Novel lactic acid bacteria, composition comprising novel lactic acid bacteria, and method for producing same

Abstract

The present invention addresses the problem of providing a bacterium belonging to Lactobacillus helveticus, which has an excellent IFN production-inducing effect. The present invention relates to a bacterium classified as Lactobacillus helveticus, which is capable of activating plasma-like dendritic cells (pDC) and / or myeloid dendritic cells (mDC) and inducing the production of type I IFN, and which induces the production of type I IFN at a level of 1.5 times or more as compared to that of Lactobacillus helveticus SBT2171 (accession number: Ferm BP-5445), and more particularly, to a bacterium classified as Lactobacillus helveticus, which is capable of activating plasma-like dendritic cells (pDC) and / or myeloid dendritic cells (mDC) and inducing the production of type I IFN at a level of 1.5 times or more than that of Lactobacillus helveticus SBT2171 (accession number: Ferm BP-5445).

Description

Technical Field

[0001] This invention relates to novel bacteria belonging to Lactobacillus helveticus and compositions containing novel bacteria, cultures of novel bacteria, or cell treatments thereof, as well as methods for manufacturing the same. Background Technology

[0002] Immunity is a biological defense mechanism possessed by many animals, its function being to eliminate pathogens such as bacteria and viruses that invade from the outside, or tumor cells that develop within the body. The immune system comprises two main systems: the innate immune system and the acquired immune system, both responsible for the organism's defense functions. The innate immune system is primarily responsible for the initial immune response to bacterial and viral infections, with dendritic cells being a powerful and crucial component.

[0003] Dendritic cells are highly plastic cells with various subtypes. However, as dendritic cells that induce type I interferon (type I IFN) to exhibit antiviral proliferation activity through activation of Toll-like receptors (TLRs), plasmacytoid dendritic cells (pDCs) and myeloid dendritic cells (mDCs) are known.

[0004] pDCs are the main cells that produce type I IFN in vivo. To induce type I IFN production using pDCs, stimulation by intracellular and in vivo TLRs such as TLR7 / TLR9 is required.

[0005] mDCs induce the production of type I IFN through activation of TLR3 and other mechanisms. In addition, mDCs induce the activation of helper T cells (CD4+ T cells) by releasing inflammatory cytokines such as interleukin-12 (IL-12) and tumor necrosis factor-α (TNF-α) through bacterial infection.

[0006] Meanwhile, recent studies have shown that lactic acid bacteria play a role in activating the innate immune system.

[0007] Patent document 1 describes that Lactococcus Lactis has the function of activating pDC to induce the production of IFN.

[0008] Patent document 2 describes that Lactobacillus helveticus SBT2171 (FERM BP-5445) has the effect of increasing pDC.

[0009] Existing technical documents

[0010] Patent documents

[0011] Patent Document 1: Japanese Patent No. 6170190

[0012] Patent Document 2: Japanese Patent No. 6705628 Summary of the Invention

[0013] The problem the invention aims to solve

[0014] As described in Patent Document 1, it is known that Lactococcus lactis has an IFN-inducing effect. However, in recent years, with the increasing awareness of preventing viral infections such as COVID-19, there is a desire for bacteria that can exert a better IFN-inducing effect.

[0015] Furthermore, the SBT2171 strain described in Patent Document 2 was selected based on the pDC proliferation-promoting effect rather than the IFN production-inducing effect. Regarding *Lactobacillus helveticus*, research on its IFN production-inducing effect is still insufficient.

[0016] In view of the above, the objective of this invention is to provide a bacterium belonging to Lactobacillus helveticus that has excellent IFN production induction activity.

[0017] Solution for solving the problem

[0018] Through in-depth and persistent research and efforts, the inventors discovered a strain of *Lactococcus lactis* that exhibits superior type I IFN production induction compared to *Lactococcus lactis* JCM5805 specifically described in Patent Document 1 and *Lactobacillus helveticus* SBT2171 described in Patent Document 2, thus completing this invention. The invention that solves the aforementioned problems is described below.

[0019] [1] A bacterium classified as Lactobacillus helveticus is a bacterium that can activate plasmacytoid dendritic cells (pDCs) and / or myeloid dendritic cells (mDCs) and induce the production of type I IFN. Compared with Lactobacillus helveticus SBT2171 (accession number: Ferm BP-5445), it induces the production of type I IFN by more than 1.5 times.

[0020] [2] A Lactobacillus helveticus MCC2430 (accession number: NITE BP-03882).

[0021] [3] A composition comprising at least one selected from the bacteria described in [1] or [2], a culture of the bacteria, and a cell treatment of the bacteria.

[0022] [4] The composition according to [3], wherein the composition is a type I IFN generation induction composition.

[0023] [5] The composition according to [3], wherein the composition is selected from at least one of the group consisting of antiviral compositions, immune-stimulating compositions, compositions for the treatment or prevention of immune diseases, anticancer compositions, anti-inflammatory compositions, and compositions for the prevention of bone loss.

[0024] [6] The composition according to any one of [3] to [5] is a food or medicine.

[0025] [7] A method of manufacturing a composition, comprising the step of adding at least one of the bacteria selected from [1] or [2], a culture of the bacteria and a cell treatment of the bacteria to a raw material.

[0026] [8] The manufacturing method according to [7] includes a step of fermenting raw materials using the bacteria, and the composition is a fermentation composition.

[0027] The effects of the invention

[0028] The bacteria of the present invention exert excellent type I IFN production induction effects by activating pDC and / or mDC.

[0029] The compositions of the present invention induce the production of type I IFN in subjects who ingest them, thereby exerting antiviral effects, immune stimulation effects, therapeutic or preventive effects for immune diseases, anticancer effects, anti-inflammatory effects, and preventive effects for decreased bone strength.

[0030] According to the manufacturing method of the present invention, it is possible to manufacture a composition that exhibits the above-mentioned effects. Attached Figure Description

[0031] Figure 1 A bar chart is presented to show the concentration of IFNα in culture supernatant determined by flow cytometry using an IFNα assay kit. The experiment was performed three times, and the average values ​​are presented graphically. Error bars in the chart represent standard deviations. The p-values ​​in the chart were obtained using a Student's t-test. It should be noted that various strains were treated with a 10-fold increase in bactericide relative to dendritic cells, except for JCM5805, which was treated with an additional 100-fold increase in bactericide. The label "JCM5805_100" in the chart indicates the result of the JCM5805 test with a 100-fold increase in bactericide for dendritic cells.

[0032] Figure 2 A bar chart is presented to show the concentration of IFNβ in culture supernatant as determined by enzyme-linked immunosorbent assay (ELISA) using an IFNβ assay kit. The experiment was performed three times, and the average values ​​are presented graphically. Error bars in the chart represent standard deviations. The p-values ​​in the chart were obtained using a Student's t-test. It should be noted that various strains were treated with a 10-fold increase in bactericide relative to dendritic cells, except for JCM5805, which was treated with an additional 100-fold increase in bactericide. The label "JCM5805_100" in the chart indicates the result of the JCM5805 test with a 100-fold increase in bactericide for dendritic cells. Detailed Implementation

[0033] Preferred embodiments of the present invention will be described. However, the present invention is not limited to the following preferred embodiments, and free modifications can be made within the scope of the present invention. In this specification, unless otherwise stated, percentages are expressed in units of mass.

[0034] <Bacteria capable of inducing type I IFN production>

[0035] This invention relates to bacteria classified as Lactobacillus helveticus, which can activate plasmacytoid dendritic cells (pDCs) and / or myeloid dendritic cells (mDCs) and induce type I IFN production.

[0036] More specifically, the bacteria of the present invention are classified as Lactobacillus helveticus, which induces more than 1.5 times the production of type I IFN compared with Lactobacillus helveticus SBT2171 (accession number: Ferm BP-5445).

