KR20220168788A - Composition for enhancing immunity comprising Curtobacterium proimmune K3 - Google Patents

Abstract

The present invention relates to a composition for enhancing immunity including a newly isolated and identified strain Curtobacterium proimmune K3, its culture medium, or its cell-free extract. The Curtobacterium proimmune K3 strain (accession number: KCTC14591BP), its culture medium, or its cell-free extract according to the present invention are can effectively promote both intrinsic and adaptive immunity in the body through expression of one or more cytokines selected from the group consisting of IL-2, IFN-γ, TNF-a, and IL-1β, and promotion of serum IgA or serum IgG production. Therefore, it can be used in a variety of ways, such as food for enhancing immunity, health functional food, feed and medicine, and skin health material.

Description

Curtobacterium proimmune K3 strain and a composition for enhancing immunity comprising the same {Composition for enhancing immunity comprising Curtobacterium proimmune K3}

The present invention relates to a composition for enhancing immunity comprising a newly isolated and identified strain, Curtobacterium proimmune K3 strain, a culture thereof, or a cell-free extract thereof.

Immunity refers to a series of biological defense reactions that occur to exclude substances other than its own components from breaking the homeostasis of the living body or threatening itself, and protecting the body from harmful substances invading through the skin, digestive tract, respiratory tract, etc. It can be said to be a homeostasis maintenance activity through a phagocytic action that removes external stimuli as a defense system, or an action that reduces severe inflammation in a wound. When the immune function is deficient or lowered, the immune response is not properly activated, so it does not respond properly to foreign substances in the body, resulting in infection. Nutrients related to the enhancement of immune function include minerals including Fe, Zn, Cu, Se, vitamins A, C, E, B vitamins, and specific amino acids and fatty acids. In addition to malnutrition, immune system imbalance can be progressed by congenital and external factors. For example, there are cases where phagocytic cells do not function properly due to congenital insufficiency of the body's immune function, and this state of immunosuppression also causes the penetration of external viruses into the human body (host) and promotes the proliferation of the infiltrated viruses, making it vulnerable to various infectious diseases. will do Macrophages are representative immune cells that respond to external viruses through phagocytosis. Phagocytosis is an activity in which cells capture solid particles from the environment in a dictionary sense. Through this phagocytic action, it is the most important primary defense function of our body that neutralizes harmful pathogens (viruses, bacteria, etc.) and becomes an effective means of protection. Phagocytes are cells produced by differentiation of monocytes, and differentiate into macrophages that exit blood vessels and enter tissues, gradually increasing in size and having active phagocytosis. Even in the case of infection by the same microorganism, a person with a weakened immune system may develop a more serious infection than a person without a weakened immune system.

Human immunity enhancement is to protect our body from various germs and viruses (bacterial infectious diseases, colds, AIDS, etc.), and is defined as a defense response of body tissues against infectious agents such as pathogenic viruses and bacteria. In recent years, many studies have reported on the immune-enhancing effect of lactic acid bacteria and vitidobacteria. There is a limit. In addition, considering the vitality in the human body, high-density products with increased content dozens of times have been produced and distributed in recent years, but side effects caused by excessive strains have been reported. There is a need.

The inventors of the present invention completed the present invention by confirming that the isolated and identified Curtobacterium proimmune K3 strain exhibits an excellent immune enhancing effect while conducting research on isolating useful strains from traditional fermented ginseng beverages.

Accordingly, an object of the present invention is to provide an immune enhancer comprising the Curtobacterium proimmune K3 strain, its culture medium or its cell-free extract, and food, health functional food composition, feed composition and pharmaceutical composition containing the same.

In order to achieve the above object, the present invention provides a Curtobacterium proimmune K3 strain (Accession Number: KCTC14591BP).

In addition, the present invention provides a composition for enhancing immunity, including a Curtobacterium proimmune K3 strain (Accession No.: KCTC14591BP), a culture thereof, or a cell-free extract thereof.

In addition, the present invention provides a food composition for enhancing immunity, including a Curtobacterium proimmune K3 strain (Accession Number: KCTC14591BP), a culture thereof, or a cell-free extract thereof.

In addition, the present invention provides a health functional food composition for enhancing immunity, including a Curtobacterium proimmune K3 strain (Accession Number: KCTC14591BP), a culture thereof, or a cell-free extract thereof.

In addition, the present invention provides a feed composition for enhancing immunity, including a Curtobacterium proimmune K3 strain (Accession No.: KCTC14591BP), a culture thereof, or a cell-free extract thereof.

In addition, the present invention provides a cosmetic composition for enhancing immunity, including a culture solution of Kurtobacterium proimmune K3 strain (Accession No.: KCTC14591BP) or a cell-free extract thereof.

In addition, the present invention provides a pharmaceutical composition for enhancing immunity, including a Curtobacterium proimmune K3 strain (Accession Number: KCTC14591BP), a culture thereof, or a cell-free extract thereof.

In addition, the present invention, the steps of inactivating the Kurtobacterium proimmune K3 strain (Accession Number: KCTC14591BP); It provides a method for producing a composition for enhancing immunity, including para-probiotics.

Curtobacterium proimmune K3 strain (accession number: KCTC14591BP), its culture medium or its cell-free extract according to the present invention is one selected from the group consisting of IL-2, IFN-γ, TNF-a and IL-1β It can effectively promote both innate and adaptive immunity in the body through the expression of abnormal cytokines and the promotion of serum IgA or serum IgG production, so it can be used as food, health functional food, feed, pharmaceuticals, and skin health materials for immune enhancement. can be utilized

