Novel lactobacillus paracasei and use thereof

Abstract

The present invention relates to a novel Lactobacillus paracasei and a use thereof.

Description

Novel Lactobacillus paracasei strain and its use

[0001] [Cross-reference with related applications]

[0002] This application claims the benefit of priority from Korean Patent Application No. 10-2023-0084068, filed June 29, 2023, the entire contents of which are incorporated herein by reference.

[0003] The present invention relates to a novel Lactobacillus paracasei strain and its use.

[0004] Influenza viruses are RNA viruses belonging to the genus Myxoviridae (Influenzavirus) of the family Orthomyxoviridae. They are 80-120 ㎛ in length, and are mostly spherical, but some are linear. They are broadly divided into three types: A, B, and C. Type A is divided into subtypes A0, A1, and A2, and type B is divided into subtypes B1, B1α, B1β, and B2, with type A showing greater mutations. Type A viruses have the characteristic of changing their antigenic structure little by little, and sometimes rapidly. This rapid change is called discontinuous variation. For example, if you are infected with a subtype of influenza virus of type A, you acquire immunity to that subtype of virus, but if a new type of virus is created due to discontinuous variation, a pandemic can occur.

[0005] Currently, attenuated influenza virus vaccines are used as preventative technologies for influenza. However, rapid vaccines for novel influenza strains, such as the novel (H1N1) influenza and avian influenza, pose challenges due to their long viral replication times and the difficulty of mass production. Furthermore, they are ineffective against variant viruses. Furthermore, most conventional influenza vaccines are administered via injection, which complicates the process and imposes significant time and space constraints. Furthermore, they are expensive and pose safety concerns due to direct virus administration.

[0006] Accordingly, there has been a continuous demand for the development of novel biological agents with antiviral effects against influenza to address the shortcomings of conventional influenza vaccines.

[0007] Also, the microbiome is a compound word made up of microbiota and genome, and can be said to be a microbial community that includes microorganisms that live or coexist in all environments, including humans, animals and plants, soil, oceans, lakes, rocks, and the atmosphere, as well as their entire genetic information. Recently, it is also used to refer to microbial communities in various environments. Research is being conducted on various microbiomes, such as plant microbiome, animal microbiome, environmental microbiome, human microbiome, gut microbiome, and skin microbiome, and among these, research on the human microbiome is the most extensive.

[0008] The human body is a complex and vibrant ecosystem, and various microorganisms exist in the stomach, skin, and other areas of the human body. In the stomach and skin, over 500 to 1,000 different microorganisms coexist, forming a microbial flora. Intestinal microorganisms in the stomach synthesize and supply substances such as vitamins to the human body, and play a role in suppressing infections caused by pathogenic microorganisms introduced through contaminated food. The present inventors confirmed that the Lactobacillus paracasei strain exhibits excellent efficacy in suppressing influenza virus infection, and discovered the possibility that Lactobacillus paracasei can be used to prevent or treat influenza virus infection, thereby completing the present invention.

[0009] One object of the present invention is to provide a novel Lactobacillus paracasei strain.

[0010] Another object of the present invention is to provide a composition comprising the novel strain.

[0011] Another object of the present invention is to provide a use of a composition comprising the novel strain.

[0012] In order to achieve the above object, one aspect of the present invention provides a Lactobacillus paracasei MI29 strain deposited under accession number KCTC 14636BP.

[0013] In addition, in order to achieve the above purpose, another aspect of the present invention provides a composition comprising at least one selected from the group consisting of the strain, a culture solution of the strain, a concentrate of the culture solution, a dried product of the culture solution, and an extract of the culture solution.

[0014] In addition, in order to achieve the above purpose, another aspect of the present invention provides a health functional food composition for improving immunity, a food composition, a probiotics composition, or a feed / feed additive composition, or a pharmaceutical composition for preventing or treating a disease related to immunodeficiency, comprising at least one selected from the group consisting of the strain, a culture solution of the strain, a concentrate of the culture solution, a dried product of the culture solution, and an extract of the culture solution.

[0015] In addition, in order to achieve the above purpose, another aspect of the present invention provides a health functional food composition for inhibiting influenza virus infection, a food composition or a feed / feed additive composition, or a pharmaceutical composition for preventing or treating influenza virus infection disease, comprising at least one selected from the group consisting of the strain, a culture solution of the strain, a concentrate of the culture solution, a dried product of the culture solution, and an extract of the culture solution.

[0016] The novel Lactobacillus paracasei strain of the present invention has the effect of increasing the expression of genes and proteins of interferon or cytokines, and exhibits the activity of inhibiting lung damage in mice when orally administered to influenza-infected mice, and thus has the advantage of being usefully used for the purpose of inhibiting influenza virus infection, or as a medicine, health functional food, food, and feed for preventing, improving, or treating influenza virus infection diseases.

[0017] However, the effects of the present invention are not limited to the effects mentioned above, and other effects not mentioned will be clearly understood by those skilled in the art from the description below.

[0018] Figure 1 shows the results of an experiment confirming the effect of increasing IFN-1 interferon by the novel Lactobacillus paracasei strain of the present invention.

[0019] Figure 2 shows the results of confirming changes in body weight and interferon-stimulated gene (ISG) expression by the novel Lactobacillus paracasei strain of the present invention.

[0020] Figure 3 shows the results of an experiment confirming the effect of reducing virus titer by the novel Lactobacillus paracasei strain of the present invention.

[0021] Figures 4a and 4b show the results of confirming the expression levels of interferon-induced genes, cytokines, and fatty acid synthesis-related genes in a mouse model orally administered the novel Lactobacillus paracasei strain of the present invention.

[0022] Figure 5 shows the results of confirming the expression of the IFN-1 gene and the number of pDCs and monocytes by the novel Lactobacillus paracasei strain of the present invention.

[0023] Figure 6 shows the results of histological analysis of the lungs in a mouse model orally administered the novel Lactobacillus paracasei strain of the present invention.

[0024] Figure 7 shows the results of analyzing the metabolic pathways appearing in the cecum and lung tissue of a mouse model orally administered the novel Lactobacillus paracasei strain of the present invention.

[0025] Figure 8 shows the results of an experiment confirming the relationship between the novel Lactobacillus paracasei strain of the present invention and fatty acids.

[0026] Hereinafter, the present invention will be described in detail.

[0027]

[0028] The terms and words used in this specification and claims should not be interpreted as limited to their usual or dictionary meanings, but should be interpreted as meanings and concepts that conform to the technical idea of ​​the present invention, based on the principle that the inventor can appropriately define the concept of the term to explain his or her own invention in the best way.

[0029]

[0030] 1. Novel Lactobacillus paracasei strain

[0031] One aspect of the present invention provides a novel Lactobacillus paracasei strain.

[0032] The novel Lactobacillus paracasei strain of the present invention includes 16S rRNA having a base sequence of SEQ ID NO: 1, and is particularly the Lactobacillus paracasei MI29 strain deposited with the Korean Collection for Type Culture (KCTC) of the Korea Research Institute of Bioscience and Biotechnology under the accession number KCTC 14636BP on July 13, 2021. The Lactobacillus paracasei strain was reclassified through whole-genome analysis by an internationally recognized organization (Microbiology Society) on April 15, 2020, and was also revised in the “Standards and Specifications for Health Functional Foods” on April 20, 2021 (Ministry of Food and Drug Safety Notice No. 2021-168), and is also called Lacticaseibacillus paracasei. Therefore, in the present invention, Lactobacillus paracasei MI29 and Lacticazeibacillus paracasei MI29 are meant to be the same strain.

[0033] The novel Lactobacillus paracasei strain of the present invention may be a strain capable of living in the intestines of humans or non-human animals, and may exert various effects on the health of humans or non-human animals while living in the intestines. The novel Lactobacillus paracasei strain of the present invention may be a safe microorganism because it does not exhibit toxicity or cause diseases to humans or non-human animals, and may act as a beneficial bacterium that helps the health of humans or non-human animals in the intestines, and thus may function as a probiotic microorganism.

[0034] The strain of the present invention may have the ability to utilize one or more sugars selected from the group consisting of ribose, D-galactose, D-glucose, D-fructose, D-mannose, L-sorbose, D-mannitol, D-sorbitol, N-acetylglucosamine, amygdalin, arbutin, esculin, salicin, D-cellobiose, D-maltose, D-lactose, D-saccharose, D-trehalose, inulin, D-melezitose, gentiobiose, D-turanose, D-tagatose, and gluconate.

