seen starting from the 6th day after influenza virus infection, and all individuals died on the 7th day after infection. In addition, in the JCM5805 strain administration group and the SHIELD lactic acid bacteria administration group, individuals who died were also seen starting from the 6th day after infection, and all individuals died on the 8th day after infection. On the other hand, in the 279 strain administration group, individuals who died were seen on the 8th day after infection, but half of the individuals still survived on the 13th day after infection. In the 279 strain administration group, a significant improvement in the survival rate was seen compared to the control group. In addition, in the SHIELD lactic acid bacteria administration group, although a significant increase in the survival rate was seen relative to the control group, the degree of improvement was not as great as that of the 279 strain administration group.

[0194] [Test Example 8]

[0195] [Evaluation of viral counts and NK activity in the lungs of influenza-infected mice (Part 2)]

[0196] The feeding of mice, the test food, the method of administering the test food, and the method of infecting with influenza virus were carried out in the same manner as in Test Examples 5 and 6. The mice were divided into groups, and 5 mice were used in each group. Five days after the virus inoculation day (the 15th day from the start of the test) (the 20th day from the start of the test), the mice were bled from the abdominal aorta under isoflurane anesthesia for euthanasia, and the number of viruses in the lungs and the evaluation of NK activity were carried out.

[0197] (Determination of the number of viruses in the lungs)

[0198] The number of viruses in the lungs was measured using the right lungs removed from mice. The number of viruses was measured in the same manner as in Test Example 6.

[0199] Fig.12 This is a graph showing the number of viruses (PFU / lung) in lung tissue on the 5th day after influenza virus infection (the 20th day after the start of the test). Each value is expressed as mean ± standard error, and when the P value is less than 0.01 when using the Wilcoxon rank sum test in comparison with the control group, it is judged to be statistically significant and marked with "**".

[0200] (Evaluation of NK activity)

[0201] The NK activity was evaluated in the same manner as in Test Example 6. Fig.13 It is a graph showing the NK activity of mouse spleen cells on the 5th day after influenza virus infection treatment (the 20th day from the start of the test). The horizontal axis represents the ratio of the number of effector cells to the number of target cells. That is, mouse spleen cells and YAC-1 cells were mixed in a ratio of 10:1, 20:1, and 40:1, and the cell damage rate was measured, and the measurement results were shown as NK activity. Among them, each value is represented by mean ± standard error, and in comparison with the control group, when the P value when using the Wilcoxon rank sum test is less than 0.05, it is determined to be statistically significantly different, and marked "* (279 strain administration group)" and "# (JCM5805 strain administration group)".

[0202] (Pathological histological examination of lung)

[0203] The left lung extracted from mice was used in the pathological histological examination. After the left lung was fixed, paraffin sections were made in all examples of each group according to the conventional method, and the paraffin sections were stained with HE. Optical microscopy was implemented, and photos of the paraffin sections were taken with a digital camera in the representative examples of each group. Each inspection result of the pathological tissue was scored as follows in five grades according to the staining ratio of the entire paraffin section.

[0204] 0: No change (-, 0% of the total),

[0205] 1: Very mild (±, 0% <~ ≤ 25%),

[0206] 2: Mild (+, 25% < ~ ≤ 50%),

[0207] 3: Moderate (++, 50% < ~ ≤ 75%),

[0208] 4: Severe (+++, 75%<~≤100%).

[0209] In addition, in the bronchi, the total score of three items, namely, hyperplasia / hypertrophy of the mucosal epithelium, degeneration / necrosis of the mucosal epithelium, and infiltration of mononuclear / polymorphonuclear leukocytes, was calculated. In addition, in the alveoli, the total score of five items, namely, airless lung, edema, hemorrhage, infiltration of mononuclear / polymorphonuclear leukocytes (alveolar septum), and infiltration of mononuclear / polymorphonuclear leukocytes, was calculated.

[0210] Figure 14 is a graph showing the scores calculated by pathological histological examination of the left lung on the 5th day after influenza virus infection treatment (the 20th day after the start of the experiment). Among them, each value is expressed as the mean ± standard error, and in the comparison with the control group, when the P value when using the Wilcoxon rank sum test is less than 0.05, it is judged to be statistically significantly different and marked with "*".