1 is Curtobacterium proimmune K3 ( Curtobacterium Proimmune K3) treatment-induced cytotoxicity through MTT analysis (A), LIVE/DEAD analysis (B), and results of confirming cell morphological changes (C) according to Kurtobacterium proimmune K3 treatment. (All values are expressed as mean ± SD, ns, not statistically significant).
Figure 2 is a diagram showing the results of confirming NO production (A) and cytokine expression changes according to Kurtobacterium proimmune K3 lysate treatment through mRNA analysis (B to D) and protein ELISA analysis (E to G). (All values are expressed as mean ± SD, *p < 0.05, **p < 0.01, ***p < 0.001 vs. con group).
Figure 3 shows the results of confirming cell viability (A) and cytokine expression changes according to treatment with Curtobacterium proimmune K3 lysate in splenocytes through mRNA analysis (B to E) and protein ELISA analysis (F to I) (All values are expressed as mean ± SD, * p <0.05, ** p <0.01, *** p <0.001 vs. con group).
Figure 4 is a diagram showing the results of confirming the effect of restoring the organ index of the spleen and thymus, which are immune-related organs, according to the treatment with Curtobacterium proimmune K3 lysate in CTX-immunosuppressed mice (all values are expressed as mean ± SD, *p < 0.05, **p < 0.01, ***p < 0.001 vs. con group. #p < 0.05, ## p < 0.01, ### p < 0.001 vs. CTX group).
Figure 5 is a diagram showing the results confirming the effect of enhancing NK cell survival according to the treatment with Curtobacterium proimmune K3 lysate in CTX-immunosuppressed mice (all values are expressed as mean ± SD, * p <0.05, * *p < 0.01, ***p < 0.001 vs. con group. #p < 0.05, ## p < 0.01, ### p < 0.001 vs. CTX group).
Figure 6 is a diagram showing the results of confirming the adaptive immunity enhancing effect through spleen cell proliferation according to the treatment with Curtobacterium proimmune K3 lysate in CTX-immunosuppressed mice (all values are expressed as mean ± SD, * p <0.05, **p <0.01, ***p <0.001 vs. con group. #p <0.05, ## p <0.01, ### p <0.001 vs. CTX group).
Figure 7 is a diagram showing the results of confirming the effect of improving adaptive immunity according to the treatment with Curtobacterium proimmune K3 lysate in CTX-immunosuppressed mice through changes in ear edema (all values are expressed as mean ± SD, * p <0.05, **p <0.01, ***p <0.001 vs. con group. #p <0.05, ## p <0.01, ### p <0.001 vs. CTX group).
Figure 8 is a view showing the results of confirming the IgA (F) and IgG (G) content of serum by ELISA analysis of the adaptive immunity enhancing effect according to the treatment with Curtobacterium proimmune K3 lysate in CTX-immunosuppressed mice. (All values are presented as mean ± SD; *p < 0.05, **p < 0.01, ***p < 0.001 vs. con group. #p < 0.05, ## p < 0.01, ### p < 0.001 vs. CTX group).
Figure 9 is a view showing the results of confirming the recovery of serum immunity-related cytokines according to the treatment with Curtobacterium proimmune K3 lysate in CTX-immunosuppressed mice. (All values are presented as mean ± SD, *p < 0.05, ** p < 0.01, *** p < 0.001 vs. con group. #p < 0.05, ## p < 0.01, ### p < 0.001 vs. .CTX Group)
Figure 10 is a diagram showing the results of confirming changes in mRNA expression levels of immune-related cytokines according to the treatment of Curtobacterium proimmune K3 lysate in the spleen and thymus of CTX-immunosuppressed mice (all values are mean ± SD). Indication, *p < 0.05, **p < 0.01, ***p < 0.001 vs. Con group. #p < 0.05, ## p < 0.01, ### p < 0.001 vs. CTX treated group).
Figure 11 shows the activation of NF-kB and MAPK signaling pathways in Kurtobacterium proimmune K3 lysates by immunoblotting of IκBα and NF-κB proteins in spleen cells (A) and ERK, JNK and p38 proteins in spleen cells. This is a diagram showing the results confirmed through immunoblotting (B) (all values are expressed as mean ± SD, * p <0.05, ** p <0.01, *** p <0.001 vs. con group. #p <0.05 , ## p < 0.01, ### p < 0.001 vs. CTX group)

The present invention Curtobacterium proimmune K3 ( Curtobacterium proimmune K3, KCTC14591BP); and Curtobacterium proimmune K3 ( Curtobacterium proimmune K3, KCTC14591BP), a composition for enhancing immunity, a health functional food composition, a food composition, a cosmetic composition, a feed composition, and a pharmaceutical composition containing a culture medium thereof or a cell-free extract thereof.

Hereinafter, the present invention will be described in detail.

"Curtobacterium proimmune K3 of the present invention ( Curtobacterium proimmune K3)" is a strain isolated from a traditional fermented ginseng beverage in Yongin, Korea, and as a result of 16s rRNA sequence-based analysis, it was confirmed that it is a new, previously unknown strain. Therefore, the strain was submitted to the Korea Research Institute of Bioscience and Biotechnology on June 1, 2021. deposited and given accession number KCTC14591BP The 16s rRNA sequence of this strain is shown as SEQ ID NO: 1.

The Curtobacterium proimmune K3 strain, its culture medium, or its cell-free extract of the present invention activates the NF-κ and MAPK signaling pathways without exhibiting toxicity, and the immune-related cytokines IL-2, IFN- Not only can it enhance the production of γ, TNF-a and IL-1β, but it can also promote the proliferation of spleen cells and the production of serum IgA and IgG, so it can show the effect of enhancing both innate and adaptive immunity. .

The Curtobacterium proimmune K3 strain of the present invention includes both live and dead cells, and can exhibit an immune enhancing effect by being included in the form of live cells or dead cells. In the present invention, "dead cells" may be used interchangeably with the same meaning as inactivated strains, lysates, and paraprobiotics.

In the present invention, "paraprobiotics" means microorganisms or cell fractions inactivated by various physical and chemical methods to provide useful effects on human health. A method for separating and purifying paraprobiotics from probiotics may be performed by one or more selected from the group consisting of, but not limited to, inactivation by heat, ultraviolet treatment, radiation, high pressure, and ultrasonic waves.