[0035] In a specific embodiment of the present invention, the culture solution of the strain of the present invention and the standard strain of Lactobacillus paracasei were each inoculated into tubes of various sugar substrates included in the API CHL50 kit, and then the sugar utilization ability of the strain was measured. As a result, the strain of the present invention can utilize ribose, D-galactose, D-glucose, D-fructose, D-mannose, L-sorbose, D-mannitol, D-sorbitol, N-acetylglucosamine, amygdalin, arbutin, esculin, salicin, D-cellobiose, D-maltose, D-lactose, D-saccharose, D-trehalose, inulin, D-melezitose, gentiobiose, D-turanose, D-tagatose and gluconate, and can utilize glycerol, erythritol, D-arabinose, L-arabinose, D-xylose, andonitol, methyl-β-D-xylopyranoside, L-rhamnose, dulcitol, inositol, methyl-α-D-mannopyranoside, It was confirmed that methyl-α-D-glucopyranoside, D-melibose, D-raffinose, amidone, xylitol, D-lyxose, D-fucose, L-fucose, D-arabitol, L-arabitol, 2-keto-gluconate and 5-keto-gluconate were not available. In addition, it was confirmed that the strain of the present invention, unlike the standard strain, metabolizes L-sorbose, D-sorbitol, amygdalin, D-maltose, D-lactose, D-saccharose and inulin.

[0036] Therefore, it was confirmed that the strain of the present invention is a novel strain different from the conventional strain, not only because it differs from the standard strain in 16S rRNA base sequence, but also because it differs in terms of the strain's sugar utilization ability depending on the type of sugar.

[0037] Probiotics are live microorganisms that provide health benefits. They play a crucial role in suppressing harmful bacteria and increasing beneficial bacteria in the intestinal flora. To be recognized as a probiotic, they must survive stomach acid and bile, reach the small and large intestines, proliferate and establish there, exhibit beneficial effects within the intestines, and be non-toxic and non-pathogenic.

[0038] The strain of the present invention may have physiological characteristics that enable it to be utilized as a probiotic.

[0039] Specifically, the strain may be acid resistant. The acid resistance may mean a property of the strain to survive well in an acidic environment, and may mean that the survival rate does not decrease even when the strain is exposed to an acidic environment of pH 5 or lower, for example, pH 4.5 or lower, pH 4 or lower, pH 3.5 or lower, and particularly, pH 3 or lower for 1 hour or longer, for example, 1.2 hours or longer, 1.3 hours or longer, 1.4 hours or longer, 1.5 hours or longer, 1.6 hours or longer, 1.7 hours or longer, 1.8 hours or longer, 1.9 hours or longer, and particularly, 2 hours or longer, or rather, the survival rate increases by 100% or more, for example, 110% or longer, 120% or longer, 130% or longer, and particularly, 140% or longer.

[0040] In a specific embodiment of the present invention, the Lactobacillus paracasei MI29 strain of the present invention was confirmed to have excellent acid resistance, as it showed a survival rate of about 142% even after being cultured for 2 hours under conditions of pH 3.

[0041] Additionally, the strain may be cholestatic. The above bile resistance refers to the property of the strain to survive well in a biliary environment where bile distributed from the gallbladder exists, and in a biliary environment where bile such as oxgall and bile salt exists at a concentration of 0.5% or less, for example, 0.45% or less, 0.4% or less, 0.35% or less, and especially 0.3% or less, the survival rate does not decrease even after 5 hours or more, for example, 6 hours or more, 7 hours or more, 8 hours or more, 9 hours or more, 10 hours or more, 11 hours or more, and especially 12 hours or more, or the survival rate is 200% or more, for example, 300% or more, 400% or more, 500% or more, 600% or more, 700% or more, 800% or more, 900% or more, 1000% or more, 1100% or more, 1200% or more, 1300% This could mean an increase of 1400% or more, 1500% or more, 1600% or more, 1700% or more, 1800% or more, 1900% or more, 2000% or more, 2100% or more, and especially 2200% or more.

[0042] In a specific embodiment of the present invention, the Lactobacillus paracasei MI29 strain of the present invention was cultured for 12 hours in a medium containing 0.3% Oxagall, and was found to have excellent bile tolerance, showing a survival rate of about 2208%.

[0043] The above Lactobacillus paracasei MI29 strain has excellent acid resistance and bile resistance, so it can survive in the stomach and reach the intestines.

[0044] In addition, the strain may not exhibit antibiotic resistance or may have low resistance to the antibiotic. The resistance may be extrinsic resistance, and extrinsic resistance refers to resistance that can be induced when an antibiotic resistance gene is introduced into another external bacterium through a mobile means such as a plasmid or transposon. Therefore, the strain of the present invention that does not exhibit extrinsic resistance to antibiotics does not horizontally transfer the resistance gene to harmful bacteria existing in the intestine, so there is no concern that harmful bacteria in the intestine will acquire extrinsic resistance and cause resistance problems to antibiotics.

[0045] The antibiotics to which the strain of the present invention does not exhibit resistance are not particularly limited, as long as they are known in the technical field to which the present invention belongs, and may be, for example, penicillin antibiotics, tetracycline antibiotics, macrolide antibiotics, sulfonamide antibiotics, amphenicol antibiotics, aminoglycoside antibiotics, etc., and specifically, may be ampicillin, gentamicin, erythromycin, tetracycline, and chloramphenicol.

[0046] In a specific embodiment of the present invention, the MI29 strain was tested for antibiotic resistance using the microdilution method (CLSI, M07-A9). As a result, it was confirmed that the strain had lower resistance to ampicillin, gentamicin, erythromycin, tetracycline, and chloramphenicol than that described in the EFSA guide (European Food Safety Authority antibiotic safety standards). In particular, in Europe, it has long been prohibited to commercialize lactic acid bacteria that can horizontally transfer antibiotic resistance genes to other bacteria, and in Korea, the Ministry of Food and Drug Safety recently inserted a clause on antibiotic resistance transfer into the standards for judging microorganisms as food ingredients. Therefore, the strain of the present invention has the advantage of being recognized as safe as a probiotic and thus advantageous for commercialization.

[0047] Therefore, the novel Lactobacillus paracasei MI29 strain of the present invention has excellent acid resistance and bile resistance, so it can survive in the stomach and reach the intestines, and has low antibiotic resistance, so it is recognized as safe, and as a probiotic, the effect of improving intestinal flora, bowel movement, and promoting intestinal health can be maintained for a long time. As described above, since the strain satisfies all the advantageous conditions that a probiotic should have, the novel Lactobacillus paracasei MI29 strain of the present invention can be usefully used as a probiotic for improving intestinal flora, bowel movement, and promoting intestinal health.

[0048] The strain of the present invention may possess physiological characteristics that make it suitable for use as a probiotic. In particular, the novel Lactobacillus paracasei strain of the present invention may enhance immunity. Therefore, since the strain of the present invention possesses the physiological characteristics necessary for enhancing immunity, it can be utilized as a probiotic for enhancing immunity.

[0049] The above-mentioned improvement in immunity refers to an enhancement of all immune activities related to killing target cells through the body's immune function. The above-mentioned immunity may refer to a mechanism for physiologically recognizing and eliminating foreign and endogenous foreign substances in the human and animal body, and maintaining homeostasis. For example, the above-mentioned immune activity may include cytokine secretion activity and immune cell production activity.

[0050] The above novel strain may enhance cytokine secretion activity.

[0051] The above cytokine may refer to a protein (~5-20 kDa) that plays a role in cell signaling. The cytokines are released by cells and influence the behavior of the immune cells and / or other cells that release the cytokines. Non-limiting examples of cytokines include chemokines, interferons, interleukins, lymphokines, tumor necrosis factor, monokines, and colony-stimulating factors. Cytokines can be produced by a wide range of cells, including, but not limited to, immune cells such as macrophages, B lymphocytes, T lymphocytes, mast cells, and monocytes, endothelial cells, fibroblasts, and stromal cells. The cytokine may be an interferon, an interleukin, or a mutant thereof. Many interleukins are synthesized by NK cells, helper CD4 T lymphocytes, as well as monocytes, macrophages, and endothelial cells. The interleukins can promote the development and differentiation of T and B lymphocytes and hematopoietic cells. Non-limiting examples of the interleukins include IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8 (CXCL8), IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, IL-18, IL-19, IL-20, IL-21, IL-22, IL-23, IL-24, IL-25, IL-26, IL-27, IL-28, IL-29, IL-30, IL-31, IL-32, IL-33, IL-35, IL-36 or mutants thereof. Interferon, a type of cytokine, is a glycoprotein synthesized and secreted by host cells when infected with various pathogens, such as viruses, bacteria, and parasites, or in the presence of cancer cells. Type 1 interferon, secreted by virus-infected cells, is widely known and helps surrounding cells exhibit antiviral defenses.