[0211] (Result 3: Effect of test food on the number of viruses in the lungs)

[0212] like Fig.12 As shown, no virus was detected in the untreated group, while in contrast, the number of viruses in the control group increased. On the other hand, in the 279-strain administration group, although it can be seen that the number of viruses in the lungs increased due to influenza virus infection, it was reduced compared to the control group. In addition, in the SHIELD lactic acid bacteria administration group, although it can be seen that the number of viruses decreased compared to the control group, the degree of improvement was not as great as that in the 279-strain administration group. In the JCM5805-strain administration group, no reduction in the number of viruses in the lungs was observed compared to the control group. Therefore, it can be seen that the 279 strains and SHIELD lactic acid bacteria have antiviral effects that reduce the number of viruses in the body, thereby improving the survival rate.

[0213] (Result 4: Effect of test food on NK activity)

[0214] like Fig.13As shown, in the 279-strain administration group, the NK activity of spleen cells increased regardless of the cell ratio of mouse spleen cells to YAC-1 cells compared with the control group. In addition, in the JCM5805-strain administration group, when the cell ratio of mouse spleen cells to YAC-1 cells was 10:1 and 20:1, the NK activity of spleen cells increased. On the other hand, in the SHIELD lactic acid bacteria administration group, no increase in NK activity was observed relative to the control group. Therefore, it can be seen that: 279 strains and JCM5805 strains have the effect of activating NK cells.

[0215] (Result 5: Effect of test food on lung lesions)

[0216] like Fig.14A and 14B As shown, in the untreated group, no lung lesions were observed in the bronchi and alveoli, while in the control group, the scores of the bronchi and alveoli increased, and lung lesions occurred. In addition, in the 279-strain administration group, although an increase in the score due to influenza virus infection was also observed, no significant change was observed compared with the control group. On the other hand, in the JCM5805-strain administration group and the SHIELD lactic acid bacteria administration group, no significant difference was observed in the bronchial scores compared with the control group, but a significant increase was observed in the alveolar scores. Therefore, it can be seen that: JCM5805 strain and SHIELD lactic acid bacteria activated immunity while aggravating the lung lesions caused by influenza virus infection. In contrast, although the 279 strain activated immunity, it did not cause inflammation of the bronchi or alveoli.

[0217] [Test Example 9]

[0218] [Study on the promotion of IFN-γ production in mouse spleen cells]

[0219] Spleen cells collected from the spleen of 9-week-old female C57BL / 6J mice were adjusted to 3.0×10 using RPMI1640 (GIBCO) medium containing 10% fetal bovine serum (FBS, BIOWEST), antibiotics (penicillin / streptomycin) 100 units / mL (GIBCO), and 50 μM mercaptoethanol (hereinafter referred to as RPMI1640 medium). 4 cells / well and seeded in a 96-well plate (IWAKI). Sake Lactobacillus MG-LAB279, Pediococcus pentosaceus MG-LAB533, and Lactobacillus plantarum MG-LAB302 were added to 10 μg / mL and cultured. The culture was carried out at 37°C and 5% CO2. The control group used RPMI medium without lactic acid bacteria.

[0220] (ELISA test)

[0221] 100 μL of the supernatant of mouse spleen cells cultured for 20 hours was collected from each 96-well culture plate and analyzed using ELISAMAX TM Deluxe Kit Mouse IFN-γ (ELISAMAX TM Deluxe Set Mouse IFN-γ, Biolegend), the production amount of each test body was quantified. Specifically, after the test body was prepared according to the attached protocol, the absorbance of each test body was measured at a wavelength of 450nm, and the IFN-γ production amount was quantified using a calibration curve prepared using the standard substance attached to the detection kit. The measurement results are shown in Fig.15 The measured values ​​are shown as the average of three consecutive measurements, and the error bars are shown as standard errors.

[0222] like Fig.15 As shown, it was found that the Lactobacillus sakei MG-LAB279 strain strongly induced IFN-γ production. It is believed that the IL-12p70-induced helper T cell type 1 (Th1) differentiated from the helper T precursor cell (Th0) enhances the production of IFN-γ, thereby increasing the activity of phagocytes such as T cells and monocytes and activating NK cells.