Therefore, the Curtobacterium proimmune K3 strain of the present invention is a paraprobiotic, a dead bacterium inactivated by at least one inactivation treatment selected from the group consisting of heat, ultraviolet rays, radiation, high pressure, and ultrasound, and is used in the composition for immunity enhancement. can be included

In the present invention, as a preferred embodiment, a Curtobacterium proimmune K3 strain inactivated by sonication is disclosed, and more specifically, 60 to 120 minutes, preferably 60 to 100 minutes, and more preferably 80 to 100 minutes. The Curtobacterium proimmune K3 strain can be inactivated by sonicating for 100 minutes.

The Curtobacterium proimmune K3 strain of the present invention, its culture medium, or its cell-free extract can enhance both innate immunity and/or adaptive immunity. More specifically, the Curtobacterium proimmune K3 strain of the present invention, its culture medium, or its cell-free extract contains at least one cytokine selected from the group consisting of IL-2, IFN-γ, TNF-a and IL-1β. expression can be enhanced, and production of serum IgA or serum IgG can be promoted.

Since the Curtobacterium proimmune K3 strain of the present invention exhibits an immune enhancing effect, it can be provided in the form of food compositions, health functional food compositions, cosmetic compositions, feed compositions and pharmaceutical compositions for the purpose of enhancing immunity.

Accordingly, the present invention provides a food composition or health functional food composition for enhancing immunity, including a Curtobacterium proimmune K3 strain (Accession No.: KCTC14591BP), a culture thereof, or a cell-free extract thereof.

The food or health functional food composition may further include additives selected from the group consisting of flavoring agents, flavoring agents, coloring agents, fillers, stabilizers, natural carbohydrates, nutrients, vitamins, thickeners, pH adjusting agents, preservatives, and mixtures thereof. can

The food composition of the present invention includes all forms such as functional food, nutritional supplement, health food and food additives. Food compositions of this type can be prepared in various forms according to conventional methods known in the art.

For example, as a health food, the composition itself may be prepared and consumed in the form of tea, juice, or drink, or may be granulated, encapsulated, or powdered to be consumed. In addition, functional foods include beverages (including alcoholic beverages), fruits and their processed foods (e.g., canned fruit, bottled fruit, jam, marmalade, etc.), fish, meat, and their processed foods (e.g., ham, sausage, corned beef, etc.), Bread and noodles (e.g. udon, buckwheat noodles, ramen, spaghetti, macaroni, etc.), fruit juice, various drinks, cookies, taffy, dairy products (e.g. butter, cheese, etc.), edible vegetable oil, margarine, vegetable protein, retort food, It can be prepared by adding the extract to frozen foods, various seasonings (eg, soybean paste, soy sauce, sauce, etc.). In addition, in order to use the composition of the present invention in the form of a food additive, it can be prepared and used in the form of a powder or concentrate.

In the food composition of the present invention, the preferred content of Curtobacterium proimmune K3, its culture medium or its cell-free extract may be 0.001 to 50%, preferably 0.01 to 30%, based on the total weight of the food composition. may be contained.

In one embodiment of the present invention, the health functional food composition of the present invention can be prepared in general formulations such as tablets, pills, granules, powders, liquids, hard capsules, soft capsules, etc., porridge, bread, beverages, bars, It can be made in any form, such as chocolate, cookies, tea, drinks, vitamin complexes, meat, sausages, candies, noodles, and jellies.

In order to prepare various formulations or forms as described above, food acceptable carriers or additives such as the above-mentioned excipients may be used, and known to be usable in the art for the preparation of the formulation or form to be prepared. Any carrier or additive may be used.

In addition, the present invention relates to a feed composition for enhancing immunity, including a Kurtobacterium proimmune K3 strain (Accession No.: KCTC14591BP), a culture thereof, or a cell-free extract thereof.

The feed composition may additionally include known feed supplements, food additives or feed additives, and may be prepared in the form of fermented feed, formulated feed, pellets and silage.

In addition, the present invention relates to a cosmetic composition for enhancing immunity, including a Curtobacterium proimmune K3 strain (Accession No.: KCTC14591BP), a culture thereof, or a cell-free extract thereof.

The cosmetic composition may include, without limitation, ingredients commonly accepted in addition to the active ingredients, and may include, for example, antioxidants, stabilizers, solubilizers, vitamins, pigments, and conventional adjuvants such as fragrances, and carriers. The cosmetic composition may be interpreted as including various materials for skin health without limitation.

In addition, the present invention relates to a pharmaceutical composition for enhancing immunity, including a Curtobacterium proimmune K3 strain (Accession No.: KCTC14591BP), a culture thereof, or a cell-free extract thereof.

The composition can be used for the prevention, treatment or improvement of immune-related diseases, such as asthma, seasonal or perennial rhinitis, allergic rhinitis, conjunctivitis, atopic dermatitis, urticaria, hemolysis of red blood cells, acute glomerulonephritis, It may include a cold, chronic fatigue, but is not limited thereto.

The pharmaceutical compositions may be formulated and used in the form of oral preparations such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, external preparations, suppositories and sterile injection solutions according to conventional methods, respectively. For formulation, appropriate carriers, excipients or diluents commonly used in the manufacture of pharmaceutical compositions may be included.

Examples of the carrier or excipient or diluent include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, undecided various compounds or mixtures including vaginal cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil, and the like.

When formulated, it can be prepared using diluents or excipients such as commonly used fillers, weighting agents, binders, wetting agents, disintegrants, and surfactants.

A solid preparation for oral administration may be prepared by mixing the strain with at least one excipient, for example, starch, calcium carbonate, sucrose or lactose, gelatin, and the like. In addition to simple excipients, lubricants such as magnesium stearate and talc may also be used.

Liquid preparations for oral administration include suspensions, solutions for oral use, emulsions, syrups, etc., and various excipients such as wetting agents, sweeteners, aromatics, preservatives, etc. may be included in addition to water and liquid paraffin, which are commonly used simple diluents. .

Formulations for parenteral administration include sterilized aqueous solutions, water-insoluble agents, suspensions, emulsions, freeze-dried preparations, and suppositories. Propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate may be used as non-aqueous solvents and suspensions. As a base material for suppositories, witepsol, macrogol, Tween 61, cacao butter, laurin paper, glycerol gelatin, and the like can be used.