[0037] Bacteria classified as *Lactobacillus helveticus* whose production of type I IFN induced by activation of pDC and / or mDC is more than 1.5 times that of SBT2171 are included within the scope of this invention. There are no particular limitations on the conditions for the test used to confirm whether a bacterium belongs to the category of type I IFN production determined by this invention. It should be noted that, for ease of explanation, the test used to confirm whether a bacterium belongs to the category of this invention is referred to as the "confirmation test" in this specification.

[0038] As a confirmatory test, an example can be an experimental system in which bacterial cells are added to a mixed culture system containing pDC and / or mDC, and the concentration of type I IFN in the culture supernatant is measured. If the concentration of type I IFN is measured to be 1.5 times or more compared to adding SBT2171 bacterial cells under the same conditions, then the bacteria used as the subject of the confirmatory test can be determined to be within the scope of the present invention.

[0039] The culture system with added bacteria can be a culture system that culturees either pDC or mDC alone, or a mixed culture system of pDC and mDC.

[0040] The scope of this invention includes any of the following forms: a form in which pDC is activated to induce the generation of type I IFN without activating mDC; a form in which mDC is activated to induce the generation of type I IFN without activating pDC; and a form in which both pDC and mDC are activated to induce the generation of type I IFN.

[0041] The bacterial cells added to the pDC and / or mDC culture system can be either live or dead. If dead bacteria are used, heat-sterilized bacteria are preferred.

[0042] There is no limit to the number of cells added to the culture system of pDCs and / or mDCs. For example, 10 to 100 times the number of cells can be added relative to the number of pDCs and / or mDCs. Specifically, examples of adding 10 times the number of cells to the culture system of pDCs and / or mDCs can be suitably exemplified.

[0043] There are no restrictions on the source tissues of pDCs and / or mDCs used for confirmatory tests. For example, pDCs and / or mDCs derived from peripheral blood mononuclear cells or bone marrow cells can be used.

[0044] There are no restrictions on the types of animals from which the source can be derived; for example, mammals such as humans, monkeys, mice, rats, rabbits, dogs, and cats can be used. Human-derived pDC and / or mDC are preferred.

[0045] There are no particular limitations on the method for determining the concentration of type I IFN in the culture supernatant of pDC and / or mDC; flow cytometry or ELISA can be used.

[0046] Commercially available kits can also be used for the assay. Examples include the IFNα assay kit (CytometricBead Array Kit, Becton Dickinson) and the IFNβ assay kit (Human IFN-beta DuoSet, R&D Systems).

[0047] Type I interferon in mammals consists of eight or more distinct classes, including IFNα, IFNβ, IFNδ, IFNε, IFNκ, IFNω, IFNτ, and IFNζ. In humans, IFNα, IFNβ, IFNε, IFNκ, and IFNω have been identified. The scope of this invention includes any form that induces the production of interferon belonging to type I IFN.

[0048] In a preferred embodiment of the present invention, the generation of IFNα and / or IFNβ is induced.

[0049] The scope of this invention includes any of the following forms: forms that induce the production of IFNα without inducing the production of IFNβ; forms that induce the production of IFNβ without inducing the production of IFNα; and forms that induce the production of both IFNα and IFNβ.

[0050] In a preferred embodiment of the present invention, the generation of both IFNα and IFNβ is induced.

[0051] The type I IFN measured in the confirmation test can be any interferon belonging to type I IFN, preferably IFNα or IFNβ. In this case, the measured object can be either IFNα or IFNβ, or both IFNα and IFNβ.

[0052] The time from adding bacterial cells to the culture system of pDC and / or mDC until measuring the concentration of type I IFN in the culture supernatant is preferably 12 to 48 hours, preferably 20 to 28 hours, and more preferably 24 hours.

[0053] The confirmation test can be conducted once or multiple times.

[0054] When conducting multiple confirmation tests, the average value of the measured concentration of type I IFN obtained from each confirmation test can be compared with SBT2171. In this case, it is preferable to conduct at least three tests.

[0055] As described above, the bacteria of the present invention induce the production of type I IFN by more than 1.5 times compared to SBT2171. In a preferred embodiment, it induces the production of type I IFN by more than 1.8 times, more preferably more than 2 times, and even more preferably more than 2.2 times compared to SBT2171.

[0056] In a preferred embodiment, the generation of IFNα and / or IFNβ is induced by 1.5 times or more compared to SBT2171. In a preferred embodiment, the generation of IFNα and / or IFNβ is induced by 1.8 times or more, more preferably 2 times or more, and even more preferably 2.2 times or more compared to SBT2171.

[0057] In a specific embodiment, compared with SBT2171, it induces the generation of IFNα by 1.5 times or more, preferably 1.8 times or more, more preferably 2 times or more, further preferably 2.2 times or more, and even more preferably 2.3 times or more.

[0058] In another specific embodiment, compared with SBT2171, it induces the generation of IFNβ by 1.5 times or more, preferably 1.8 times or more, more preferably 2 times or more, even more preferably 2.2 times or more, even more preferably 2.4 times or more, even more preferably 2.6 times or more, and even more preferably 2.7 times or more.

[0059] In a more specific embodiment, compared to SBT2171, it induces the generation of IFNα by 1.5 times or more, preferably 1.8 times or more, more preferably 2 times or more, further preferably 2.2 times or more, and even more preferably 2.3 times or more, and...

[0060] Inducing the generation of IFNβ by 1.5 times or more, preferably 1.8 times or more, more preferably 2 times or more, even more preferably 2.2 times or more, even more preferably 2.4 times or more, even more preferably 2.6 times or more, even more preferably 2.7 times or more.

[0061] The bacteria of the present invention can be in the form of strains that have been isolated and cloned into a genetically homogeneous bacterial group, or in the form of a genetically heterogeneous bacterial group that has not been cloned.

[0062] Even genetically heterogeneous bacterial communities are naturally included within the scope of this invention, as long as their characteristics as a whole community can induce the production of type I IFN as described above.

[0063] Among the bacteria of this invention, Lactobacillus helveticus MCC2430 (accession number: NITE BP-03882) is an example of a strain that has been isolated and cloned into a genetically homogeneous bacterial population. This strain will be described below.

[0064] The bacteria of the present invention can be easily proliferated through culture. As for the culture method, there is no particular limitation as long as *Lactobacillus helveticus* can proliferate; methods commonly used for culturing lactic acid bacteria can be appropriately adjusted as needed. For example, a culture temperature of 25–50°C is acceptable, preferably 35–42°C. Furthermore, culture is preferably carried out under anaerobic conditions, for example, by bubbling anaerobic gas such as carbon dioxide. Alternatively, culture can be carried out under microaerophilic conditions such as liquid static culture.

[0065] The culture medium for culturing the bacteria of this invention is not particularly limited, and can be used by appropriately adjusting the culture medium commonly used for culturing lactic acid bacteria as needed. Specifically, as a carbon source, for example, the following sugars can be used based on their assimilation properties: galactose, glucose, fructose, mannose, cellobiose, maltose, lactose, sucrose, trehalose, starch, starch hydrolysate, and molasses waste. As a nitrogen source, ammonium salts or nitrates such as ammonia, ammonium sulfate, ammonium chloride, and ammonium nitrate can be used. Additionally, as inorganic salts, for example, sodium chloride, potassium chloride, potassium phosphate, magnesium sulfate, calcium chloride, calcium nitrate, manganese chloride, and ferrous sulfate can be used. Furthermore, organic components such as peptone, soybean flour, defatted soybean meal, meat extract, and yeast extract can also be used. Furthermore, as a pre-prepared culture medium, MRS medium can be appropriately used, for example.

[0066] The bacteria of the present invention can be provided in the form of biologically pure cultures.