[0223] [Test Example 10]

[0224] [Study on the promotion of IL-10 production in human monocytic leukemia cells]

[0225] Human monocytic leukemia THP-1 cells provided by JCRB Cell Bank were cultured in RPMI1640 (GIBCO) medium containing 10% fetal bovine serum (FBS, BIOWEST) and antibiotics (penicillin / streptomycin) 100 units / mL (GIBCO) (hereinafter referred to as RPMI1640 medium) at a concentration of 1.0×10 6 The cells were cultured at 37°C and 5% CO2 at a concentration of 10 cells / mL. Then, PMA (phorbol 12-myristate 13-acetate) was added to a final concentration of 5 ng / mL to differentiate them into macrophages, and the cells were cultured for 24 hours. Differentiation into macrophages was confirmed under a microscope, and the culture supernatant was removed and washed, and then new PRMI medium was added and cultured for 24 hours. The cells were recovered using 0.25 w / v% trypsin-1 mmol / L EDTA·4Na solution (Wako Pure Chemical Industries, Ltd.) and the concentration was adjusted to 2.0×10 4cells / well and seeded in a 96-well plate (IWAKI). One hour after seeding, the dried bacterial powders of Lactobacillus sakei MG-LAB279, Pediococcus pentosaceus MG-LAB533 and Lactobacillus plantarum MG-LAB302 were suspended in RPMI1640 medium and added to a final concentration of 100ug / mL for culture. After 24 hours of culture, the culture medium was recovered and the IL-10 concentration in the culture medium was determined using an IL-10 Human Uncoated ELISA Kit (IL-10 Human Uncoated ELISA Kit, Invitrogen). The measurement results are shown in Fig.16 The measured values ​​are shown as the average of three consecutive measurements, and the error bars are shown as standard deviations.

[0226] like Fig.16 As shown, MG-LAB279 strain promotes the production of IL-10 in human monocytic leukemia cells. Therefore, it is known that MG-LAB279 strain has a high ability to induce the production of anti-inflammatory cytokines. Therefore, it is believed that it may be effective in the prevention and treatment of inflammatory diseases including those caused by viral infection.

[0227] [Test Example 11]

[0228] [Anti-inflammatory test of dextran sulfate sodium using colitis model mice]

[0229] (Raising mice)

[0230] The test used 8-week-old female C57BL / 6J mice (Charles River, Japan) after 1 week of pre-feeding. Animals were kept in a breeding room maintained at a management temperature of 20-26°C, a management humidity of 40-70% RH, and a light and dark cycle of 12 hours each (lighting: 7:00-19:00). During the pre-feeding period, a sterilized polycarbonate flat-bottomed cage (213mm×324mm×131mm) with a floor mat (Pal Soft, Oriental Yeast Industry) was used. In addition, considering the breeding environment, the floor mat was replaced twice a week.

[0231] (Grouping of mice)

[0232] The mice were divided into three groups, namely, a group given a normal diet (hereinafter referred to as the "control group"), a group given a normal diet and induced inflammation using dextran sulfate sodium (DSS) (hereinafter referred to as the "DSS group"), and a group administered with the lactic acid bacteria Lactobacillus sakei MG-LAB279 strain and induced inflammation (hereinafter referred to as the "279 strain administration group"), with 4, 8, and 8 mice in each group.

[0233] (Test food and administration method)

[0234] The test food for the control group and the DSS group was powdered feed (MF, Oriental Yeast Industry Co., Ltd.). The test food for the 279-strain administration group was the above-mentioned powdered feed mixed with 0.5% dead bacteria of the lactic acid bacteria Lactobacillus sakei MG-LAB279 strain. After grouping, the powdered feed (MF) or the feed mixed with the test substance was placed in a feeding machine (MF-4S, SHINFACTORY Co., Ltd.) and allowed the mice to ingest it freely. For the feed, the feed was supplemented or replaced with new feed twice a week. The administration of the test food continued until the end of the experiment.

[0235] (Methods of Inflammation Induction and Body Weight Measurement)

[0236] The 8-week-old C57BL / 6J mice in each group were allowed to freely eat the above feed until the end of the experiment. For the DSS group and the 279 strain administration group, the drinking water was replaced with a 2.5% DSS aqueous solution on the 8th day of feeding, and the mice were allowed to drink water freely to induce colitis. The body weight of each mouse was measured before the start of DSS administration (8th day) and on the 11th, 13th, and 14th days. Fig.12 The middle graph shows the body weight change rate on the 11th, 13th, and 14th days relative to the body weight before the start of DSS ingestion (Day 8).