[0052] In particular, the novel strain may activate the signaling of type 1 interferon.

[0053] The types of type 1 interferons include IFN-α, IFN-β, IFN-ε, IFN-κ, and IFN-ω. Type 1 interferons are generally produced in response to viral invasion and are primarily produced by fibroblasts and monocytes. Furthermore, type 1 interferons activate various molecules that inhibit viral nucleic acid replication. Furthermore, type 1 interferons can enhance the expression of interferon-stimulated genes by binding to the IFN-α receptor and promoting the transcription of ISGs, which encode various proteins with antiviral functions.

[0054] The above type 1 interferon is produced by the host and is mainly mediated by plasmacytoid dendritic cells (pDCs) or Ly6C, which are special sensors of viral nucleic acids. hi It is an antiviral substance produced by monocytes. In relation to the activation of type 1 interferon, the novel strain is a plasmacytoid dendritic cell or Ly6C hi It may further activate the signal of the interferon by promoting the production of monocytes.

[0055] Accordingly, the novel Lactobacillus paracasei strain of the present invention may have an activity of inhibiting infection by microorganisms such as viruses or bacteria. The virus may be, for example, SI (swine flu), avian influenza virus, rhinovirus, adenovirus, coronavirus, parainfluenza virus, respiratory syncytial virus, herpesvirus (HSV), acquired immunodeficiency syndrome virus (HIV), hepatitis virus, etc., and may be influenza virus in particular, but is not limited thereto. Specifically, it includes a virus that is killed or inhibited by increasing the secretion of IFN-γ in NK cells. The above bacteria may include, but are not limited to, Escherichia Coli, Staphylococcus Aureus, Pseudomonas Aeruginosa, Aspergillus niger, and Candida albicans.

[0056] In particular, the novel strain of the present invention may have antiviral activity against influenza viruses.

[0057] The above influenza virus is an RNA virus belonging to the family Orthomyxoviridae, and is divided into three serotypes: A, B, and C. Of these, types B and C have only been confirmed to infect humans, while type A has been confirmed to infect humans, horses, pigs, other mammals, and various types of poultry and wild birds. The serotypes of influenza A viruses are classified according to the types of two proteins on the surface of the virus, hemagglutinin (HA) and neuraminidase (NA), and can be classified into 144 types (16 types of HA and 9 types of NA) depending on the serotype. The HA protein plays a role in attaching the virus to somatic cells, and the NA protein enables the virus to penetrate into cells. Examples of the influenza virus include, but are not limited to, PR8, H1N1 or H3N2.

[0058] The above novel strain may reduce the titer of the influenza virus.

[0059] Additionally, the novel strain may increase the production of fatty acids. Specifically, the novel strain may increase the production of palmitic acid.

[0060]

[0061] *The above palmitic acid is a type of solid fatty acid that is widely distributed in the plant and animal kingdoms and is also contained in most oils and fats. It may have an antiviral effect against influenza viruses by activating cytokine secretion or type 1 interferon signaling.

[0062] The increase in the production of the above fatty acids can protect the host from influenza virus infection in a G protein-coupled receptor (Gpr)-dependent manner, and specifically, can protect the host from influenza virus infection in a Gpr40 and / or Gpr120-dependent manner. The protection of the host from influenza virus infection in a G protein-coupled receptor (Gpr)-dependent manner may mean that cytokine secretion or type 1 interferon signaling is activated when the G protein-coupled receptor can function normally at the same time as the production of the fatty acids is increased, or that cytokine secretion or type 1 interferon signaling is not activated when an antagonist for the G protein-coupled receptor is treated even when the production of the fatty acids is increased.

[0063] In a specific embodiment of the present invention, the expression of genes related to fatty acid synthesis was measured and metabolites were analyzed in the lung tissue of mice orally administered the novel strain. As a result, it was confirmed that administration of the novel strain of the present invention increased the expression of genes related to fatty acid synthesis, increased palmitic acid production, increased cytokine secretion, and enhanced type 1 interferon signaling.

[0064] In addition, in a specific embodiment of the present invention, the degree of IFN-I signal activation was measured when palmitic acid was treated and when palmitic acid and a G protein-coupled receptor antagonist were treated. As a result, it was confirmed that the IFN-I signal was amplified when palmitic acid was treated, but the IFN-I signal was not amplified when the antagonist was treated together, indicating that the increased production of fatty acids protects the host from influenza virus infection in a manner dependent on G protein-coupled receptors.

[0065] Therefore, the novel strain of the present invention can be effectively used for the purpose of inhibiting, preventing, or treating influenza virus infection by enhancing the antiviral activity of the host against the influenza virus.

[0066] In a specific embodiment of the present invention, after treating the reporter cell lines of IFN-α and IFN-β with the novel strain of the present invention, the absorbance value of the reporter cell lines was measured. As a result, it was confirmed that the interferon signals of IFN-α and IFN-β were increased when the novel strain of the present invention was treated. In addition, after orally administering the strain of the present invention to influenza-infected mice, the changes in the expression of ISG, cytokine genes, and fatty acid production-related genes were measured. As a result, when the strain of the present invention was orally administered, the expression of genes and proteins of Oas1, Cxcl10, Ifit1, Ifit3, Rsad2, Irf7, Oasl2, Mx1, IL-1β, IL-6, IL-18, accA, accB, accC, accD, fabD, fabH, and fabZ was found to increase, confirming that the strain of the present invention enhances the interferon signal, activates cytokine secretion, and increases fatty acid production.

[0067]

[0068] 2. Composition comprising the novel Lactobacillus paracasei strain of the present invention

[0069] Another aspect of the present invention provides a composition comprising, as an active ingredient, at least one selected from the group consisting of the novel Lactobacillus paracasei strain of the present invention described above, a culture solution of the strain, a concentrate of the culture solution, a dried product of the culture solution, and an extract of the culture solution.

[0070] The above culture medium is obtained by culturing the novel Lactobacillus paracasei strain of the present invention, and may be the culture medium itself containing cells of the strain, or a culture supernatant obtained by removing cells therefrom, or may also be a filtrate, concentrate, or dried product thereof. The culture medium from which the cells have been removed contains components produced and secreted by the novel strain of the present invention, such as metabolites, and thus may have a probiotic effect with excellent activity in improving immunity.

[0071] The above concentrate increases the solid concentration of the culture medium, and may be a concentrate of a culture medium containing cells of the novel Lactobacillus paracasei strain of the present invention, or a concentrate of a culture supernatant from which cells of the novel Lactobacillus paracasei strain of the present invention have been removed. The concentrate may be concentrated by vacuum concentration, plate concentration, thin film concentration, etc., but is not limited thereto, and may be performed at a temperature of 40 to 60 degrees Celsius using, for example, a known concentrator. Depending on the concentration of the concentrate, the content of the culture medium included in the composition of the present invention can be appropriately adjusted.

[0072] The above dried product includes, but is not limited to, a product dried through methods such as freeze drying, vacuum drying, hot air drying, spray drying, reduced pressure drying, spray drying, foam drying, high-frequency drying, and infrared drying.

[0073] The above extract means an extract obtained from the above culture solution or its concentrate, and may include an extract, a diluted or concentrated extract, a dried product obtained by drying the extract, or a controlled or purified product thereof, or a fraction obtained by fractionating the same.

[0074] In addition, the composition may further include other known ingredients or lactic acid bacteria that are suitable for ingestion together with the novel Lactobacillus paracasei strain of the present invention and have an activity of improving immunity or an antiviral activity against influenza virus when ingested.

[0075] The composition comprising the novel Lactobacillus paracasei strain of the present invention may be manufactured in a unit dosage form or may be manufactured by placing it in a multi-dose container by formulating it using a carrier, excipient and / or additive according to a method that can be easily performed by a person of ordinary skill in the art to which the present invention pertains. In this case, the formulation may be in the form of a solution, suspension or emulsion in an oil or aqueous medium, or in the form of an extract, powder, granules, tablets, capsules, gels (e.g., hydrogels) or lyophilizer, and may additionally include a dispersant, stabilizer or cryoprotectant as the additives.