[0067] <Lactobacillus helveticus MCC2430>

[0068] This invention relates to Lactobacillus helveticus MCC2430 (accession number: NITEBP-03882). Hereinafter, it is simply referred to as MCC2430.

[0069] Based on the following bacteriological properties and characteristics, MCC2430 of the present invention was deposited as a novel strain as *Lactobacillus helveticus* (accession number: NITE BP-03882) on April 13, 2023, at the Patent and Microbial Collection Center of the Technical Base for Product Evaluation (NPMD) (Address: Room 122, 2-5-8 Kazusa-Kamazu, Kisarazu City, Chiba Prefecture, Japan 292-0818, Japan). This strain can be routinely obtained from the aforementioned collection institution.

[0070] The MCC2430 of the present invention is not limited to the above-mentioned preserved strain, but can be a strain that is substantially equivalent to the preserved strain.

[0071] As substantially equivalent strains, strains that meet at least one of (1) to (3) below can be listed.

[0072] (1) Strains identified as the same strain by the Randomly Amplified Polymorphic DNA (RAPD) method or the Pulsed-field gelelectrophoresis (PFGE) method (FAO / WHO (2006) Probiotics in Food: Health and Nutritional Properties and Guidelines for Evaluation. Report of aJoint FAO / WHO Expert Consultation on Evaluation of Health and NutritionalProperties of Probiotics in Food Including Powder Milk with Live Lactic AcidBacteria, Cordoba, Argentina, 1-4 October 2001 [and] Report of a Joint FAO / WHO Working Group on Drafting Guidelines for the Evaluation of Probiotics in Food, London, Ontario, Canada, 30 April-1 May 2002. FAO Food and Nutrition Paper 85, Food and Agriculture Organization of the United Nations, World Health Organization, Rome. Page 43 record)

[0073] (2) Strains that carry only genes from the preserved strain, do not have exogenous genes, and have DNA homology of more than 95%.

[0074] (3) A strain bred from this strain, or a strain that has undergone genetic engineering modification, mutation, or natural mutation, and which, when tested through the confirmation test described above, exhibits the same level of type I IFN induction as the strain preserved in this application.

[0075] Furthermore, strains with excellent fermentability and / or strains that are fermentable within the temperature range of conventional lactic acid bacteria fermentation are suitable.

[0076] The MCC2430 strain of the present invention can be readily propagated, for example, by culturing this strain. The culture conditions and culture medium are as described above.

[0077] The MCC2430 of the present invention can be provided in the form of a biologically pure culture.

[0078] <Application of Lactobacillus helveticus in this application>

[0079] The bacteria and MCC2430 of the present invention described above exert a probiotic effect when administered to a subject. Hereinafter, for ease of explanation, the bacteria and MCC2430 of the present invention will be collectively referred to as "Lactobacillus helveticus of this application".

[0080] The *Lactobacillus helveticus* of this application can be used in the form of the bacteria themselves, a culture, or a processed bacterial culture. That is, after culturing, the obtained culture can be used directly, or it can be diluted or concentrated for use, or the bacterial cells recovered from the culture can be used. Furthermore, the *Lactobacillus helveticus* of this application can be live bacteria or dead bacteria, or it can contain both live and dead bacteria simultaneously. Further, it can be a processed bacterial culture of the *Lactobacillus helveticus* of this application.

[0081] As for dead bacteria, examples include those sterilized through heating, freeze-drying, etc. Other methods for preparing dead bacteria include spray drying, boiling sterilization, freeze-drying, UHT sterilization, pressure sterilization, high-pressure steam sterilization, dry heat sterilization, flowing steam sterilization, electromagnetic wave sterilization, electron beam sterilization, high-frequency sterilization, radiation sterilization, ultraviolet sterilization, ethylene oxide gas sterilization, hydrogen peroxide plasma sterilization, and chemical sterilization methods (alcohol sterilization, formalin fixation, water electrolysis).

[0082] Examples of bacterial cell treatment materials include immobilized bacterial cells fixed with acrylamide, carrageenan, etc.; cell fragments of *Lactobacillus helveticus* cells with partially or completely broken cell walls and cell membranes; centrifuged supernatant (water-soluble fraction); fractions of the supernatant partially purified by treatment with ammonium sulfate, etc.; or substances obtained by concentrating the supernatant. Cell fragments can be fragments of live or dead bacteria, or substances that have been subjected to heating, freeze-drying, etc., after fragmentation.

[0083] Alternatively, the crushing can be carried out using methods and instruments known in the art, such as physical crushing, enzyme dissolution treatment, chemical treatment, autolysis treatment, etc.

[0084] Physical disruption can be performed in any form, whether in a bacterial suspension or a bacterial powder state. Examples of physical disruption include: disruption using agitation methods such as ultrasonic homogenizers, homogenizers, ball mills, bead mills, Dyno mills, and planetary mills; disruption using pressure methods such as jet mills, Freund's crushers, and cell disruptors; and disruption that damages the bacterial cells through filtration.

[0085] As an enzymatic dissolution treatment, enzymes such as lysozyme can be used to destroy the cell structure of bacteria.

[0086] As a chemical treatment, surfactants such as soybean phospholipids and glycerol fatty acid esters can be used to destroy the cell structure of bacteria.

[0087] As an autolysis treatment, the bacterial cells can be dissolved using some of the lactic acid bacteria's own enzymes.

[0088] At least one selected from *Lactobacillus helveticus*, a culture of *Lactobacillus helveticus*, and a processed form of *Lactobacillus helveticus* may be used as an active ingredient. Hereinafter, for ease of explanation, "at least one selected from *Lactobacillus helveticus*, a culture of *Lactobacillus helveticus*, and a processed form of *Lactobacillus helveticus*" will sometimes be referred to as "the ingredient of this application".

[0089] The ingredients of this application can be used in humans or non-human animals (preferably mammals), preferably in humans, pets and livestock, and more preferably in humans.

[0090] Furthermore, the target audience for the ingredients in this application is not specifically limited to anyone who desires the effects of probiotics, including infants, children, adults, healthy individuals, middle-aged and elderly people, the elderly, and those with suboptimal gut health. This technology is preferably intended for use by infants, adults, and the elderly.

[0091] In addition, the ingredients in this application have few side effects and high safety, so they can be ingested continuously over a long period of time.

[0092] The ingredients in this application can be effectively used for symptoms or diseases that can be prevented, improved, or treated by probiotics.

[0093] The components of this application have the effect of inducing the production of type I IFN by activating pDC and / or mDC in the body of the applicable subject.

[0094] Therefore, the components of this application can be used to induce the production of type I IFN. More specifically, the components of this application can be used to induce the production of IFNα and / or IFNβ. It should be noted that the uses are not limited to therapeutic purposes, but can also be used for non-therapeutic purposes.

[0095] In other words, the present invention also relates to a method for inducing the production of type I IFN, comprising administering the component of this application to an applicable subject. More specifically, the present invention also relates to a method for inducing the production of IFNα and / or IFNβ, comprising administering the component of this application to a subject for which the production of type I IFN needs to be induced. Additionally, a non-therapeutic administration method is also provided, comprising administering the component of this application to a subject for which the production of type I IFN needs to be induced.

[0096] In addition, the components of this application can be used to manufacture type I IFN generation inducers.

[0097] There are no specific limitations on the subjects who need to induce the production of type I IFN, as long as they are the ones who wish to induce the production of type I IFN. For example, subjects who need to prevent or treat viral infections, subjects who need immune stimulation, subjects who need to treat or prevent immune diseases, subjects who need to treat or prevent cancer, subjects who need to treat or prevent inflammation, and subjects who need to prevent bone loss, etc.

[0098] The type I IFN induced by the components of this application has the following functions: (1) enhancing the virus resistance of cells by inhibiting viral replication; (2) promoting the expression of MHCI molecules in uninfected cells and protecting them from NK cell attack; and (3) activating NK cells to clear virus-infected cells.