The preferred dosage of the pharmaceutical composition varies depending on the patient's condition, weight, disease severity, drug type, administration route and period, but can be appropriately selected by those skilled in the art. However, for desirable effects, it may be administered at 0.0001 to 2,000 mg/kg per day, preferably 0.001 to 2,000 mg/kg. Administration may be administered once a day or divided into several times. However, the scope of the present invention is not limited by the dosage.

The pharmaceutical composition may be administered to mammals such as rats, mice, livestock, and humans through various routes. All modes of administration can be administered, for example, by oral, rectal or intravenous, intramuscular, subcutaneous, intrauterine dural or intracerebroventricular injection.

In addition, the present invention comprises the steps of inactivating the Kurtobacterium proimmune K3 strain (Accession Number: KCTC14591BP); It provides a method for producing a composition for enhancing immunity, including para-probiotics.

The manufacturing method is for preparing Kurtobacterium proimmune K3 having an immune enhancing effect, particularly in the form of dead cells, that is, in the form of paraprobiotics, and the inactivation treatment is ultraviolet light, radiation, high pressure, and ultrasound. One selected from the group consisting of More than one inactivation treatment may be used.

Paraprobiotics, dead cells, or lysates, which are inactivated forms of Curtobacterium proimmune K3 of the present invention, have the advantage of being safer than probiotics and at the same time exhibiting excellent immune enhancing effects.

Hereinafter, the action and effect of the invention will be described in more detail through specific examples of the invention. However, these embodiments are only presented as examples of the invention, and the scope of the invention is not determined thereby.

experimental material

LB broth and agar were purchased from BECTON DICKINSON & COMPANY (B.D., Franklin Lakes, New Jersey, USA). RAW264.7 and YAC-1 cell lines were purchased from the Korea Cell Line Bank (Seoul, Korea) and used. RPMI 1640 culture medium, DMEM medium, penicillin (100 mg/mL)-streptomycin (100 U/mL) and fetal bovine serum (FBS) were purchased from GenDEPOT (Katy, TX, USA) and used. MTT, DMSO, CTX (Cyclophosphamide) and LMS (Levamisole hydrochloride) were purchased from Sigma (St. Louis, MO, USA), and LIVE/DEAD cell viability assay stain was purchased from Thermo Fisher Scientific (Thermo Scientific, USA). All primers were designed by Macrogen (Seoul, Korea). All chemicals and reagents were of reagent grade quality and commercially available.

Cell and animal testing methods

For in vitro experiments, RAW 264.7 cells were cultured in DMEM medium supplemented with penicillin (100 mg/mL)-streptomycin (100 U/mL) and 10% FBS. The cultured cells were incubated at 37 °C in a 5% CO ₂ humidifier.

For ex vivo experiments, spleens were aseptically removed from BALB/c mice (4 weeks old) by cervical dislocation. In addition, the tissue was gently homogenized using the plunger of a 10 ml injection syringe and passed through 6 layers of gauze to become single cells. Red blood cells were removed using red blood cell lysis buffer (Sigma-Aldrich, MO, USA), and floating spleen cells were washed three times with PBS and centrifuged at 1200 rpm for 3 minutes. Proliferation of Kurtobacterium proimmune K3 in spleen cells was evaluated using the WST-8 cell viability assay kit purchased from BIOMAX (Seoul, Korea). Splenocytes were cultured 1x10 ⁶ cells/well with concanavalin A (ConA) or various concentrations of Curtobacterium proimmune K3 lysate (25, 50, 100, 200, 300, 400, 500, 600 μg/mL). were seeded in 96-well plates at a density of 48 hours and incubated for 48 hours. 10 μL of WST-8 solution was added to 100 μL cell culture medium, and the plate was incubated for 2 hours. Optical density values were recorded at 450 nm using a SpectraMax ABS Plus microplate reader.

Male ICR mice (6 weeks old, 25-27 g) of SPF grade were purchased from OrientBio and used. All mice were maintained in an environment maintained with a 12-hour light: 12-hour dark cycle at a temperature of 23-27° C. and a humidity of 50%-70%. All mice were continuously supplied with standard feed and water, and all experiments were conducted in accordance with the Laboratory Animal Care Principles (NIH Publication # 80-23, revised in 1996) and the Kyung Hee University Animal Care and Use Guidelines (approval number KHGASP-20-375). The procedure was followed. After a 1-week acclimatization period, mice were randomly divided into 7 groups as follows:

Control (Con) CTX untreated group using sterile saline Model group (CTX) Sterile saline and CTX treatment group C.proimmune K3-L Kurtobacterium proimmune K3 10 ⁵ CFU/mL treatment group C.proimmune K3-H Curtobacterium proimmune K3 10 ⁶ CFU/mL treatment group C.proimmune K3 Ly-L Kurtobacterium proimmune K3 lysate 10 ⁵ CFU/mL treatment group C.proimmune K3 Ly-H Curtobacterium proimmune K3 lysate 10 ⁶ CFU/mL treated group LMS CTX treatment group treated with levamisole hydrochloride (40mg/kg) (positive control group)

Immunosuppression was induced by intraperitoneal injection of CTX in sterile saline at 80 mg/kg of body weight to all mice except for the Con group once a day for 3 consecutive days. Mice were orally administered with Curtobacterium proimmune K3 strain or a lysate thereof, or LMS, once daily for 20 days. Thereafter, all mice were euthanized, blood was collected, and serum was collected by centrifugation at 3000 rpm and 4° C. for 15 minutes, and stored at -80° C. for biochemical analysis. Meanwhile, fresh spleen and thymus tissue samples were harvested and maintained at -80°C for biochemical marker analysis.

ELISA assay method

For in vitro and ex vivo experiments, RAW264.7 cells and spleen cells were dispensed into a 96-well plate, and then treated with Kurtobacterium proimmune K3 lysates and LPS or ConA at various concentrations, respectively. 100 microliters of the culture supernatant was obtained from the cell medium and the secretion of TNF-α, IL-1βIL-2 and INF-γ was evaluated using ELISA according to the instructions of the manufacturer (R & D Systems).