[0076] Specifically, in the case of the freeze-drying agent, it includes using the strain in the form of a powder by freeze-drying it together with a cryoprotectant, and the cryoprotectant may be skimmed milk powder, maltodextrin, dextrin, trehalose, maltose, lactose, mannitol, cyclodextrin, glycerol, and / or honey. In addition, it includes using it by mixing it with a preservation carrier, adsorbing it, and then drying it to solidify it, and the preservation carrier may be diatomaceous earth, activated carbon, and / or defatted steel.

[0077] The composition comprising the novel Lactobacillus paracasei strain of the present invention can be prepared through a step of mixing at least one selected from the group consisting of a strain, a culture solution of the strain, a concentrate of the culture solution, a dried product of the culture solution, and an extract of the culture solution with any one of the carriers, excipients, or additives.

[0078] The description of the strain, carrier, excipient, and additive is as described above. When using a cryoprotectant as the additive, the novel Lactobacillus paracasei strain of the present invention and the cryoprotectant may be mixed, the mixture may be frozen at -45 degrees Celsius to -30 degrees Celsius, dried at 30 degrees Celsius to 40 degrees Celsius, ground in a mixer, and manufactured in the form of a freeze-dried powder. Specifically, the freezing process may be a vacuum freezing process at a temperature of -45 degrees Celsius to -30 degrees Celsius and a pressure of 5 to 50 mTorr for 65 to 75 hours.

[0079] When a cryoprotectant is further included in the composition of the present invention, damage or death of the strain can be suppressed during the freeze-drying process of the composition, and the composition in freeze-dried form has advantages during storage, distribution, and preservation. Furthermore, the composition in freeze-dried form can be ingested in powder form, and in this case, the strain in the composition can exhibit its activity as it grows or metabolizes in the body.

[0080]

[0081] 3. Use of the novel Lactobacillus paracasei strain of the present invention or a composition containing the same

[0082] Another aspect of the present invention provides a use of the novel Lactobacillus paracasei strain of the present invention or a composition comprising the same for improving immunity.

[0083] Another aspect of the present invention provides a use of the novel Lactobacillus paracasei strain of the present invention or a composition comprising the same for preventing or treating a disease associated with immunodeficiency.

[0084] The meaning of the above improvement in immunity can be understood in the same way as described in the table of contents of “1. New Lactobacillus paracasei strain”, so it is not described again.

[0085] The above immunodeficiency-related disease refers to a disease in which all immune activity or immune function related to killing target cells through the body's immune function is reduced, and the immune activity or immune function may be regulated by actions such as cytokine secretion and immune cell production. Therefore, the above immunodeficiency-related disease may refer to a decrease in the body's immunity or a decrease in immune activity or immune function compared to a normal person.

[0086] Another aspect of the present invention provides a use of the novel Lactobacillus paracasei strain of the present invention or a composition comprising the same for inhibiting influenza virus infection.

[0087] Another aspect of the present invention provides a use of the novel Lactobacillus paracasei strain of the present invention or a composition comprising the same for preventing or treating influenza virus infection.

[0088] The above influenza virus can be understood in the same way as described in the table of contents of “1. New Lactobacillus paracasei strain”, so it is not described again.

[0089] The above influenza virus infection may refer to a state in which the influenza virus pathogenic microorganism invades the body of a host organism and develops or multiplies. Inhibition of the above influenza virus infection may refer to suppressing the proliferation of the influenza virus or reducing the number of multiplied viruses.

[0090] The above influenza virus infection disease may refer to a disease caused by infection in which the influenza virus invades the body of a host organism.

[0091] In particular, the composition may be a food, feed, or pharmaceutical. If the composition is a food, it may be a health functional food composition for inhibiting influenza virus infection or a general food composition. If the composition is a feed, it may be a feed composition for inhibiting influenza virus infection or a feed additive composition. If the composition is a pharmaceutical, it may be a pharmaceutical composition for preventing or treating influenza virus infection.

[0092] In one embodiment of the present invention, when the composition is a health functional food composition or a general food composition for inhibiting influenza virus infection, the health functional food composition or food composition can increase the expression of at least one gene or protein selected from the group consisting of Oas1, Cxcl10, Ifit1, Ifit3, Rsad2, Irf7, Oasl2, Mx1, IL-1β, IL-6, IL-18, accA, accB, accC, accD, fabD, fabH, and fabZ in intestinal or lung cells.

[0093] Another aspect of the present invention provides a use of the novel Lactobacillus paracasei strain of the present invention or a composition comprising the same for improving immunity.

[0094] When the health functional food composition of the present invention is used as a food additive, the health functional food composition may be added directly to food or used together with other foods or food ingredients, and may be used appropriately according to a conventional method. The amount of the active ingredient may be appropriately used depending on the purpose of use (prevention or improvement). Generally, when manufacturing food or beverage, the health functional food composition of the present invention is added in an amount of 15 parts by weight or less, preferably 10 parts by weight or less, based on the raw material. However, when consumed for a long period of time for health purposes, the amount may be below the above range, and since there is no problem in terms of safety, the active ingredient may be used in an amount exceeding the above range.

[0095] There are no special restrictions on the types of the above health functional foods or foods. Examples of the above health functional foods or foods include meat, sausages, bread, chocolate, candy, snacks, confectionery, pizza, ramen, other noodles, gum, dairy products including ice cream, various soups, beverages, tea drinks, alcoholic beverages, vitamin complexes, dairy products, fermented milk, etc., and all health functional foods or foods in the conventional sense are included.

[0096] In particular, the above health functional food is a food with high medical and healthcare effects that is processed to efficiently exhibit a bioregulatory function in addition to providing nutrition, and can obtain useful effects for health purposes such as regulating nutrients for the structure and function of the human body or physiological functions. The above health functional food can be manufactured by a method commonly used in the technical field of the present invention, and can be manufactured by adding raw materials and ingredients commonly added in the art. In addition, the formulation of the above health functional food can be manufactured in various forms without limitation as long as it is a formulation recognized as a health functional food, and unlike general drugs, it has the advantage of not having side effects that may occur when taking drugs for a long time because it uses food as a raw material, and has the advantage of excellent portability.

[0097] The health functional food of the present invention includes ingredients commonly added during food manufacturing, such as proteins, carbohydrates, fats, nutrients, and seasonings. For example, when manufactured as a drink, it may include natural carbohydrates or flavoring agents as additional ingredients in addition to the active ingredients. The natural carbohydrates are preferably monosaccharides (e.g., glucose, fructose, etc.), disaccharides (e.g., maltose, sucrose, etc.), oligosaccharides, polysaccharides (e.g., dextrin, cyclodextrin, etc.), or sugar alcohols (e.g., xylitol, sorbitol, erythritol, etc.). The flavoring agents may be natural flavoring agents (e.g., thaumatin, stevia extract, etc.) or synthetic flavoring agents (e.g., saccharin, aspartame, etc.).

[0098] In addition to the above health functional food composition, various nutrients, vitamins, electrolytes, flavoring agents, coloring agents, pectic acid and its salts, alginic acid and its salts, organic acids, protective colloid thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohol, carbonating agents used in carbonated beverages, etc. may be further contained. The ratio of these added components is not particularly important, but is generally selected within the range of 0.01 to 0.1 parts by weight per 100 parts by weight of the health functional food composition of the present invention.

[0099] In addition, in another embodiment of the present invention, when the composition is a feed composition or feed additive composition for suppressing influenza virus infection, the feed composition or feed additive composition can be used in the diet of animals for the purpose of suppressing influenza infection. The feed additive of the present invention corresponds to a supplementary feed under the Feed Management Act.

[0100] In addition, in another embodiment of the present invention, when the composition is a feed composition or feed additive composition for improving immunity, the feed composition or feed additive composition can be used in the diet of animals for the purpose of improving immunity. The feed additive of the present invention corresponds to a supplementary feed under the Feed Management Act.