[0099] That is, the component of this application, which has the inducing effect of type I IFN production, can be used for the prevention or treatment of viral infections. It should be noted that the use is not limited to therapeutic purposes, but can also be for non-therapeutic purposes. In other words, the present invention also relates to a method for preventing or treating viral infections, comprising administering the component of this application to a subject requiring prevention or treatment of a viral infection. Furthermore, it also relates to a non-therapeutic administration method, comprising administering the component of this application to a subject requiring prevention or treatment of a viral infection.

[0100] In addition, the ingredients in this application can be used to manufacture preventive or therapeutic agents for viral infections.

[0101] The preventive or therapeutic effect of the ingredients in this application on viral infections can be confirmed, for example, by ingesting a composition containing the ingredients in this application, thereby reducing the incidence of viral infections, but the confirmation method is not limited to this.

[0102] There are no specific limitations on the individuals who need to prevent or treat viral infections, as long as they desire the desired preventive or therapeutic effect. For example, individuals infected with a virus can be listed.

[0103] Type I IFNs participate in the functional expression of macrophages and T cells, and also promote the activation of NK cells and neutrophils. Type I IFNs play an important role in the innate immune system, and their production can be induced to achieve immunostimulatory effects.

[0104] That is, the component of this application, which has the effect of inducing the production of type I IFN, can be used for immune stimulation. It should be noted that the use is not limited to therapeutic purposes, but can also be for non-therapeutic purposes. In other words, the present invention also relates to an immune stimulation method, which includes administering the component of this application to a subject requiring immune stimulation. Furthermore, a non-therapeutic administration method is also provided, which includes administering the component of this application to a subject requiring immune stimulation.

[0105] In addition, the ingredients in this application can be used to manufacture immunostimulants.

[0106] The immunostimulatory effect of the components of this application can be confirmed, for example, by ingesting a composition containing the components of this application, thereby activating NK cells and macrophages in peripheral blood, but the confirmation method is not limited to this.

[0107] There are no specific limitations on the individuals who require immune stimulation, as long as they desire the desired effect of immune stimulation. For example, individuals whose immune cell function declines due to aging can be listed.

[0108] The usefulness of type I IFN in the treatment of autoimmune diseases such as multiple sclerosis has been recognized and it has been used clinically. Furthermore, its therapeutic effects on various autoimmune diseases such as colitis, encephalomyelitis, arthritis, and neonatal inflammation have also been confirmed.

[0109] That is, the components of this application that induce the production of type I IFN can be used to treat or prevent immune diseases. It should be noted that the uses are not limited to therapeutic purposes, but can also be for non-therapeutic purposes.

[0110] In other words, the present invention also relates to a method for treating or preventing immune diseases, comprising administering the component of this application to a subject requiring treatment or prevention of an immune disease. Additionally, it also relates to a non-therapeutic administration method, comprising administering the component of this application to a subject requiring treatment or prevention of an immune disease.

[0111] In addition, the ingredients in this application can be used to manufacture treatments or preventative agents for immune diseases.

[0112] The therapeutic or preventive effects of the components of this application on immune diseases can be confirmed, for example, by ingesting a composition containing the components of this application, thereby inhibiting the activation of T cells in peripheral blood, but the confirmation method is not limited to this.

[0113] There are no specific limitations on the individuals who need treatment or prevention of immune diseases, as long as they expect treatment or prevention of immune diseases. For example, individuals with high levels of autoantibodies in their peripheral blood can be listed.

[0114] Type I IFNs have the effects of inhibiting cell proliferation and activating NK cells. Therefore, their anti-cancer effects, which involve indirect action through immune responses and direct action on tumor cells, have been recognized and are already in clinical application.

[0115] That is, the component of this application that induces the production of type I IFN can be used to treat or prevent cancer. It should be noted that the use is not limited to therapeutic purposes, but can also be for non-therapeutic purposes.

[0116] In other words, the present invention also relates to a method for treating or preventing cancer, comprising administering the component of this application to a subject requiring treatment or prevention of cancer. Additionally, it relates to a non-therapeutic administration method, comprising administering the component of this application to a subject requiring treatment or prevention of cancer.

[0117] In addition, the ingredients in this application can be used to manufacture drugs for the treatment or prevention of cancer.

[0118] The therapeutic or preventive effects of the components of this application on cancer can be confirmed, for example, by ingesting a composition containing the components of this application, thereby prolonging the activation of NK cells in peripheral blood or progression-free survival (PFS) (the time from the start of treatment to cancer progression or the death of the subject), but the confirmation method is not limited to this.

[0119] There are no specific limitations on who needs cancer treatment or prevention, as long as they are people who expect cancer treatment or prevention. For example, people with renal cell carcinoma can be listed.

[0120] Type I IFN mediates the inhibition of products of inflammation-inducing genes by inducing the activity of interleukin-10 (IL-10), an immunosuppressive cytokine. It also induces other immunosuppressive mediators, such as SOCS-1 (a cytokine signaling inhibitor-1) and Tristetraprolin (TTP), which act on various mechanisms to restore immune system homeostasis.

[0121] That is, the component of this application that induces the production of type I IFN can be used to treat or prevent inflammation. It should be noted that the use is not limited to therapeutic purposes, but can also be for non-therapeutic purposes.

[0122] In other words, the present invention also relates to a method for treating or preventing inflammation, comprising administering the component of this application to a subject requiring treatment or prevention of inflammation. Additionally, it also relates to a non-therapeutic method of administration, comprising administering the component of this application to a subject requiring treatment or prevention of inflammation.

[0123] In addition, the ingredients in this application can be used to manufacture anti-inflammatory agents.

[0124] The therapeutic or preventive effect of the components of this application on inflammation can be confirmed, for example, by ingesting a composition containing the components of this application, thereby increasing the proportion of regulatory T cells or the production of IL-10 in peripheral blood, but the confirmation method is not limited to this.

[0125] There are no specific limitations on the individuals who need treatment or prevention of inflammation, as long as they desire the desired therapeutic or preventative effects. For example, individuals at high risk of chronic inflammation due to aging, obesity, or other factors can be listed.

[0126] Osteoclasts play a role in bone metabolism, but excessive increases in their numbers can lead to decreased bone strength. Type I IFN has been shown to inhibit osteoclast formation, helping to prevent bone loss.

[0127] That is, the component of this application that induces the production of type I IFN can be used to prevent bone loss. It should be noted that the use is not limited to therapeutic purposes, but can also be for non-therapeutic purposes.

[0128] In other words, the present invention also relates to a method for preventing bone loss, comprising administering the component of this application to a subject requiring prevention of bone loss. Additionally, it relates to a non-therapeutic administration method comprising administering the component of this application to a subject requiring prevention of bone loss.

[0129] In addition, the ingredients in this application can be used to manufacture a bone strength reduction preventive agent.

[0130] The preventive effect of the ingredients in this application on reducing bone strength can be confirmed, for example, by ingesting a composition containing the ingredients in this application to inhibit the decrease in bone density, but the confirmation method is not limited to this.

[0131] There are no specific limitations on who needs to prevent bone loss, as long as they are the ones who want to prevent bone loss. For example, people over 50 years old with bone loss can be listed.

[0132] The preferred method of administering or ingesting the ingredients of this application is to consume them for at least one week, more preferably for at least four weeks, with the expectation of daily intake.

[0133] The amount of the ingredient used in this application is not particularly limited due to safety concerns; for example, 1×10⁻⁶ is preferred. 6 ~1×10 12cfu / kg body weight / day, more preferably 1×10 7 ~1×10 11 cfu / kg body weight / day, further optimized to 1×10 8 ~1×10 10 cfu / kg body weight / day

[0134] Furthermore, the preferred dosage (giving amount) for each individual (body weight) is 10. 7 ~10 14 cfu / day, preferably 10 8 ~10 13 cfu / day, further optimized to 10 9 ~10 12 CFU / day.