For the in vivo experiment, mouse serum was separated from whole blood at 1000 rpm for 20 minutes, and TNF-α, IL-1β, IL-2, INF-γ (R & D Systems, Minneapolis, MN, USA) and The content of IgG and IgA (Elabscience Biotechnology Co., Texas, USA) was measured using an ELISA kit.

qRT - PCR analysis method

Total mRNA was obtained from RAW264.7 cells, splenocytes and mouse spleen and thymic organs by Trizol reagent kit instructions (Invitrogen, Carlsbad, CA, USA), respectively. First, 500ng of total RNA was reverse transcribed with amfiRivert cDNA Synthesis Platinum Enzyme Mix (GenDEPOT, Katy, TX, USA), and the reaction was performed using a CFX96TM Real-Time RT-PCR system with SYBR PremixExTaqTM II (TaKaRa). qRT-PCR was performed using 50 ng cDNA in a 20 μL reaction volume using amfiSure qGreen Q-PCR Master Mix (GenDEOT, TX, USA), and gene-specific primer sequences for qRT-PCR are shown in Table 1. . Gene expression was normalized using GAPDH.

Primer Sequence GAPDH Forward 5'-ACC ACA GTC CAT GCC ATC AC-3' Reverse 5'-CCA CCA CCC TGT TGC TGT AG-3' iNOS Forward 5'-AAT GGC AAC ATC AGG TCG GCC ATC ACT-3' Reverse 5′-GCT GTG TGT CAC AGA AGT CTC GAA CTC-3′ TNF-α Forward 5′-AGCCCACGTCGTAGCAAAACCACCAA-3′ Reverse 5'-AAC ACC CAT TCC CTT CAC AGA GCA AT-3' IL-1β Forward 5'-TGC AGA GTT CCC CAA CTG GTA CAT C-3' Reverse 5′-GTG CTG CCT AAT GTC CCC TTG AATC-3′ IFN-γ Forward 5'-TAT CTC TTT CTA CCT CAG AC-3' Reverse 5′-GCA ATC ACA GTC TTG GCT AAT TAG-3′ IL-2 Forward 5′-CCT GAG CAG GAT GGA GAA TTA-3′ Reverse 5'-TCC AGA ACA TGC CGC AGA G-3'

immunoblot analyze

Spleens were lysed for 1 hour using Pierce™ RIPA Buffer (Thermo Fisher Scientific, Rockford, USA). Splenocyte lysates were centrifuged at 12,000 rpm and 4° C. for 20 minutes, and equal amounts (50 μg) of total protein were loaded on 10% SDS-PAGE and transferred to PVDF membranes. Membranes were blocked with 5% skim milk in PBST for 1 hour and then incubated overnight at 4°C with primary antibodies. After washing, the membrane was incubated with each secondary antibody for 1 hour. Immunoreactive bands were identified and quantified using Image J software.

statistical analysis

All experimental data were presented as mean ± SD, and statistically significant differences between the four groups were evaluated using one-way analysis of variance (ANOVA) followed by the Tukey-Kramer test. Statistical graphs were presented using GraphPad Prism 8.

Example One. Kurtobacterium proimmune of K3 Separation purification and lysate Produce

The strain was isolated from a traditional fermented ginseng beverage in Yongin, Korea. As a result of phylogenetic tree analysis based on the 16S rRNA base sequence of the isolated strain, it was confirmed that the isolated strain belonged to the genus Curtobacterium, and it was confirmed that it was a new strain having a 16S rRNA sequence different from previously known strains. The isolated strain, Curtobacterium proimmune K3, was deposited with the Korea Research Institute of Bioscience and Biotechnology on 2021.06.01 and assigned the number KCTC14591BP. The 16S rRNA sequence subjected to genetic analysis of the strain is shown in SEQ ID NO: 1.

A lysate of Kurtobacterium proimmune K3 was prepared as follows.

First, they were cultured in 100 mL LB medium at 36 °C for 24 hours until an optical density of 1.2 Å at 600 nm was reached. After reaching, suspended in the medium, the strain was sonicated for about 90 minutes and then centrifuged at 4000 rpm for 20 minutes. The supernatant was removed and 5 ml of PBS was added to the pellet and gently stirred until dissolved, followed by centrifugation at 4000 rpm for 20 minutes. PBS washes were repeated 3 times and then suspended as pellets for lyophilization.

Example 2. Kurtobacterium proimmune of K3 Confirmation of induction of NO production and induction of cytokine secretion ( in vitro )

To investigate the cytotoxicity of Curtobacterium proimmune K3 lysate on RAW264.7 cells, cells were treated with Curtobacterium proimmune K3 lysate (25 μg/mL, 50 μg/mL and 100 μg/mL) and LPS (1 μg/mL). mL) for 24 hours, and the effects on cytotoxicity, NO production, and cytokine secretion were confirmed.

2.1 Confirmation of cytotoxicity and induction of cell differentiation

After treatment of Kurtobacterium proimmune K3 lysate to RAW264.7 cells, cell death was confirmed through MTT assay and fluorescence image. Cytotoxicity experiments were specifically performed as follows. After treating RAW264.7 cells with Kurtobacterium proimmune K3 lysate using LIVE/DEAD staining kit (L-3224, Invitrogen, Carlsbad, CA, USA), cytotoxicity was confirmed. The mixture of Calcein AM and ethidium homodimer-1 in the kit appears in red (Ex 528 nm, Em 617 nm) for dead cells and green (Ex 495 nm; Em 515 nm) for live cells to analyze differences between cells. RAW264.7 cells were plated and cultured after treatment with Curtobacterium proimmune K3 lysate at 25 μg/mL, 50 μg/mL and 100 μg/mL, and LPS (1 μg/mL). After 24 hours of incubation, the dye was dissolved in the medium and incubated for 30 minutes. Cytotoxicity was confirmed by checking the cells with a Leica DMLS Clinical Microscope (Leica, Wetzlar, Germany). In addition, the cell morphology after treatment with Curtobacterium proimmune K3 and LPS was observed, and the results are shown in FIG. 1 .