[0101] In the present invention, the term "feed" may mean any natural or artificial diet, meal, etc., or components of said meal, which are suitable for or intended for eating, ingesting, and digesting by animals. The type of said feed is not particularly limited, and feed commonly used in the relevant technical field may be used. Non-limiting examples of said feed include plant feeds such as grains, roots, fruits, food processing by-products, algae, fibers, pharmaceutical by-products, oils, starches, meal, or grain by-products; and animal feeds such as proteins, inorganic substances, oils, minerals, oils, single-cell proteins, zooplankton, or food. These may be used alone or in combination of two or more.

[0102] In another embodiment of the present invention, when the composition is a pharmaceutical composition for preventing or treating an immunodeficiency-related disease, a human or non-human animal having an immunodeficiency-related disease may have a state in which the immune response does not function at a normal level due to insufficient immune activity compared to a human or non-human animal having a normal level of immunity.

[0103] In another embodiment of the present invention, when the composition is a pharmaceutical composition for preventing or treating an influenza virus infection disease, compared to a human or non-human animal to which the composition is administered, the composition may not be administered, and the immune response may not function at a normal level due to insufficient antiviral activity against the influenza virus.

[0104] In addition, the above prevention means any act of suppressing or delaying the onset of an immunosuppressed disease or an infectious disease caused by an influenza virus by administering the above pharmaceutical composition.

[0105] The above treatment means any action that improves or beneficially changes the symptoms of an individual suspected of having or developing an immunosuppressed disease or an infectious disease caused by an influenza virus by administering the above pharmaceutical composition.

[0106] Meanwhile, the pharmaceutical composition may be manufactured in a unit dosage form or may be manufactured by placing it in a multi-dose container by formulating it using a pharmaceutically acceptable carrier and / or excipient according to a method that can be easily performed by a person having ordinary skill in the art to which the present invention pertains, and the like. At this time, the formulation may be in the form of a solution, suspension or emulsion in an oil or aqueous medium, or in the form of an extract, powder, granules, tablets, capsules or gel (e.g., hydrogel), and may additionally include a dispersant or stabilizer.

[0107] Additionally, the strain contained in the pharmaceutical composition may be delivered in a pharmaceutically acceptable carrier such as a colloidal suspension, powder, saline solution, lipid, liposome, microspheres, or nano-spheres. These may be complexed or associated with a carrier, and may be delivered in vivo using carrier systems known in the art, such as lipids, liposomes, microparticles, gold, nanoparticles, polymers, condensation agents, polysaccharides, polyamino acids, dendrimers, saponins, adsorption enhancing substances, or fatty acids.

[0108] In addition, pharmaceutically acceptable carriers may include, but are not limited to, lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia, gum, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, polyvinyl pyrrolidone, cellulose, water, syrup, methyl cellulose, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate, and mineral oil, which are commonly used in formulations. In addition, lubricants, wetting agents, sweetening agents, flavoring agents, emulsifiers, suspending agents, preservatives, etc. may be further included in addition to the above ingredients. Suitable pharmaceutically acceptable carriers and formulations are described in detail in Remington's Pharmaceutical Sciences (19th ed., 1995).

[0109] The pharmaceutical composition according to the present invention can be administered orally or parenterally during clinical administration, and can be used in the form of a general pharmaceutical preparation. That is, the pharmaceutical composition of the present invention can be administered in various oral and parenteral dosage forms during actual clinical administration, and when formulated, it is prepared using diluents or excipients such as commonly used fillers, bulking agents, binders, wetting agents, disintegrants, and surfactants. Solid preparations for oral administration include tablets, pills, powders, granules, capsules, etc., and these solid preparations are prepared by mixing a herbal extract or fermented herbal product with at least one excipient, such as starch, calcium carbonate, sucrose or lactose, gelatin, etc. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used. Liquid preparations for oral administration include suspensions, solutions, emulsions, and syrups. In addition to commonly used simple diluents such as water and liquid paraffin, they may contain various excipients such as wetting agents, sweeteners, fragrances, and preservatives. Preparations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, and suppositories. Non-aqueous solvents and suspensions can include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate. Suppository bases can include withepsol, macrogol, Tween 61, cacao butter, laurin butter, glycerol, and gelatin.

[0110] The pharmaceutical composition of the present invention can be used alone or in combination with methods using surgery, radiation therapy, hormone therapy, chemotherapy, and biological response modifiers for the prevention and treatment of diseases related to immunodeficiency or infectious diseases caused by influenza virus.

[0111] The concentration of the active ingredient included in the composition of the present invention can be determined in consideration of the therapeutic purpose, the patient's condition, the required period, etc., and is not limited to a specific range of concentrations. The pharmaceutical composition of the present invention is administered in a pharmaceutically effective amount. In the present invention, the 'pharmaceutically effective amount' means an amount sufficient to treat a disease with a reasonable benefit / risk ratio applicable to medical treatment, and the effective dosage level can be determined according to the type and severity of the patient's disease, the activity of the drug, the sensitivity to the drug, the time of administration, the route of administration and the excretion rate, the treatment period, factors including concurrently used drugs, and other factors well known in the medical field. The pharmaceutical composition according to the present invention can be administered as an individual therapeutic agent, or in combination with other therapeutic agents for the prevention and treatment of diseases related to immunosuppression or infectious diseases caused by influenza virus, and can be administered simultaneously, separately, or sequentially with conventional therapeutic agents, and can be administered singly or in multiple doses. It is important to administer an amount that can achieve the maximum effect with the minimum amount without side effects by taking all of the above factors into consideration, and this can be easily determined by those skilled in the art.

[0112] Specifically, the effective amount of the pharmaceutical composition of the present invention may vary depending on the patient's age, sex, condition, weight, absorption rate of the active ingredient in the body, inactivation rate, excretion rate, type of disease, and concomitantly administered drugs, and may increase or decrease depending on the route of administration, severity of influenza virus infection, sex, weight, age, etc.

[0113] Another aspect of the present invention provides a method for preventing or treating an immunosuppressed disease, comprising administering the pharmaceutical composition to a subject.

[0114] Another aspect of the present invention provides a method for preventing or treating influenza infection, comprising administering the pharmaceutical composition to a subject.

[0115] The subject may be a human or a non-human animal. Furthermore, the subject may be a human or non-human animal with a lower than average level of immunity.

[0116] The subject may be a human or a non-human animal. Furthermore, the subject may be a human or non-human animal with a level of anti-influenza activity lower than the average level.

[0117] The description of the formulation, administration method, dosage, and concentration of the active ingredient contained in the composition of the above pharmaceutical composition is the same as above.

[0118]

[0119] Hereinafter, the present invention will be described in detail by examples.

[0120] However, the following examples specifically illustrate the present invention, and the content of the present invention is not limited by the following examples.

[0121]

[0122] [Example 1]

[0123] Establishment of a novel Lactobacillus paracasei strain of the present invention

[0124] Samples were collected from healthy individuals' stool, suspended in anaerobic PBS, and diluted decimal. The samples were spread on MRS (BD, USA) solid media and cultured at 37°C for 48 hours, and the MI29 strain was purified. The MI29 strain isolated as described above was subjected to PCR using the universal primer set 5'-AGAGTTTGATCMTGGCTCAG-3' (SEQ ID NO: 2) and 5'-TACGGYTACCTTGTTACGACTT-3' (SEQ ID NO: 3) to analyze the base sequence of the 16S rRNA of the MI29 strain.

[0125] As a result, the 16S rRNA base sequence of the MI29 strain was the same as sequence number 1, and it was confirmed to have 99.8% homology with the previously reported Lactobacillus paracasei standard strain Lactobacillus paracasei KCTC3510 (L. paracaseiKCTC3510). At this time, sequence comparison with the standard strain was performed in NCBI BLAST (https: / blast.ncbi.nlm.nih.gov / Blast.cgi).

[0126] Accordingly, the MI29 strain was named as shown in Table 1 below, and deposited at the Korean Collection for Type Culture (KCTC) of the Korea Research Institute of Bioscience and Biotechnology on July 13, 2021, and was assigned a deposit number as shown in Table 1 below.

[0127] Strain name Accession number MI29 Lactobacillus paracasei MI29KCTC 14636BP

[0128] The novel strain of the present invention as described above was inoculated into MRS liquid medium, cultured at 37°C for 24 hours to reach the stationary phase, and then the culture was centrifuged at 4,000xg for 20 minutes to obtain the supernatant, passed through a 0.45um filter, stored at -80°C, and used for experiments.

[0129]

[0130] [Example 2]

[0131] Characterization of new strains

[0132] For the novel strain isolated and identified in Example 1 above, sugar utilization ability and antibiotic resistance were confirmed in MRS medium.