[0135] In addition, the preferred dosage of the component in this application is 0.01 to 100 mL / kg body weight / day, and more preferably 0.1 to 10 mL / kg body weight / day.

[0136] It should be noted that in this specification, CFU refers to Colony Forming Unit. When the bacteria described are dead bacteria, CFU can be replaced with cells.

[0137] <Composition>

[0138] The components of this application can be used directly. Alternatively, they can be mixed with physiologically, pharmaceutically, or dietaryally acceptable conventional carriers or diluents for use. Specifically, this invention also relates to compositions containing at least one of the following: bacteria selected from the present invention, cultures of said bacteria, and cell treatments of said bacteria; and MCC2430 of the present invention, cultures of said strains, and cell treatments of said strains. Hereinafter, for ease of explanation, such compositions will be described as compositions of the present invention.

[0139] The components contained in the compositions of the present invention exert the various pharmacological effects described above. Specifically, the compositions of the present invention can be provided in the form of a composition for inducing type I IFN production. Furthermore, the compositions of the present invention can be provided in the form of at least one composition selected from the group consisting of antiviral compositions, immune-stimulating compositions, compositions for the treatment or prevention of immune diseases, anticancer compositions, anti-inflammatory compositions, and compositions for the prevention of decreased bone strength.

[0140] The intended recipients of the compositions of the present invention are not particularly limited, and various recipients as described above can be listed. That is, the compositions of the present invention can, for example, be provided to recipients who desire an induction effect of type I IFN. Furthermore, the compositions of the present invention can, for example, be provided to recipients who desire preventive or therapeutic effects against viral infections, recipients who desire immune stimulation effects, recipients who desire therapeutic or preventive effects against immune diseases, recipients who desire therapeutic or preventive effects against cancer, recipients who desire therapeutic or preventive effects against inflammation, recipients who desire preventive effects against decreased bone strength, etc.

[0141] The ingredients described herein can also be included as active ingredients in the composition. Furthermore, due to their high safety profile, they can be used in a wide range of products, including pharmaceuticals and food products. These products can be manufactured by appropriately using any ingredients suitable for each application and by known manufacturing methods suitable for each application.

[0142] In this specification, the term "composition" has the meaning of including food and beverage compositions, pharmaceutical compositions, feed, etc.

[0143] Hereinafter, embodiments of the compositions of the present invention provided in pharmaceutical form and in food and beverage form will be described in detail.

[0144] <Pharmaceutical Compositions>

[0145] The compositions of the present invention can be provided in the form of pharmaceutical compositions. There are no particular limitations on the pharmaceutical compositions of the present invention as long as they contain the ingredients of this application.

[0146] The pharmaceutical compositions of the present invention may be in the form of directly using the components of this application, or they may be in the form of formulation by mixing with physiologically acceptable liquid or solid formulation carriers.

[0147] The pharmaceutical compositions of the present invention can be safely administered to patients suffering from various diseases. It is anticipated that even with prolonged and continuous administration, the present invention is unlikely to produce side effects. Furthermore, the present invention can also be safely administered to infants and children. Therefore, the present invention is also applicable to the prevention, improvement, and / or treatment of diseases or their symptoms in infants and children.

[0148] This invention can be used for therapeutic purposes or for non-therapeutic purposes.

[0149] "Non-therapeutic purposes" refers to the concept of actions that do not involve medical treatment, i.e., actions that treat the human body. Examples include health promotion and cosmetic procedures.

[0150] "Improvement" refers to the improvement of a disease, symptom, or condition; the prevention or delay of the worsening of a disease, symptom, or condition; and the reversal, prevention, or delay of the progression of a disease or symptom.

[0151] "Prevention" refers to preventing or delaying the onset of diseases or symptoms in the target population, or reducing the risk of diseases or symptoms in the target population.

[0152] The dosage form of the pharmaceutical compositions of the present invention is not particularly limited. Specifically, examples include tablets, pills, powders, liquids, suspensions, emulsions, granules, capsules, syrups, suppositories, injections, ointments, patches, eye drops, and nasal drops. Furthermore, during formulation, additives commonly used as formulation carriers, such as excipients, binders, disintegrants, lubricants, stabilizers, flavoring and odor-correcting agents, diluents, surfactants, or solvents for injections, can be used.

[0153] Furthermore, during formulation, the pharmaceutical compositions of this invention may use excipients, pH adjusters, colorants, flavoring agents, and other ingredients commonly used in formulation. Additionally, provided it does not impair the efficacy of this invention, the pharmaceutical compositions of this invention may also use known or potentially future-discovered ingredients that have preventative, ameliorative, and / or therapeutic effects on diseases or symptoms related to this invention.

[0154] Based on this, formulation can be carried out using appropriate and well-known methods according to the dosage form. During formulation, a suitable formulation carrier can also be appropriately mixed in.

[0155] The content of *Lactobacillus helveticus* in the pharmaceutical composition of this invention can be appropriately set according to the dosage form, usage, patient's age, sex, type of disease, severity of disease, and other conditions, but is generally preferred to be 1 × 10⁻⁶. 6 ~1×10 12 cfu / g or 1×10 6 ~1×10 12 More preferably, it is in the range of cfu / mL, or more preferably in the range of 1×10⁻� 7 ~1×10 11 cfu / g or 1×10 7 ~1×10 11 Within the range of cfu / mL. When the *Lactobacillus helveticus* in this application is a dead bacterium, cfu / g or cfu / mL can be replaced with cells / g or cells / mL.

[0156] The timing of administration of the pharmaceutical composition of the present invention is not particularly limited, and the timing can be appropriately selected according to the treatment method for the target symptoms or disease. Furthermore, it can be administered prophylactically or used for maintenance therapy. Additionally, the administration method is preferably determined based on the formulation, the patient's age, sex, other conditions, and the severity of the patient's symptoms. It should be noted that the pharmaceutical composition of this technology can be administered once a day or in multiple divided doses under any circumstances, and can also be administered every few days or weeks.

[0157] <Food and beverage composition>

[0158] Furthermore, the compositions of the present invention can be used as food and beverage compositions. The food and beverage compositions of the present invention can be manufactured by adding the ingredients of the present application to known food and beverages, or by mixing the ingredients of the present application into the raw materials of food and beverages to create a new food and beverage composition.

[0159] There are no particular limitations on the food and beverage compositions of this invention as long as they contain the ingredients of this application. Examples of food and beverage compositions include beverages such as refreshing drinks, carbonated drinks, nutritional drinks, fruit juice drinks, and lactic acid bacteria drinks (including concentrated stock solutions and powders for preparation of these beverages); frozen desserts such as ice cream, sherbet, and shaved ice; confectionery such as candy, chewing gum, candy, gum, chocolate, compressed candy, snacks, biscuits, jelly, jam, cream, and baked goods; dairy products such as processed milk, milk beverages, fermented milk, drinkable yogurt, and butter; bread; enteral nutrition foods, liquid foods such as porridge, weaning foods, infant formula, and sports drinks; and other functional foods. Additionally, the food and beverage composition can also be a nutritional supplement, such as a tablet-form nutritional supplement. When used as a nutritional supplement, the *Lactobacillus helveticus* of this application can be ingested without affecting other foods, depending on the daily food intake and calorie consumption.

