KR102546973B1 - Composition for enhancing immunity comprising Bacillus velenzensis Kh2-2 - Google Patents

Abstract

The present invention relates to a Bacillus valergensis Kh2-2 strain, and more particularly, to an immuno-enhancing use of the strain. The Bacillus balegensis Kh2-2 strain according to the present invention promotes the expression of at least one cytokine selected from the group consisting of IL-2, IFN-γ, TNF-a and IL-1β and the production of serum IgA or serum IgG Since it can effectively promote both innate and adaptive immunity in the body, it can be used in various ways such as food for immunity enhancement, health functional food, feed, medicine, and skin health material.

Description

Composition for enhancing immunity comprising Bacillus velenzensis Kh2-2 strain {Composition for enhancing immunity comprising Bacillus velenzensis Kh2-2}

The present invention relates to a Bacillus valergensis Kh2-2 strain, and more particularly, to an immuno-enhancing use of the strain.

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, in recent years, high-density products with increased content dozens of times have been produced and distributed in consideration of vitality in the human body, but side effects caused by excessive strains have been reported, so there is a need for new strains for immunity enhancement that can solve these problems. There is a need.

The inventors of the present invention completed the present invention by confirming that the isolated and identified Bacillus valerensis Kh2-2 strain exhibits an excellent immune enhancing effect while conducting research on isolating useful strains from traditional fermented foods.

Accordingly, an object of the present invention is to provide a Bacillus velenzensis Kh2-2 strain (accession number: KCTC14642BP).

Another object of the present invention is to provide a composition for enhancing immunity comprising the Bacillus valerensis Kh2-2 strain, its culture medium, its cell body or its cell-free extract.

In order to achieve the above object, the present invention provides a Bacillus velenzensis Kh2-2 strain (Accession Number: KCTC14642BP).

In addition, the present invention provides a pharmaceutical composition for enhancing immunity comprising the Bacillus valergensis Kh2-2 strain, its culture medium, its cell body or its cell-free extract.

In addition, the present invention provides a food composition for enhancing immunity comprising the Bacillus valergensis Kh2-2 strain, its culture medium, its cell body or its cell-free extract.

In addition, the present invention provides a health functional food composition for enhancing immunity comprising the Bacillus valergensis Kh2-2 strain, its culture medium, its cell body or its cell-free extract.

In addition, the present invention provides a feed composition for enhancing immunity comprising the Bacillus valergensis Kh2-2 strain, its culture medium, its cells or its cell-free extract.

The Bacillus balegensis Kh2-2 strain according to the present invention promotes the expression of at least one cytokine selected from the group consisting of IL-2, IFN-γ, TNF-a and IL-1β and the production of serum IgA or serum IgG Since it can effectively promote both innate and adaptive immunity in the body, it can be used in various ways such as food for immunity enhancement, health functional food, feed, medicine, and skin health material.

Figure 1A is a diagram showing the results of confirming the acid resistance of the Bacillus strain under pH 2.0 conditions.
Figure 1B is a diagram showing the results of confirming the bile resistance in the 0.5% bile salt condition of the Bacillus strain.
Figure 1C is a diagram showing the results of confirming the beta-glucosidase activity of Bacillus strains.
Figure 1D is a diagram showing the results of confirming the cytotoxicity of Bacillus strain cells in RAW264.7 cells.
Figure 1E is a diagram showing the results of confirming NO production according to the treatment of Bacillus strain cells in RAW264.7 cells.
Figure 1F is a diagram showing the results of confirming the amount of cytokine expression according to the treatment of Bacillus cells in RAW264.7 cells.
Figure 2A is a view showing the results of analyzing the induction of cell differentiation according to the treatment of Bacillus valerensis Kh2-2 cells in macrophages.
Figure 2B is a diagram showing the results of cytokine mRNA expression analysis according to the treatment of Bacillus ballegensis Kh2-2 cells in RAW264.7 cells.
Figure 2C is a diagram showing the target cytokine protein expression analysis results according to the treatment of Bacillus balegensis Kh2-2 cells through ELISA.
3A and B are views showing the results of confirming the expression of MAPK and NF-κB signaling pathway-related proteins according to the treatment of Bacillus valerensis Kh2-2 cells in macrophages.
Figure 4A is a diagram showing the results of examining the cytotoxicity of Bacillus valerensis Kh2-2 cells in mouse splenocytes.
Figure 4B is a diagram showing the results of analyzing the amount of NO production according to the treatment of Bacillus bellegensis Kh2-2 cells in mouse splenocytes.
Figure 4C is a diagram showing the results of analyzing the amount of cytokine mRNA production according to the treatment of Bacillus bellegensis Kh2-2 cells in mouse splenocytes.
Figure 4D is a diagram showing the results of analyzing the protein expression levels of the cytokine proteins TNF-α, IFN-γ, and IL-2 according to the treatment of Bacillus bellegensis Kh2-2 cells in mouse splenocytes.
5A is a diagram showing a control group and an experimental group for in vivo experimentation.
Fig. 5B is a diagram showing organ indices of the spleen and thymus according to the treatment of the Bacillus balegensis Kh2-2 strain or cells in immunosuppressed mice.
Figure 5C is a diagram showing the results of confirming the NK cell toxicity of the Bacillus ballegensis Kh2-2 strain or its cells.
Figure 5D is a diagram showing the result of confirming the effect of splenocyte proliferation according to the treatment of Bacillus valerensis Kh2-2 strain or its cells in immunosuppressed mice.
Figure 5E is a view showing the results of confirming the adaptive immune effect according to the treatment of Bacillus valerensis Kh2-2 strain or its cells in immunosuppressed mice.
Figure 5F is a diagram showing the results of confirming the serum IgA and IgG contents according to the treatment of the Bacillus valergensis Kh2-2 strain or its cells in immunosuppressed mice.
6A to C are diagrams showing the expression levels of cytokines IL-2, IL-6, and IFN-γ in serum according to the treatment of Bacillus valergensis Kh2-2 strain or its cells in immunosuppressed mice.
7A and B are views showing the results of confirming the expression of proteins related to MAPK and NF-κB signaling pathways according to the treatment of Bacillus valergensis Kh2-2 strain or its cells in immunosuppressed mice.
Figure 8A is a diagram showing the results of measuring the colon length according to the treatment of Bacillus valergensis Kh2-2 strain or its cells in immunosuppressed mice.
Figure 8B is a diagram showing the mRNA expression analysis results for targeted cytokines according to the treatment of the Bacillus valergensis Kh2-2 strain or its cells in the colon tissue of immunosuppressed mice.
Figure 8C is a view showing the results of Venn diagram analysis on the shared abundance of the intestinal microflora according to the treatment of the Bacillus valergensis Kh2-2 strain or its cells in immunosuppressed mice.
FIG. 8D is a diagram showing the result of confirming the taxonomic composition distribution at the phylum level of each group of intestinal microflora based on the Venn diagram of FIG. 8C.
Figure 8E is a diagram showing the results of comparing the averages of the top three abundant intestinal microflora at the phylum level, based on Figure 8D.
8F is a diagram showing the results of analyzing the taxonomic compositional distribution of the gut microbiota at the genus level of each group based on the analysis of the shared abundance of the gut microbiota.