[0133] [2-1] Sugar availability

[0134] To determine the biochemical properties of the novel strain of the present invention, such as the presence of enzymes and metabolic properties produced, or the ability to utilize specific carbohydrates with acid formation, numerous tests were performed simultaneously using the API CHL50 kit (BioMerieux, France). As comparison targets, the standard strain of Lactobacillus paracasei, Lactobacillus paracasei KCTC3510 (L. paracaseiKCTC3510), and the control strain Lactobacillus paracasei MI3 (L. paracaseiMI3), which mitigates influenza infection, were used.

[0135] As a result, as shown in Table 2, the novel strain of the present invention was confirmed to utilize the sugars of ribose, D-galactose, D-glucose, D-fructose, D-mannose, L-sorbose, D-mannitol, D-sorbitol, N-acetylglucosamine, amygdalin, arbutin, esculin, salicin, D-cellobiose, D-maltose, D-lactose, D-saccharose, D-trehalose, inulin, D-melezitose, gentiobiose, D-turanose, D-tagatose, and gluconate. In addition, unlike the standard strain, the novel strain of the present invention was confirmed to metabolize L-sorbose, D-sorbitol, amygdalin, D-maltose, D-lactose, D-saccharose, and inulin. In addition, it was confirmed that the novel strain of the present invention, unlike the influenza infection-alleviating control strain, does not undergo andonitol and D-lyxose metabolism, but undergoes amygdalin metabolism.

[0136] Substrate reactionSubstrate reactionKCTC3510MI3MI29KCTC3510MI3MI29Blank---Esculin+++Glycerol---Salicinw++Erythritol---D-Cellobiose+++D-Arabinose---D-Maltose-w+L-Arabinose---D-Lactose-++Ribose+++D-Mellibose---D-Xylose---D-Saccharose-++L-Xylose---D-Trehalose+++Andonitol-w-Inulin-++Methyl-β-D-Xylopyranoside---D-Melezitosew++D-Galactose+++D-Raffinose---D-Glucose+++Amidone-- -D-fructose+++glycogen---D-mannose+++xylitol---L-sorbose-++genthiobiose+++L-rhamnose---D-turanose+++dulcitol---D-lyxose-w-inositol---D-tagatose+++D-mannitol+++D-fucose---D-sorbitol-++L-fucose---methyl-α-D-mannopyranoside---D-arabitol---methyl-α-D-glucopyranoside---L-arabitol---N-acetylglucosamine+++gluconatewwwAmygdalin--w2-keto-gluconate---Arbutinw++5-keto-gluconate---

[0137] The above w stands for weak positive.

[0138] [2-2] Confirmation of enzyme activity

[0139] The enzyme activity of the Lactobacillus paracasei MI29 strain of the present invention isolated in Example 1 was confirmed using the API ZYM kit (BioMerieux, France). As control strains, Lactobacillus paracasei KCTC3510 (L. paracaseiKCTC3510), a standard strain of Lactobacillus paracasei, and Lactobacillus paracasei MI3 (L. paracaseiMI3), an influenza infection mitigation control strain, were used. Specifically, the bacterial solution was inoculated into the cuple of the API ZYM strip and cultured at 37°C for 4 hours. Thereafter, ZYM A and ZYM B reagents were sequentially added one drop at a time, and a score of 0 to 5 was recorded based on the intensity of the color that appeared after 5 minutes.

[0140] As a result, as shown in Table 3, the novel strain of the present invention was confirmed to have β-galactosidase and β-glucosidase enzyme activities, unlike the standard strain. In addition, the novel strain of the present invention was confirmed to have stronger esterase lipase (C8) and naphthol-AS-BI-phosphohydrolase activities and lower β-galactosidase activity, compared to the influenza infection-mitigating control strain.

[0141] Substrate reactionSubstrate reactionKCTC3510MI3MI29KCTC3510MI3MI29Blank000Acid phosphatase122Alkaline phosphatase111Naphthol-AS-BI-phosphohydrolase345Esterase(C4)233α-galactosidase000Esterase lipase(C8)212β-galactosidase052Lipase(C14)111β-glucuronidase000Leucine arylamidase555α-glucosidase344Valine arylamidase555β-glucosidase011Christine arylamidase111N-acetyl-β-glucosaminidase000Trypsin000α-mannosidase000α-chymotrypsin000α-fucosidase000

[0142] [2-3] Confirmation of acid resistance and bile resistance

[0143] In order to evaluate the intestinal suitability of the novel strain of the present invention, the acid resistance and bile resistance of the strain were confirmed. As control strains, Lactobacillus paracasei KCTC3510 (L. paracaseiKCTC3510), a standard strain of Lactobacillus paracasei, and Lactobacillus paracasei MI3 (L. paracaseiMI3), an influenza infection mitigation control strain, were used. The acid resistance was evaluated by measuring the survival rate of the strain under pH 3.0 conditions. Specifically, the strain was inoculated at 1% in a 0.05% L-cysteine ​​hydrochloride MRS (BD, USA) liquid medium adjusted to pH 3.0. First, for the measurement of the initial cell count, the strain was diluted decimalally in PBS, and then the viable cells were counted on the MRS solid medium supplemented with L-cysteine ​​hydrochloride. Afterwards, the inoculum of the above strain was cultured at 37°C for 2 hours, and the viable cell count after 2 hours of culture (CFU / ml 2h) was compared with the initial strain (CFU / ml 0h) to calculate the survival rate (%).

[0144] Acid resistance (survival rate, %) = [(CFU / ml 2h ÷CFU / ml 0h )]×100

[0145] Next, the strains were inoculated at 1% each in a 0.05% L-cysteine ​​hydrochloride MRS (BD, USA) liquid medium supplemented with 0.3% Oxgall. Then, the strain inoculum was diluted decimalally in PBS to measure the initial bacterial count, and the viable cells were counted on the MRS solid medium supplemented with L-cysteine ​​hydrochloride. After that, the strain inoculum was cultured at 37°C for 12 hours, and the growth rate (%) was calculated by comparing the viable cell count after 12 hours of culture (CFU / ml 12h) to the initial cell count (CFU / ml 0h).

[0146] Bile tolerance (growth rate, %) = [(CFU / ml 12h ÷CFU / ml 0h )]×100

[0147] As a result, as shown in Table 4 below, it was found that the Lactobacillus paracasei MI29 strain of the present invention exhibited acid resistance of 142% and bile resistance of 2.208%.

[0148] Strain Acid resistance (%) Bile resistance (%) L. paracasei KCTC351096284 L. paracasei MI31121,557 L. paracasei MI291422,208

[0149] [2-4] Confirmation of antibiotic resistance

[0150] To confirm the intestinal compatibility and safety of Lactobacillus paracasei MI29 of the present invention, antibiotic resistance was confirmed using the microdilution method (CLSI, M07-A9). As control strains, Lactobacillus paracasei KCTC3510 (L. paracaseiKCTC3510), a standard strain of Lactobacillus paracasei, and Lactobacillus paracasei MI3 (L. paracaseiMI3), an influenza infection mitigation control strain, were used. LAB susceptibility test medium (LSM) medium was used as a medium to confirm the minimum inhibitory concentrations (MICs) of antibiotics of Lactobacillus paracasei MI29. After dispensing 100 μL of the diluted inoculum in the LSM medium onto a microplate, 100 μL of each diluted solution of five antibiotics (ampicillin (AMP), gentamicin (GEN), erythromycin (ERY), tetracycline (TET), and chloramphenicol (CHL)) was dispensed. The diluted solutions of the antibiotics were prepared by stepwise dilution from 1 mg / L to 128 mg / L. Afterwards, the diluted inoculum of the antibiotics was cultured at 37°C for 24 hours, and the MIC was measured.

[0151] As a result, as shown in Table 5, the Lactobacillus paracasei MI29 strain of the present invention was confirmed to exhibit resistance below the European Food Safety Authority (EFSA) standards for the above five antibiotics. Therefore, since the MI29 strain of the present invention does not horizontally transfer resistance genes to harmful bacteria existing in the intestines, there is no concern that harmful bacteria in the intestines will acquire external resistance and cause resistance problems to antibiotics, and thus it has an advantage in that it is advantageous for commercialization as a probiotic.