[0160] The food and beverage compositions of the present invention may contain various ingredients without impairing the effects of the present invention. For example, the food and beverage compositions of the present invention may contain various proteins such as whey protein, casein, soy protein, or pea protein, or mixtures or decomposed products thereof; amino acids such as leucine, valine, isoleucine, or glutamine; sugars such as galactose, glucose, fructose, mannose, cellobiose, maltose, lactose, sucrose, trehalose, dextrin, and starch, or human oligosaccharides such as 2'-fucosylated lactose, 3'-fucosylated lactose, lactose-N-fucopentose I, lactose-N-difucohexasose I, lactose-N-tetrasaccharide, and lactose-N-neotetrasaccharide; vitamins such as vitamin B6 or vitamin C; creatine; citric acid; or fish oil, etc.

[0161] The food and beverage composition of the present invention can be sold as a food and beverage composition or food product labeled for use inducing type I IFN production, inhibiting viral infection, antiviral use, immune stimulation, treatment or prevention of immune diseases, anticancer use, anti-inflammatory use, and prevention of bone loss. Furthermore, the food and beverage composition of the present invention can be sold as a food and beverage composition or food product labeled for use in preventing or treating diseases or symptoms by inducing type I IFN production. Additionally, the food and beverage composition or food product of the present invention can be labeled with phrases such as "desiring to inhibit viral infection," "desiring to maintain immune function," "acting on plasma-like dendritic cells to help maintain the immune function of healthy individuals," "desiring to inhibit allergies," "relieving nasal discomfort caused by allergens," "relieving eye discomfort caused by allergens," "desiring to inhibit inflammation," "desiring to maintain bone composition," "desiring to maintain strong bones," and "contributing to bone health by maintaining bone composition." Furthermore, any description expressing an effect incidentally produced by inducing type I IFN production is also permitted.

[0162] Furthermore, the food and beverage composition of the present invention can be provided / sold as a food and beverage composition or food and beverage labeled for use with probiotics or the like (including health care purposes). Additionally, as a target of consumption, the food and beverage composition or food and beverage can be provided / sold labeled as "those who wish to live in symbiosis with Lactobacillus," "those who wish to live in symbiosis with lactic acid bacteria," "those who wish to improve their intestinal environment," "those who wish to regulate their gastrointestinal tract," "those who wish to form a healthy intestinal environment," or "those who wish to improve their intestinal environment," etc.

[0163] The term "labeling" refers to all actions taken to inform the user of the aforementioned uses. Any labeling that evokes or analogizes the aforementioned uses is equivalent to the "labeling" of this invention, regardless of its purpose, content, object, or medium. However, it is preferable to label in a way that directly conveys the aforementioned uses to the user.

[0164] Specifically, examples include: acts of describing the above-mentioned uses on the food and beverage composition of the present invention or on the goods or packaging of the goods; acts of transferring, delivering, displaying or importing articles on the goods or packaging that describe the above-mentioned uses; acts of describing and displaying or publishing the above-mentioned uses in advertisements, price lists or transaction documents related to the goods, or providing such information by means of electromagnetic methods (Internet, etc.), etc., and especially preferably, indicating them on packaging, containers, product catalogs, brochures, POPs and other sales materials, and other documents.

[0165] Furthermore, as a label, it is preferable to have a label that has obtained administrative or other permission (for example, a label that has been granted permission based on various administrative regulations and is issued in the form of such permission). Examples include labels for health functional foods, and more specifically, labels for health functional foods, health foods, functional foods, enteral nutrition foods, foods for special purposes, nutritional functional foods, and quasi-drugs. Other labels permitted by the Consumer Affairs Agency can also be listed, such as labels for foods for designated health use, nutritional functional foods, functional labeled foods, and labels permitted under similar regulations. Examples of the latter include labels for foods for designated health use, labels for foods for designated health use with additional conditions, labels that aim to influence the structure and function of the body, labels that reduce the risk of disease, and functional labels based on scientific evidence. In further detail, labels for foods for designated health use (especially labels for health purposes) as stipulated in the Cabinet Office Ordinance (Cabinet Office Ordinance No. 57 of August 31, 2008) regarding permission for special purposes as stipulated in the Health Promotion Law, and similar labels, can be listed.

[0166] The content of *Lactobacillus helveticus* in the food and beverage composition of this invention can be appropriately set according to the form of the food and beverage composition, but it is generally preferred to be 1 × 10⁻⁶. 6 ~1×10 12 cfu / g or 1×10 6 ~1×10 12 More preferably, it is in the range of cfu / mL, or more preferably in the range of 1×10⁻� 7 ~1×10 11 cfu / g or 1×10 7 ~1×10 11 Within the range of cfu / mL.

[0167] As an example of the food and beverage composition involved in this invention, infant formula milk powder (also known as baby formula milk powder) can be cited. Baby formula milk powder refers to infant formula milk powder for infants aged 0-12 months, follow-up milk powder for infants aged 6-9 months and above (under 3 years old), formula milk powder for low birth weight infants (newborns with a birth weight of less than 2500g), and various therapeutic milk powders for treating infants with conditions such as milk allergy and lactose intolerance. Furthermore, this composition can be used in health functional foods and patient foods. The health functional food system is established based on domestic and international dynamics and in coordination with previous specific health food systems. It covers not only conventional foods but also foods in the form of tablets, capsules, etc., and includes two types: specific health foods (individually licensed type) and nutritional functional foods (standard specification type).

[0168] It should be noted that, in this invention, the form of infant formula can be liquid milk in addition to milk powder.

[0169] <Manufacturing Method>

[0170] The method for manufacturing the composition of the present invention will be described below. The method for manufacturing the composition of the present invention includes a step of adding the component of this application to raw materials. In the present invention, the step of adding the component of this application can be performed at any stage of the manufacturing process.

[0171] Alternatively, the invention may include a step of adding *Lactobacillus helveticus* (as described in this application) to the raw material to ferment it. In this case, the composition of the invention may be provided in the form of a fermentation composition.

[0172] The following are examples of methods for manufacturing the compositions of the present invention, but are not limited thereto.

[0173] As an example of a manufacturing method, a method for manufacturing a composition that includes at least one of the steps (a) or (b) below can be listed.

[0174] (a) The process of mixing the ingredients of this application with prebiotics

[0175] (b) The process of mixing the ingredients of this application with the milk ingredients

[0176] At this point, a composition containing the components of this application may be used as an ingredient. Furthermore, the composition may also contain probiotics such as Lactobacillus, lactic acid bacteria, and Bifidobacterium.

[0177] The process of mixing the prebiotic and / or milk components can be performed at any stage of the manufacturing process. For example, in the case of mixing the milk components, it can be performed at the same time as step (a), or it can be performed before or after that step.

[0178] In addition, it is suitable for the milk component to be in powder form, and it is even more suitable to mix it with the Lactobacillus helveticus powder of this application.

[0179] In addition, as a method for manufacturing the composition of the present invention, a method for manufacturing the composition including the following steps (A) and (B) can also be listed.

[0180] We can provide:

[0181] (A) A process of culturing *Lactobacillus helveticus* of this application in a culture medium to obtain a culture or product containing *Lactobacillus helveticus* of this application;

[0182] (B) The process of drying the culture or product to obtain bacterial powder or dried product.

[0183] Freeze-drying or spray drying is suitable for this drying process.

[0184] The milk component is not particularly limited, and examples include cow's milk, buffalo milk, goat's milk, mare's milk, skim milk, skim milk concentrate, skim milk powder, concentrated milk, whole milk powder, cream, butter, buttermilk, condensed milk, lactooligosaccharides, and milk proteins, etc., and one or more selected from the group consisting of them can be used. Furthermore, the milk protein is not particularly limited, and examples include whey, casein, and their hydrolysates, etc., and one or more selected from the group consisting of them can be used. Among the milk components, those derived from cow's milk are preferred.

[0185] The lactooligosaccharides are not limited to bovine milk oligosaccharides; for example, they can also be human milk oligosaccharides. There are no particular limitations on human milk oligosaccharides; examples include 2'-fucosylated lactose, 3'-fucosylated lactose, lactose-N-fucopentose I, lactose-N-difucohexasaccharide I, lactose-N-tetrasaccharide, lactose-N-neotetrasaccharide, etc., and one or more from the group consisting of these can be used.