As shown in FIGS. 1A and B, it was confirmed that no cell death was observed in all experimental groups treated with Curtobacterium proimmune K3. Also, as shown in FIG. 1C , it was confirmed that Curtobacterium proimmune K3 induces differentiation by increasing the cell size and elongating the pseudopods (yellow arrows in FIG. 1C ) in RAW264.7 cells. These results show that Kurtobacterium proimmune K3 can induce differentiation without cytotoxicity to RAW264.7 cells.

2.2 Confirmation of NO production and cytokine secretion effects

Paraprobiotics can modulate the anti-inflammatory response and induce a positive immune response. Macrophages are known to play an important role in the immune response, and mucosal macrophages are known to play particularly important functions in bacterial elimination, homeostasis maintenance, and protective immunity. When macrophages are exposed to inflammatory stimuli, they secrete cytokines such as TNF-α, IL-1βIFN-γ, and other molecules (NO). In order to confirm whether Kurtobacterium proimmune K3 can induce cytokine and NO production, experiments were performed to confirm mRNA cytokine gene expression of TNF-α, IL-1β and IFN-γ and NO production.

First, RAW264.7 cells were plated in a 96-well plate (1 x 10 ⁴ cells/well) for 24 hours, and various concentrations of Kurtobacterium proimmune K3 lysates 25, 50, 100, 200, 300, 400, 500, 600 μg/ml and LPS (1 μg/mL) were treated for 24 hours and then cultured. 100 μL of culture supernatant was added and reacted with 100 μL of Griess reagent. After incubation for 15 minutes, absorbance was observed with a microplate reader at 575 nm. A standard curve of sodium nitrite was used to estimate the concentration of nitrite. In addition, ELISA analysis was performed to quantify cytokine expression. As a result, an increase in NO production and cytokine secretion was induced in all groups treated with Kurtobacterium proimmune K3 for 24 hours, confirming the effect of enhancing immune activity, especially at a concentration of 100 μg/ml or less, excellent concentration-dependent immunity An activity appeared, which is shown in FIG. 2 .

As shown in FIG. 2, it was confirmed that NO production and cytokine secretion were increased in a concentration-dependent manner in the group treated with Curtobacterium proimmune K3 for 24 hours. This is a result showing that Kurtobacterium proimmune K3 can enhance cytokine secretion in macrophages of RAW264.7 cells.

2.3 Confirmation of cytokine secretion effect in spleen cells

An experiment was performed to confirm whether Kurtobacterium proimmune K3, as a paraprobiotic, could also induce immune-related cytokine production in spleen cells. To this end, rat splenocytes were treated with Kurtobacterium proimmune K3 lysate, and then WST-8 analysis was performed. Splenocytes of BALB/c mice were treated with 25 to 100 μg/mL of Kurtobacterium proimmune K3 lysate for 24 hours, and ConA (1 μg/mL) was used as a positive control. The results of WST-8 analysis and ELISA analysis for quantification are shown in FIG. 3 .

As shown in Figure 3, the increase in cytokine release compared to the control group was confirmed in the ConA-treated group known to secrete the immune regulatory cytokines TNF-α, IL-1β, IL-2, and IFN-γ as T cell mitogens. In the experimental group treated with the Curtobacterium proimmune K3 of the present invention, a significant increase in TNF-α, IL-1β, IL-2, and IFN-γ secretion was confirmed without cell death. These results show that the Kurtobacterium proimmune K3 lysate can promote cytokine expression in splenocytes.

ELISA analysis for protein expression quantification also confirmed that the expression of TNF-α, IL-1β, IL-2, and IFN-γ was increased in a dose-dependent manner in the Curtobacterium proimmune K3 lysate-treated group. These results show that the Kurtobacterium proimmune K3 lysate can exhibit an immunostimulatory effect by activating various immune regulatory cytokines in splenocytes.

Example 3. In immunosuppressed mice Kurtobacterium proimmune of K3 Confirmation of immune enhancing effect

3.1 Organ index and confirmation of innate immunity effect

Based on the results showing that the Kurtobacterium proimmune K3 lysate can exhibit an immunomodulatory effect, its immunomodulatory effect was further confirmed in vivo. For in vivo immunity experiments, cyclophosphoamine (CTX)-induced immunosuppressed mice were used, and LMS 40mg/kg treated group was used as a positive control group. Curtobacterium proimmune K3 lysate treatment group was C.proimmune K3 Ly-L (10 ⁵ CFU/mL treatment group) or C.proimmune K3 Ly-H (10 ⁶ CFU/mL treatment group) according to the treatment concentration. split was carried out. The body weight of the mouse was measured every day during the experiment, and no significant change was observed in the initial body weight after 1 week of adaptation period. However, it was confirmed that the remaining 5 immunosuppressed mouse groups, excluding the control group, lost weight after CTX treatment compared to the control group. However, unlike the CTX-treated group, no change in body weight was observed in the Kurtobacterium proimmune K3 lysate-treated group during the entire experimental period. The organ index was confirmed in the spleen and thymus, which are immune-related organs, and the results are shown in FIG. 4 .

As shown in Figure 4, it was confirmed that the indices of the spleen and thymus, which can be considered as indirect indicators for immunological disorders caused by CTX, decreased after CTX treatment. However, in the Kurtobacterium proimmune K3 lysate-treated group This decrease was recovered in a dose-dependent manner, and a better degree of spleen and thymus index recovery effect than that of the LMS-treated group used as a positive control was confirmed. These results show that the Kurtobacterium proimmune K3 lysate has an effect on the atrophic immune system induced by CTX in mice.

3.2 NK Confirmation of cell activity enhancement effect

An experiment was performed to determine whether the Kurtobacterium proimmune K3 lysate could also affect the activity of NK cells, which are effector lymphocytes of the innate immune system that control microbial infection and play an immune regulatory role. Splenocyte suspensions were prepared as described above, then suspended in complete RPMI-1640 medium solution to a final density of 1 x 10 ⁶ cells/mL and placed on 96-well plates. For the NK cell activity assay, YAC-1 cells were used as target cells and cultured with spleen cells (effector cells). The splenocyte suspension was added to YAC-1 cells (1 x 10 ⁵ cells/mL) to obtain an effector to target cell ratio of 10:1 in a 96-well plate. Cell viability was assessed using the WST-8 detection kit and microplate reader at 450 nm. RPMI 1640 medium was used as a control. NK cell toxicity was calculated through the following formula, and the results are shown in FIG. 5 .