[0152] MIC (mg / L) Strain AMPGENERYTETCHLL. paracasei MI2928 ≤ 1 ≤ 12 EFSA432144

[0153] [Example 3] Confirmation of the antiviral effect of the novel Lactobacillus paracasei strain of the present invention against influenza virus.

[0154] [3-1] Confirmation of IFN-Ⅰ signal activation

[0155] Using an IFN-α / β reporter cell line, it was confirmed whether the novel strain of the present invention activates type I interferon (hereinafter, IFN-I) signaling. To measure IFN-I signaling activity, the degree of interferon signaling was measured through a colorimetric assay. At this time, Lactobacillus paracasei KCTC3510 (L. paracaseiKCTC3510), a standard strain of Lactobacillus paracasei, and Lactobacillus paracasei MI3 (L. paracaseiMI3), an influenza infection-mitigating control strain, were used as control strains. Specifically, the IFN-α / β reporter HEK cell line (source: Invivogen) was cultured overnight in DMEM medium containing 10% fetal bovine serum (FBS), 1% penicillin-streptomycin, recombinant IFN-β, and 4% of the supernatant of the MI29 strain culture medium. The culture medium was centrifuged at 8,000 rpm for 10 minutes, and the supernatant was passed through a 0.45-μm syringe filter to prepare a supernatant of the culture medium of the strain. The supernatant of the culture medium of the strain prepared in this way was treated to the HEK-Blue IFN-α / β reporter cell line, and after 24 hours, the absorbance (OD) displayed by reaction with QUANTI-Blue solution (Invivogen, USA) 640 ) was measured.

[0156] As a result, as can be confirmed in Fig. 1 and Table 6, it was found that the IFN-I signaling activity of the Lactobacillus paracasei MI29 strain was higher than that of other strains. In addition, as shown in Fig. 1, when the MI29 strain of the present invention was treated, the absorbance was approximately 2 times greater than that of the standard strain and the influenza infection alleviation control strain, confirming that the novel strain of the present invention has an excellent function of activating the IFN-I signal.

[0157] StrainOD640L. paracaseiKCTC35100.159±0.010L. paracaseiMI30.162±0.019L. paracaseiMI290.303±0.031

[0158] [3-2] Confirmation of the effect of IFN-I on the antiviral activity of the strain of the present invention against influenza virus

[0159] In order to confirm what effect the above IFN-I has on the antiviral activity of the strain of the present invention against the influenza virus, IFNAR1 gene deletion (IFNAR1 - / - ) mice were used. At this time, IFNAR1 administered PBS as a control group - / - I used a mouse.

[0160]

[0161] As a result, as shown in Fig. 2, when the MI29 strain was administered, the IFNAR1 - / - The body weight of the mice was reduced to a lesser extent, and the expression of ISG genes was not increased, confirming that the antiviral activity of the strain of the present invention against influenza virus was mediated by IFN-I.

[0162] [3-3] Confirmation of the effect of oral administration of the strain of the present invention

[0163] In order to confirm the effect of oral administration of the MI29 strain of the present invention, the MI29 strain was orally administered to A / PR8 influenza-infected C57BL / 6 (B6) mice (source: OrientBio) for 2 weeks, and then the expression of antiviral genes and the protective effect against influenza virus were investigated. At this time, mice orally administered the MI3 strain were used as a control group. Specifically, the MI3 strain and the MI29 strain were cultured in MRS medium and BD GasPak TMThe strains were cultured under anaerobic conditions at 37°C using a 100 system. The cultures of the strains were centrifuged, and the resulting culture pellets were suspended in anaerobic PBS. The suspended pellets were administered at 1 x 10 of each strain per dose. 9 After oral administration of the above mice daily for 2 weeks at a dose of 1 x 10 CFU, a sublethal dose (1 x 10 3 Mice were induced intranasally with 100 PFU.

[0164] [3-3-1] Confirmation of the virus titer reduction effect of the strain of the present invention

[0165] For the mouse lung tissue isolated as described above, a viral plaque assay was performed to measure the number of plaques, thereby measuring the number (quantity, unit) of viruses present in the cells, i.e., the viral titer. Specifically, the isolated lung cells were homogenized, and lung extracts were prepared in PBS. Subsequently, the lung cell samples were centrifuged at 12,000 rpm for 5 minutes, and the supernatant was obtained. MDCK cells cultured in MEM medium supplemented with 10% FBS and 1% penicillin-streptomycin were cultured at 1 x 10 6 The collected supernatant sample was inoculated into a dog. The collected supernatant sample was incubated with the MDCK cells at room temperature for 1 hour. Thereafter, the culture was washed with PBS and cultured in MEM medium containing 1% agarose and 10 μg / ml trypsin at 37°C and 5% CO2 for 3 days. The cultured cells were fixed in 4% formalin overnight at room temperature, and the number of lytic plaques was measured.

[0166] As a result, as shown in Fig. 3, it was confirmed that mice orally administered the MI29 strain showed less weight loss and a decrease in the virus titer in the lungs compared to mice treated with PBS or MI3 after influenza virus infection.

[0167] [3-3-2] Confirmation of expression levels of interferon-induced genes (ISGs), cytokines, and fatty acid synthesis-related genes

[0168] For the mouse lung cells separated as described above, RNA was extracted and the changes in the expression levels of ISG, cytokine genes and fatty acid synthesis-related genes after oral administration of the strain of the present invention were measured. The ISG genes were Oas1, Cxcl10, Ifit1, Ifit3, Rsad2, Irf7, Oasl2 and Mx1, the cytokine genes were IL-1β, IL-6 and IL-18, and the fatty acid synthesis-related genes were acetyl-CoA carboxylase (Acc) genes such as accA, accB, accC and accD and fatty acid biosynthesis (Fab) genes such as fabD, fabH and fabZ, and their expression levels were measured. Specifically, total RNA was extracted from the mouse lung cells using the RNeasy Mini Kit, and the RNA of the novel strain of the present invention was extracted using Trizol (Thermo Fisher Scientific). Subsequently, cDNA was synthesized from the RNA using a gDNA remover and ReverTra Ace qPCR RT Master Mix. Real-time PCR was then performed using the cDNA as a template. The following primer sets were used for the real-time PCR:

[0169] Oas1: 5'-CGCACTGGTACCAACTGTGT-3' and 5'-CTCCCATACTCCCAGGCATA-3';

[0170] Cxcl10: 5'-TTTCTGCCCTCATCCTGCTG-3' 및 5'-CTCATCATTCTTTTTCATCGTG-3';

[0171] Ifit1: 5'-CAGAAGCACACATTGAAGAA-3' 및 5'-TGTAAGTAGCCAGAGGAAGG-3';

[0172] Ifit3: 5'-GCCGTTACAGGGAAATACTGG-3' 및 5'-CCTCAACATCGGGGCCT-3';

[0173] Rsad:, 5'-AACAGGCTGGTTTGGAGAAG-3' 및 5'-TGCCATTGCTCACTATGCTC-3';

[0174] Irf7: 5'-GCCAGGAGCAAGACCGTGTT-3' 및 5'-TGCCCCACCACTGCCTGTA-3';

[0175] Oasl2: 5'-GGATGCCTGGGAGAGAATCG-3' 및 5'-TCGCCTGCTCTTCGAAACTG-3';

[0176] Mx1: 5'-AAGATGGTCCAAACTGCCTTCG-3' 및 5'-GCCTTGGTCTTCTCTTTCTCAGC-3';

[0177] IL-1β: 5'-GTTGACGGACCCCAAAAGAT-3' 및 5'-AAGGTCCACGGGAAAGACAC-3';

[0178] IL-6: 5'-ATGGATGCTACCAAACTGGA-3' 및 5'-TTGGATGGTCTTGGTCCTTA-3';

[0179] IL-18: 5'-GGCTGCCATGTCAGAAGACT-3' 및 5'-ATCTTCCTTTTGGCAAGCAA-3';

[0180] accA: 5'-GCACGCGGGAACTGATTAAC-3' 및 5'-TCGATGAAACTTGGCTGCCT-3';

[0181] accB: 5'-ATCTGGTCAAAGCAAGTGCG-3' and 5'-AGTACGGATCAGCGTCAGGT-3';

[0182] accC: 5'-CTCTGTGCGGTGTTCTCAA-3' and 5'-AATCCGGCATCGGTTACGAG-3';

[0183] accD: 5'-AGCAAGGCAGCTATCGTGAG-3' and 5'-CACCAGATCGGCATCCCATT-3';

[0184] fabD: 5'-CAGGTTGAAGCGTGGTTGTC-3' and 5'-TTAGTCGGTTCAGCCACCTG-3';

[0185] fabH: 5'-CGAATCACAGTCCGTTTGCC-3' and 5'-CTGGTGTTGCCATGCCTCATC-3';

[0186] fabZ: 5'-CTTGGCGGCATTAAGAAGGC-3' and 5'-TTGCCCAAACCTGCATTGTC-3'.