[0186] The hydrolysate can be produced by hydrolyzing the milk components (suitably milk proteins) to produce whey hydrolysate, casein hydrolysate, etc. Examples of such hydrolysis include acid / alkali hydrolysis and enzymatic hydrolysis. Enzymatic hydrolysis is suitable, and proteolytic enzymes are preferred. Examples of proteolytic enzymes include proteases, trypsin, chymotrypsin, plasmin, pepsin, papain, peptidase, and aminopeptidase. The pH of the hydrolysis reaction is appropriately adjusted to the pH most suitable for the enzyme used, for example, pH 2 to 6. The temperature of the hydrolysis reaction is not particularly limited, but it is generally suitable to carry it in the range of 30 to 60°C. The reaction time is preferably 2 to 10 hours.

[0187] The form of *Lactobacillus helveticus* or its culture is not particularly limited in this application; it may be liquid or solid. However, from the viewpoint of ease of handling during manufacturing and storage, a dried form (e.g., bacterial powder, dried culture, etc.) is suitable. Furthermore, as mentioned above, examples of such a culture include the bacterial cells themselves after separation from the culture medium, a culture containing the bacterial cells, and a culture without the bacterial cells.

[0188] The drying method is not particularly limited and may include spray drying, boiling sterilization, freeze drying, UHT sterilization, pressure sterilization, high-pressure steam sterilization, dry heat sterilization, flowing steam sterilization, electromagnetic wave sterilization, electron beam sterilization, high-frequency sterilization, radiation sterilization, ultraviolet sterilization, ethylene oxide gas sterilization, hydrogen peroxide plasma sterilization, and chemical sterilization methods (alcohol sterilization, formalin fixation, and water electrolysis).

[0189] In addition, the bacterial cells can be broken down. The broken material can be bacterial cells that have been broken down into live bacteria or dead bacteria, or bacterial cells that have been subjected to heating, freeze-drying, or other processes after being broken down.

[0190] From the viewpoint of increasing the viability of bacteria, it is suitable to freeze-dry the culture; from the viewpoint of increasing production efficiency, it is suitable to spray-dry the culture.

[0191] Furthermore, when manufacturing nutritional supplements or tablets, the composition of the present invention may include: a step of mixing a culture containing *Lactobacillus helveticus* of this application with excipients to obtain a mixture; and a step of molding it into the specified shape. As a molding step, examples include tableting, and as an example of tableting, a mixture of powders and granules may be compressed into tablets.

[0192] As an example of the manufacturing method of the present invention, a method for manufacturing a fermented food and beverage (suitably fermented milk) containing the present application's Lactobacillus helveticus can be cited.

[0193] The process involves adding the bacterial powder obtained in the aforementioned manufacturing method to fermented milk raw materials to obtain fermented milk using *Lactobacillus helveticus* as described in this application. Alternatively, the process involves mixing the bacterial powder with fermented milk to obtain fermented milk containing *Lactobacillus helveticus* as described in this application. The amount of *Lactobacillus helveticus* cells in the composition is adjusted by regulating the fermentation conditions and mixing amount to achieve a predetermined amount. Thus, fermented food and beverages (suitably fermented milk or fermented products) containing *Lactobacillus helveticus* as described in this application can be provided.

[0194] Example

[0195] The present invention will now be described in further detail based on embodiments. It should be noted that the embodiments described below are merely examples of representative embodiments of the present invention and are not intended to limit the scope of the invention.

[0196] (1) Preparation of heat-sterilized forms of lactic acid bacteria

[0197] The four lactic acid bacteria strains listed in Table 1 were cultured statically at 30°C or 37°C for 16 hours on MRS or M17 medium, respectively. After culture, the bacteria were collected, washed twice with sterile water, and then sterilized in an autoclave at 90°C for 15 minutes. Then, the cultures were prepared into 1×10⁻⁶ cultures. 8 Cells / mL or 1×10 9 Each strain was adjusted with sterile water at a concentration of cells / mL to obtain heat-sterilized bodies for testing.

[0198] [Table 1]

[0199]

[0200] (2) Preparation of dendritic cells

[0201] Dendritic cells (DCs) used in the experiment were isolated from human peripheral blood mononuclear cells (PBMCs) purchased from STEMCELL Technologies using the EasySep Human Pan-DC Pre-Enrichment Kit (STEMCELL Technologies). The DCs were then suspended in RPMI-1640 medium supplemented with antibiotics and a final concentration of 10% heat-inactivated fetal bovine serum (FBS) to achieve a density of 1 × 10⁻⁶ cells / mL. 6 The concentration was adjusted at a rate of cells / mL. The prepared suspension contained a mixture of plasmacytoid dendritic cells (pDCs) and myeloid dendritic cells (mDCs).

[0202] (3) Measurement of type I IFN production in dendritic cells induced by bactericidal stimulation

[0203] Add bactericides of various strains prepared in (1) to the dendritic cell suspension prepared in (2), and culture in a CO2 incubator at 37°C and 5% CO2.

[0204] It should be noted that the bactericidal agents of the above four strains were added at a rate of 10 times the number of dendritic cells. In addition, for JCM5805, an additional test was conducted in which the bactericidal agent was added at a rate of 100 times the number of dendritic cells.

[0205] The culture supernatant was recovered 24 hours after the start of culture. The concentration of IFNα was determined by flow cytometry using an IFNα assay kit (Cytometric BeadArray Kit, Becton Dickinson). The concentration of IFNβ was determined by ELISA using an IFNβ assay kit (Human IFN-beta DuoSet, R&D Systems).

[0206] (4) Results

[0207] The results of measuring the production of INFα and INFβ in dendritic cells induced by bactericidal stimulation of various lactic acid bacteria are shown in the figure. Figure 1 and Figure 2 .

[0208] Lactobacillus helveticus MCC2430 exhibited the highest induction capacity for both INFα and INFβ production. It should be noted that Lactobacillus helveticus SBT2171, as described in Japanese Patent No. 6705628 (Patent Document 2), is known to contribute to the increase of pDCs. Furthermore, Lactobacillus lactis subsp. Lactis JCM5805, as described in Japanese Patent No. 6170190 (Patent Document 1), is known to activate pDCs and promote IFNα production. This demonstrates that MCC2430 possesses a significantly higher induction capacity for type I IFN production compared to these known strains.

[0209] [Manufacturing Example]

[0210] Examples of manufacturing pharmaceutical and food compositions of the present invention are shown below, but the compositions of the present invention are not limited thereto.

[0211] [Manufacturing Example 1]

[0212] Lactobacillus helveticus MCC2430 was added to MRS liquid medium and anaerobically cultured at 37°C for 16 hours. The culture was then concentrated and freeze-dried to obtain a lyophilized bacterial powder (bacterial powder). The bacterial powder was uniformly mixed with whey protein concentrate (WPC) to obtain a composition. 20g of this composition was dissolved in 200g of water to obtain a composition containing Lactobacillus helveticus MCC2430 of the present invention.

[0213] [Manufacturing Example 2]

[0214] Lactobacillus helveticus MCC2430 was added to MRS liquid medium and anaerobically cultured at 37°C for 16 hours. After centrifugation and three washings with distilled water, the bacterial cells were resuspended in distilled water at a concentration of 10 mg (based on bacterial weight) / ml and then sterilized by heating at 100°C for 15 minutes to prepare a solution containing the heat-sterilized cells. The solution containing the heat-sterilized cells was concentrated and freeze-dried to obtain a lyophilized powder (bacterial powder). The bacterial powder was then uniformly mixed with oligosaccharides to obtain a composition. Oligosaccharides such as 2'-fucosylated lactose, 3'-fucosylated lactose, lactose-N-fucopentose I, lactose-N-difucohexasose I, lactose-N-tetrasaccharide, lactose-N-neotetrasaccharide, etc., or isomaltooligosaccharides, lactulose, raffinose, fructooligosaccharides, galactooligosaccharides, and soybean oligosaccharides can be used.