NK cytotoxicity (%) = (OD _T - (OD _S -OD _E )) / OD _T

- where OD _T is the absorbance of the target cell control, OD _S is the absorbance of the test sample, and OD _E is the absorbance of the effector cell control.

As shown in FIG. 5, compared to control (Con) mice, CTX-treated mice had significantly reduced NK cell activity, and in mice treated with Curtobacterium proimmune K3 lysate, reduced NK cell activity was evident confirmed improvement.

These results show that the Kurtobacterium proimmune K3 lysate can exhibit an effect on CTX-induced suppression of innate immunity.

3.3 splenocytes Confirm proliferative effect

Curtobacterium proimmune K3 lysate in CTX-treated mice treated with C.proimmune K3 Ly-L (10 ⁵ CFU/mL treatment group) or C.proimmune K3 Ly-H (10 ⁶ CFU/mL treatment group) After that, an experiment was performed to confirm whether they could induce spleen proliferation, and the results are shown in FIG. 6 .

As shown in Figure 6, it was confirmed that the proliferation of splenocytes was significantly inhibited in the CTX-treated group, but recovered in a dose-dependent manner in the experimental group treated with Kurtobacterium proimmune K3 lysate.

3.3 Confirmation of adaptive immunity effect

Delayed hypersensitivity (DTH) refers to a typical cell-mediated immune response that reflects an individual's memory T cell function. In order to evaluate the adaptive immune effect through delayed hypersensitivity response, the abdominal hair of the mice was removed and the mice were sensitized with 20 μL of 1% dinitrofluorobenzene (DNFB), and the sensitization process was repeated 24 hours later. . DNFB was applied to the right ear after 4 days. After 24 hours of reaction, both ears of all mice were punched with 5 mm holes in the corresponding part, and the weight of each ear part was recorded. The degree of ear swelling was expressed as the difference in weight between the left and right ears of each mouse. In order to evaluate the adaptive immune effect, changes in ear edema were confirmed in each experimental group, and the results are shown in FIG. 7 .

As shown in FIG. 7 , the DTH response confirmed by ear edema was downregulated in the CTX-injected group compared to the control group (Con). The degree of ear edema was recovered in a concentration-dependent manner in the Kurtobacterium proimmune K3 lysate-treated group, which was confirmed to show a better effect than the LMS-treated group.

Through the above results, it was confirmed that Kurtobacterium proimmune K3 can play a role in enhancing the DTH response and enhancing the immune response.

3.4 Serum IgA and IgG content comparison

The immune response is regulated by B-lymphocytes, which are known to differentiate into plasma cells to produce immunoglobulins (IgA and IgG) and to bind specific antigens. Through the measurement of serum IgA and IgG content using ELISA, the effect of the humoral immune response was confirmed, and the results are shown in FIG. 8 .

As shown in FIG. 8, serum IgA and IgG levels were significantly reduced in CTX-treated mice compared to control mice, but for the Curtobacterium proimmune K3 lysate-treated group, especially for high-concentration Curtobacterium proimmune K3 Serum IgA and IgG levels increased in the seafood-treated group. This indicates that Kurtobacterium proimmune K3 can enhance humoral immunity in CTX-treated mice.

3.5 Confirmation of cytokine secretion enhancement effect

An experiment was performed to confirm whether the Curtobacterium proimmune K3 treatment of the present invention affects the level of serum cytokines. First, a Curtobacterium proimmune K3 lysate was administered to mice immunosuppressed by CTX treatment, and the levels of TNF-α, IL-1β, IFN-γ, and IgA and IgG (F) in serum were confirmed. Results are shown in FIG. 9 . In addition, changes in the mRNA expression level of each cytokine were measured in the spleen and thymus, which are immune-related organs, and the results are shown in FIG. 10 .

As shown in FIG. 9, although serum cytokines were significantly suppressed due to the immunosuppressive response by CTX injection, TNF-α (A) and IL-1β (B) in the group administered with Kurtobacterium proimmune K3 lysate ), IFN-γ (C), IgA (E) and IgG (F) levels increased in a dose-dependent manner.

The same result was also confirmed in FIG. 10 . Specifically, CTX injection induced a significant decrease in the levels of cytokines (IL-2, TNF-α, IFN-γ, and IL-1β) in spleen and thymus tissues, but in the group treated with Kurtobacterium proimmune K3 lysate, In all cases, the levels of IL-2, TNF-α, IFN-γ, and IL-1β were significantly increased compared to the CTX group mice in a dose-dependent manner. This result shows that gene expression of cytokines can be restored in tissues.

Example 4. NF - κB and MAPKs Effects on signaling pathways

NF-κB signaling is an important regulator of cytokine gene expression and plays an important role in the intervention of anti-inflammatory therapies, and is known to affect cell growth and differentiation by activating MAPKs. It also regulates the cellular response to cytokines and the related expression of genes such as TNF-α, IL-2, and IFN-γ. Therefore, in order to confirm the effect of Kurtobacterium proimmune K3 lysate on the signaling pathway in regulating cytokine expression, NF-κB and MAPK proteins including phosphorylated NF-κB and IκBα and phosphorylated JNK, ERK and The amount of p38 was analyzed, respectively, and the results are shown in FIG. 11 .

As shown in FIG. 11, NF-κB signaling and activation of MAPK protein were greatly suppressed in the CTX-treated group, but phosphorylated NF-κB, IκBα, and phosphorylated JNK were significantly inhibited in the Kurtobacterium proimmune K3 lysate-treated group. , it was confirmed that the decreased protein expression of ERK and p38 was restored. These results show that Curtobacterium proimmune K3 can activate NF-κB and MAPK signaling pathways to restore immune suppression induced by CTX.