[0237] (Measurement of large intestine length and colitis symptom score)

[0238] On the 14th day after the start of the experiment, autopsy was performed under isoflurane anesthesia, and the length of the large intestine (distal cecum to anus) was measured. The colitis symptom score was measured at the beginning of inflammation induction (8th day) and on the 11th and 13th days. The colitis symptom score table in Table 6 was used to calculate the total score of diarrheal symptoms and bloody stool symptoms observed in the stool. Fig.19 Colitis symptom scores are shown. Fig.19 This is a graph showing the colitis symptom scores at the start of inflammation induction (8th day) and on the 11th and 13th days over time. Each score value is expressed as mean ± standard error. The test was conducted by comparing the DSS group with the 279 strain administration group (without corresponding t-test). The significance level was 5% risk rate ("*" in the figure).

[0239] [Table 6]

[0240] score diarrhea bloody stool 0 normal normal 1 Soft stools (formed stools with a lot of water) Blood mixed in stool when visually inspected 2 Diarrhea (watery, unformed stools) Blood in the anus 3 Watery stools (liquid stools) Frequent anal bleeding

[0241] (Result 1: Effect on weight change rate)

[0242] like Fig.17As shown, the control group had an increase in body weight, whereas the DSS group had a decrease in body weight. On the other hand, in the 279-strain-administered group, although a decrease in body weight due to DSS ingestion was also observed, it was less severe than in the DSS group.

[0243] (Result 2: Effect on colon length and colitis symptom score)

[0244] like Fig.18 As shown in Figure 2, the large intestine of the DSS group was shortened compared to the control group. In the 279-strain administration group, although shortening of the large intestine due to DSS ingestion was also observed, it was suppressed compared to the DSS group. Fig.19 As shown in the figure, no colitis symptoms appeared at the beginning of inflammation induction (day 8), but on the 11th and 13th days, the colitis symptom scores of the DSS group increased. On the other hand, in the 279-strain administration group, although the colitis symptom score increased due to the intake of DSS, it was alleviated compared with the DSS group. Therefore, it was confirmed that the lactic acid bacteria Lactobacillus sakei MG-LAB279 strain can delay and alleviate the onset of DSS-induced colitis symptoms.

[0245] [Production Example 1] (Cultivation of lactic acid bacteria)

[0246] The lactic acid bacteria Lactobacillus sakei MG-LAB279 strain was cultured in a commercially available culture medium. After the bacteria were collected by centrifugation, water was added for re-suspending and centrifugation was performed to obtain a concentrated bacterial solution. The solution was sterilized at 105°C or above for 30 minutes. The concentrated bacterial solution was then spray-dried to obtain a dead bacterial powder. The powder was mixed with dextrin (NSD300 manufactured by San-ei Sucrochemical Co., Ltd.) to obtain a 10% dead bacterial powder by mass.

[0247]

[0248]

[0249] To be filled in by the receiving office

[0250]

[0251] To be filled in by the International Bureau

[0252] 0-5 The International Bureau receives this form 0-5-1 Authorized Officer

Claims

1. A Lactobacillus sakei MG-LAB279 strain, with the accession number being NITE BP-03645.

2. A composition, wherein Containing the strain according to claim 1 and / or its bacterial components.

3. An immune activation composition, in, Contains the strain according to claim 1 and / or its bacterial components as an active ingredient.

4. The immunostimulating composition according to claim 3, wherein The immunostimulating composition does not cause inflammation.

5. A composition for preventing or treating viral infection, in, Contains the strain according to claim 1 and / or its bacterial components as an active ingredient.

6. A composition for activating NK cells, in, Contains the strain according to claim 1 and / or its bacterial components as an active ingredient.

7. A composition for promoting interferon λ production, in, Contains the strain according to claim 1 and / or its bacterial components as an active ingredient.

8. A composition for promoting interleukin-12p70 production, in, Contains the strain according to claim 1 and / or its bacterial components as an active ingredient.

9. A composition for relieving stress, in, Contains the strain according to claim 1 and / or its bacterial components as an active ingredient.

10. A composition for suppressing inflammation, wherein: Contains the strain according to claim 1 and / or its bacterial components as an active ingredient.

11. A composition for improving skin, in, Contains the strain according to claim 1 and / or its bacterial components as an active ingredient.

12. A food or beverage, wherein: A composition comprising the MG-LAB279 strain according to claim 1 or any one of claims 2 to 11.

13. A pharmaceutical product, wherein: A composition comprising the MG-LAB279 strain according to claim 1 or any one of claims 2 to 11.

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