[0187] As a result, as shown in Fig. 4, mice orally administered the MI29 strain were confirmed to have increased expression of ISGs (Oas1, Cxcl10, Ifit1, Ifit3, Rsad2, Irf7, Oasl2, and Mx1) in lung tissues compared to mice treated with PBS or MI3 strain. In addition, it was confirmed that the expression of proinflammatory cytokines (IL-1β, IL-6, and IL-18) was increased in lung tissues of mice orally administered the MI29 strain. In addition, the mRNA expression levels of Acc (accA, accB, accC, and accD) and Fab (fabD, fabH, and fabZ) genes in the MI29 strain were higher than those in the MI3 strain, confirming that oral administration of the strain of the present invention increases fatty acid synthesis.

[0188] [3-3-3] Quantification of IFN-α, IFN-β, dendritic cells, and monocytes

[0189] To determine the amount of IFN-α and IFN-β in the isolated mouse lung tissue, enzyme-linked immunosorbent assay (ELISA) experiments were performed according to the manufacturer's instructions (R&D Systems). In addition, IFN-Ⅰ and IL-1β were mainly expressed in plasmacytoid dendritic cells (pDCs) and Ly6C hi Since it is produced by monocytes, the number of pDCs and monocytes was measured in mice orally administered the MI29 strain of the present invention. The number of pDCs and monocytes was measured using a flow cytometry method. For cell separation and flow cytometry, mouse lung tissue was cut, and the tissue was suspended in 5 ml of digestion buffer consisting of RPMI-1640 medium, 10% FBS, 2 mM L-glutamine, 1 mM sodium pyruvate, 20 mM hydroxyethyl piperazine ethane sulfonic acid (HEPES), 1% penicillin-streptomycin, 2 mg / ml collagenase D, and 15 μg / ml DNase I. The suspension was cultured at 37°C for 1 hour, centrifuged, and the resulting pellet was suspended in 1 ml of red blood cell lysis buffer and incubated at room temperature for 1 minute. Thereafter, the cultured cells were washed with PBS. The cells were cultured with anti-CD16 / 32 antibodies and stained using a Live / Dead cell staining kit. Specifically, eBio927 was used for anti-PDCA-1, 30-F11 for anti-CD45, M1 / 70 for anti-CD11b, N418 for anti-CD11c, HK 1.4 for anti-Ly6C, and 1A8 for anti-Ly6G. The number of pDCs was CD45 + CD11c + PDCA-1 +The number of monocytes was measured by counting the number of CD45 + CD11b + Ly6G - Ly6C + The measurement was made by counting human cells.

[0190] As a result, as shown in Fig. 5, in the lungs of mice orally administered the MI29 strain, IFN-α and IFN-β were found to increase, and the number of pDCs and monocytes also increased. Therefore, it was confirmed that oral administration of the MI29 strain of the present invention can enhance the immune response.

[0191] [3-3-4] Histological analysis of mouse lungs

[0192] Lung tissues from mice administered MI29 orally were dissected, fixed with 4% paraformaldehyde (PFA), and embedded in paraffin. Lung tissues were then stained with hematoxylin-eosin (H&E). Pathological scores were calculated blindly using a scoring system based on the level of lung tissue destruction, epithelial cell layer damage, and polymorphonuclear cell infiltration. Mice administered PBS or MI3 served as controls.

[0193] As a result, as shown in Fig. 6, it was confirmed that the degree of lung damage was lower when MI29 was administered orally than when PBS or MI3 was administered.

[0194] [3-3-5] Checking ambassador profile changes

[0195] Since microbial metabolites play a crucial role in the intestines and lungs, non-targeted analysis and principal component analysis (PCA) were performed on MI29- and MI3-treated mice to identify metabolites in cecum and lung tissues. Furthermore, metabolite enrichment analysis was performed to determine which metabolites were functionally affected by strain treatment.

[0196] As a result, as shown in Fig. 7, fatty acid-related metabolic pathways were confirmed to be enhanced in the cecum and lung tissues of MI29-treated mice compared to MI3-treated mice. These results confirmed that the MI29 strain has superior fatty acid synthesis ability compared to the MI3 strain.

[0197] [3-4] Confirmation of the relationship between the strain of the present invention and fatty acids

[0198] As confirmed in the above Experimental Example 3-3-5, when the strain of the present invention was treated, it was confirmed that the synthesis of fatty acids such as palmitic acid increased. Therefore, in order to investigate the effect of fatty acids on IFN-I signaling, palmitic acid was treated to the IFN-α / β reporter cell line of Experimental Example 3-1, and then the IFN-I signal was measured. Specifically, bovine serum albumin-conjugated palmitic acid was added to DMEM medium, and then the IFN-α / β reporter cell line was cultured for 24 hours. In addition, to inhibit G-protein coupled receptor 40 (Gpr40) and G-protein coupled receptor 120 (Gpr120), 100 μM of GW1100, a Gpr40 antagonist, and 100 μM of AH 7614, a Gpr120 antagonist, were treated. For the IFN-α / β reporter cell line cultured as above, a colorimetric assay was performed using QUANTI-Blue solution to measure the absorbance (OD 640 ) was measured.

[0199] As a result, as illustrated in Fig. 8, when the palmitic acid was treated, the IFN-I signal was amplified, but when the Gpr40 / 120 antagonist was treated, the signal amplification effect as described above disappeared. Therefore, it was confirmed that the fatty acid-related metabolites enhanced by administration of the MI29 strain were related to the activation of the IFN-I signal.

[0200]

[0201] [Manufacturing example]

[0202] Preparation of a crude composition containing a novel Lactobacillus paracasei strain

[0203] A crude product of a composition comprising the novel Lactobacillus paracasei strain of the present invention was prepared.

[0204] The novel Lactobacillus paracasei strain of the present invention was cultured using an optimized enrichment medium and culture conditions. After cell recovery, 5-50 (vol / weight)% of maltodextrin, 5-50 (vol / weight)% of trehalose, or 5-50 (vol / weight)% of cellulose as a cryoprotectant was added to the concentrate, followed by freeze-drying and grinding to produce lactic acid bacteria powder.

[0205]

[0206] Although representative embodiments of the present invention have been described above as examples, the scope of the present invention is not limited to the specific embodiments described above, and those skilled in the art will be able to make appropriate changes within the scope described in the claims of the present application.

[0207]

[0208] [Accession number]

[0209] Name of depositor: Korea Research Institute of Bioscience and Biotechnology, Biological Resource Center (KCTC)

[0210] Accession number: KCTC 14636BP

[0211] Date of acceptance: 20210713

[0212]

Claims

1. Lactobacillus paracasei MI29 strain deposited under accession number KCTC 14636BP.

2. In claim 1, Lactobacillus paracasei MI29 strain comprising 16S rRNA having the base sequence of sequence number 1.

3. A composition comprising at least one selected from the group consisting of Lactobacillus paracasei MI29 strain deposited under accession number KCTC 14636BP, a culture solution of the strain, a concentrate of the culture solution, a dried product of the culture solution, and an extract of the culture solution.

4. In claim 3, The composition is a composition that enhances the expression of at least one gene selected from the group consisting of Oas1, Cxcl10, Ifit1, Ifit3, Rsad2, Irf7, Oasl2, Mx1, IL-1β, IL-6, IL-18, accA, accB, accC, accD, fabD, fabH, and fabZ in intestinal or lung cells.

5. In claim 3, The composition above has antiviral activity against influenza virus.

6. In any one of claims 3 to 5, The composition above is a health functional food composition for improving immunity.

7. In any one of claims 3 to 5, The composition above is a pharmaceutical composition for preventing or treating a disease related to immunodeficiency.

8. In any one of claims 3 to 5, The composition above is a health functional food composition for inhibiting influenza virus infection.

9. In any one of claims 3 to 5, The composition above is a pharmaceutical composition for preventing or treating influenza infection.

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