Through the above results, the Curtobacterium proimmune K3 of the present invention stimulates the immune activation of RAW264.7 macrophages (in vitro) and spleen cells (ex vivo) by promoting the mRNA expression and secretion of immune-related cytokines. It was confirmed that In addition, through in vivo experiments, Curtobacterium proimmune K3 restores innate and adaptive immunity, relieves immune suppression, and significantly enhances the production of cytokines such as IL-2, IFN-γ, TNF-a, and IL-1β. By confirming that it can be done, it was confirmed that Kurtobacterium proimmune K3 can exhibit an immune enhancing effect.

Korea Research Institute of Bioscience and Biotechnology KCTC14591BP 20210601

<110> University-Industry Cooperation Group of Kyung Hee University <120> Composition for enhancing immunity comprising Curtobacterium proimmune K3 <130> GKH1-65 <160> 1 <170> KoPatentIn 3.0 <210> 1 <211> 1450 <212> DNA <213> artificial sequence <220> <223> Curtobacterium proimmune K3 16s rRNA <400> 1 ttttgagttt ttggtnccgg ctcaggacga acgctggcgg cgtgcttaac acatgcaagt 60 cgaacgatga tcaggagctt gctcttgtga ttagtggcga acgggtgagt aacacgtgag 120 taacctgccc ctgactctgg gataagcgtt ggaaacgacg tctaatactg gatacgactg 180 ccggccgcat ggtctggtgg tggaaagatt ttttggttgg ggatggactc gcggcctatc 240 agcttgttgg tgaggtaatg gctcaccaag gcgacgacgg gtagccggcc tgagagggtg 300 accggccaca ctgggactga gacacggccc agactcctac gggaggcagc agtggggaat 360 attgcacaat gggcgaaagc ctgatgcagc aacgccgcgt gagggatgac ggccttcggg 420 ttgtaaacct cttttagtag ggaagaagcg aaagtgacgg tacctgcaga aaaagcaccg 480 gctaactacg tgccagcagc cgcggtaata cgtagggtgc aagcgttgtc cggaattatt 540 gggcgtaaag agctcgtagg cggtttgtcg cgtctgctgt gaaatcccga ggctcaacct 600 cgggcttgca gtgggtacgg gcagactaga gtgcggtagg ggagattgga attcctggtg 660 tagcggtgga atgcgcagat atcaggagga acaccgatgg cgaaggcaga tctctgggcc 720 gtaactgacg ctgaggagcg aaagcgtggg gagcgaacag gattagatac cctggtagtc 780 cacgccgtaa acgttgggcg ctagatgtag ggacctttcc acggtttctg tgtcgtagct 840 aacgcattaa gcgccccgcc tggggagtac ggccgcaagg ctaaaactca aaggaattga 900 cgggggcccg cacaagcggc ggagcatgcg gattaattcg atgcaacgcg aagaacctta 960 ccaaggcttg acatacaccg gaaacggcca gagatggtcg cccccttgtg gtcggtgtac 1020 aggtggtgca tggttgtcgt cagctcgtgt cgtgagatgt tgggttaagt cccgcaacga 1080 gcgcaaccct cgttctatgt tgccagcgcg ttatggcggg gactcatagg agactgccgg 1140 ggtcaactcg gaggaaggtg gggatgacgt caaatcatca tgccccttat gtcttgggct 1200 tcacgcatgc tacaatggcc ggtacaaagg gctgcgatac cgtaaggtgg agcgaatccc 1260 aaaaagccgg tctcagttcg gattgaggtc tgcaactcga cctcatgaag tcggagtcgc 1320 tagtaatcgc agatcagcaa cgctgcggtg aatacgttcc cgggccttgt acacaccgcc 1380 cgtcaagtca tgaaagtcgg taacacccga agccggtggc ctaaccctgg gaagagcctc 1440 aaggatgacc 1450

Claims (14)

Curtobacterium proimmune K3 (Curtobacterium proimmune K3) strain (accession number: KCTC14591BP).
Curtobacterium proimmune K3 strain (accession number: KCTC14591BP), a culture thereof or a cell-free extract thereof, comprising a composition for enhancing immunity.
The composition for enhancing immunity according to claim 2, wherein the Kurtobacterium proimmune K3 strain is live or dead.
3. The immune Enhancement composition.
The composition for enhancing immunity according to claim 2, wherein the immune enhancement is innate immunity or adaptive immunity enhancement.
The method of claim 2, wherein the Curtobacterium proimmune K3 strain (accession number: KCTC14591BP), its culture medium or its cell-free extract is in the group consisting of IL-2, IFN-γ, TNF-a and IL-1β To enhance the expression of one or more selected cytokines, immune enhancing composition.
The composition for enhancing immunity according to claim 2, wherein the Curtobacterium proimmune K3 strain (Accession No.: KCTC14591BP), its culture medium or its cell-free extract promotes the production of serum IgA or serum IgG.
Curtobacterium proimmune K3 strain (accession number: KCTC14591BP), a culture thereof, or a food composition for enhancing immunity, including a cell-free extract thereof.
Curtobacterium proimmune K3 strain (accession number: KCTC14591BP), a culture thereof, or a health functional food composition for immune enhancement, including a cell-free extract thereof.
Curtobacterium proimmune K3 strain (accession number: KCTC14591BP), a culture thereof, or a feed composition for immune enhancement, including a cell-free extract thereof.
Curtobacterium proimmune K3 strain (accession number: KCTC14591BP) containing a culture medium or a cell-free extract thereof, a cosmetic composition for enhancing immunity.
Curtobacterium proimmune K3 strain (accession number: KCTC14591BP), a pharmaceutical composition for immunity enhancement comprising a culture medium thereof or a cell-free extract thereof.
Inactivating the Kurtobacterium proimmune K3 strain (accession number: KCTC14591BP); A method for producing a composition for enhancing immunity comprising para-probiotics.
[Claim 14] The method of claim 13, wherein the inactivation treatment is at least one inactivation treatment selected from the group consisting of ultraviolet rays, radiation, high pressure, and ultrasonic waves.

Images