[0215] [Manufacturing Example 3]

[0216] Lactobacillus helveticus MCC2430 was added to MRS liquid medium and cultured at 37°C for 16 hours. The culture was then concentrated and freeze-dried to obtain a lyophilized bacterial powder. The bacterial powder was uniformly mixed with a dried powder of milk protein concentrate (MPC480, manufactured by Fonterra, protein content 80% by mass, casein:whey protein = approximately 8:2) to obtain a composition. 20g of this composition was dissolved in 200g of water to obtain a composition containing Lactobacillus helveticus MCC2430 of the present invention.

[0217] [Manufacturing Example 4]

[0218] Lactobacillus helveticus MCC2430 was added to MRS liquid medium and cultured at 37°C for 16 hours. The culture was then concentrated and freeze-dried to obtain a freeze-dried powder (bacterial powder) containing Lactobacillus helveticus MCC2430 of the present invention. Next, the bacterial powder was mixed with crystalline cellulose in a stirred granulator. Then, purified water was added for granulation, and the granules were dried to obtain a granulated product (pharmaceutical composition) containing the bacterial fermentation components and excipients. Thus, a granulated product containing Lactobacillus helveticus MCC2430 of the present invention can be obtained.

[0219] [Manufacturing Example 5]

[0220] The method for producing fermented milk with added Lactobacillus helveticus MCC2430 is as follows.

[0221] First, the milk raw material is mixed with water and other ingredients as needed, preferably homogenized, and then heat-sterilized. Homogenization and heat sterilization can be performed using conventional methods. Lactic acid bacteria starter culture is added (inoculated) into the heat-sterilized milk preparation, and fermentation is maintained at a specified temperature to obtain fermented material. Curd is formed through fermentation.

[0222] As a lactic acid bacteria starter, bacteria commonly used in yogurt making, such as *Lactobacillus bulgaricus*, *Lactococcus lactis*, and *Streptococcus thermophilus*, can be used. When the pH reaches the target value, the formed curd is broken up by stirring and cooled to below 10°C to obtain the fermented product. Cooling to below 10°C reduces the activity of the lactic acid bacteria and inhibits acid formation.

[0223] Next, the fermented product obtained in the fermentation process is heated to obtain a heated fermented product (heat-treated fermented product). By moderately heating the fermented product, the acid production caused by lactic acid bacteria in the heated fermented product can be suppressed. This, in turn, can suppress the decrease in pH during subsequent manufacturing processes and / or storage.

[0224] Next, *Lactobacillus helveticus* MCC2430 is added to the post-fermentation product obtained in the heat treatment process. The preferred amount of *Lactobacillus helveticus* MCC2430 added relative to the post-fermentation product is 1 × 10⁻⁶. 7 ~1×10 11 cfu / mL, more preferably 1×10⁻⁶ 8 ~1×10 10 cfu / mL. When Lactobacillus helveticus MCC2430 is a dead bacterium, cfu / mL can be replaced with cells / mL.

[0225] After adding *Lactobacillus helveticus* MCC2430 to the heated fermentation product, concentration is carried out. The concentration process can be performed using well-known concentration methods, such as centrifugation or membrane separation.

[0226] In centrifugation, whey is removed from the concentrate, resulting in concentrated fermented milk with a higher concentration of solid components.

[0227] As described above, fermented milk containing Lactobacillus helveticus MCC2430 of the present invention can be produced.

[0228] By ingesting or administering fermented milk containing Lactobacillus helveticus MCC2430 of the present invention as described above, it is expected to obtain effects such as type I IFN production induction, viral infection inhibition, antiviral effects, immune stimulation, treatment or prevention of immune diseases, anticancer effects, anti-inflammatory effects, and prevention of bone loss.

[0229] [Manufacturing Example 6]

[0230] The manufacturing method of formula milk powder containing Lactobacillus helveticus MCC2430 is as follows.

[0231] 10 kg of desalted bovine whey protein powder (Mirai), 6 kg of bovine casein powder (Fonterra), 48 kg of lactose (Mirai), 920 g of mineral mixture (Tomita Pharmaceutical), 32 g of vitamin mixture (Tanabe Pharmaceutical), 500 g of lactulose (Morinaga Milk Industry), 500 g of raffinose (Nippon Beet Sugar), and 900 g of galactooligosaccharide syrup (Yakult Pharmaceutical Industry Co.) were dissolved in 300 kg of warm water and then heated at 90°C for 10 minutes. 28 kg of modified fat (Taiyo Oils) was added for homogenization. Then, sterilization and concentration were performed, followed by spray drying to prepare approximately 95 kg of formula milk powder. 1.8 × 10⁻⁶ Lactobacillus helveticus MCC2430 bacterial cell powder dispersed in starch was added to this powder. 11 100g of (CFU / g, manufactured by Morinaga Milk Industry Co., Ltd.) is used to prepare approximately 95kg of lactic acid bacteria / oligosaccharide blended formula milk powder. The resulting formula milk powder is dissolved in water, and when prepared into a milk preparation with a total solids concentration of 14% (w / v) as a standard formula milk concentration, the lactic acid bacteria count in the milk preparation can be 2.7 × 10⁻⁶. 9 cfu / 100mL.

[0232] The above-mentioned formula milk powder may also contain human human oligosaccharides such as 2'-fucosylated lactose, 3'-fucosylated lactose, lactose-N-fucopentose I, lactose-N-difucohexasaccharide I, lactose-N-tetrasaccharide, and lactose-N-neotetrasaccharide, or probiotics such as Lactobacillus bacteria, lactic acid bacteria, and Bifidobacterium bacteria other than Lactobacillus helveticus MCC2430.

[0233] By ingesting or administering formula milk containing Lactobacillus helveticus MCC2430 of the present invention as described above, it is expected to obtain effects such as type I IFN induction, viral infection inhibition, antiviral effects, immune stimulation, treatment or prevention of immune diseases, anticancer effects, anti-inflammatory effects, and prevention of bone loss.

[0234] Industrial availability

[0235] This invention can be applied to food and pharmaceutical products containing lactic acid bacteria.

Claims

1. A bacterium classified as *Lactobacillus helveticus*, which is capable of activating plasmacytoid dendritic cells (pDCs) and / or myeloid dendritic cells (mDCs) and inducing the production of type I IFN, inducing more than 1.5 times the production of type I IFN compared to *Lactobacillus helveticus* SBT2171 (accession number: Ferm BP-5445).

2. A Lactobacillus helveticus MCC2430 (accession number: NITE BP-03882).

3. A composition comprising at least one selected from the bacteria of claim 1 or 2, a culture of the bacteria, and a cell treatment of the bacteria.

4. The composition according to claim 3, wherein, The composition is a composition for inducing the generation of type I IFN.

5. The composition according to claim 3, wherein, The composition is selected from at least one of the following groups: antiviral compositions, immune-stimulating compositions, compositions for the treatment or prevention of immune diseases, anticancer compositions, anti-inflammatory compositions, and compositions for the prevention of decreased bone strength.

6. The composition according to claim 3, wherein it is a food or beverage or a pharmaceutical product.

7. A method for manufacturing a composition, comprising the step of adding at least one selected from the bacteria of claim 1 or 2, a culture of the bacteria, and a bacterial cell treatment to a raw material.

8. The manufacturing method according to claim 7, comprising a step of fermenting the raw material using the bacteria, wherein the composition is a fermentation composition.

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