Hereinafter, the present invention will be described in detail.

According to an aspect of the present invention, the present invention provides a Bacillus velenzensis Kh2-2 strain (Accession Number: KCTC14642BP).

The Bacillus balegensis Kh2-2 strain according to the present invention is a strain isolated from a traditional fermented ginseng beverage and is a safe strain when ingested into the body. The Bacillus balegensis Kh2-2 strain was patented at the Biological Resources Center on July 22, 2021, and was given accession number KCTC14642BP.

In an embodiment of the present invention, the strain preferably includes the nucleotide sequence of SEQ ID NO: 1. The nucleotide sequence of SEQ ID NO: 1 is a 16S rDNA gene, and is a site used when identifying a strain.

In an embodiment of the present invention, the strain is characterized in that it has acid resistance or bile resistance.

In an embodiment of the present invention, the strain preferably has an immune enhancing activity.

In the present invention, immunostimulation is one of the important therapeutic strategies for reinforcing the body's defense mechanism against various diseases such as cancer and inflammatory diseases, and increases the activity of immune cells to stimulate the immune response to enhance the immune system. can be obtained. For example, phagocytes play a major role in the immune response. Phagocytosis, a major role in macrophages, absorbs microorganisms and other pyrogenic particles, and also inhibits tumor necrosis factor-α (TNF-α) and interleukin-α. It stimulates the immune response by secreting a number of cytokines such as 1β (interleukin; IL-1β) and interleukin-12 (interleukin; IL-12) and cytotoxic and inflammatory substances such as nitric oxide (NO). stimulate Therefore, increasing macrophage activity can be one means for enhancing immunity. Since the occurrence of infections and diseases mainly occurs in a state of reduced immune function, various studies have been conducted to enhance these immune responses through immune enhancing substances when the function of the body's immune system is reduced.

In a preferred embodiment of the present invention, the immunity is preferably innate immunity or adaptive immunity, and more preferably may mean both innate immunity and adaptive immunity. The scope is not limited.

According to another aspect of the present invention, the present invention provides a composition for enhancing immunity comprising the Bacillus velenzensis Kh2-2 strain, a culture thereof, a cell cell thereof, or a cell-free extract thereof.

In the present invention, the culture medium means that the strain is inoculated and cultured in a liquid medium, and the culture medium may be removed and only the cells are resuspended in a buffer such as PBS.

The composition for enhancing immunity of the present invention may be a pharmaceutical composition, a food composition, a health functional food composition, or a feed composition.

In the case of the pharmaceutical composition according to the present invention, the composition may include a pharmaceutically effective amount of the cells and excipients or diluents.

The pharmacologically effective amount in the above refers to an amount sufficient to exert an immune enhancing effect. The term "pharmaceutically acceptable" refers to a composition that is physiologically acceptable and does not usually cause allergic reactions such as gastrointestinal disorders and dizziness or similar reactions when administered to humans.

In addition, the pharmaceutical composition according to the present invention is formulated according to conventional methods into oral formulations such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, external preparations, suppositories and sterile injection solutions. can Suitable formulations known in the art are preferably those disclosed in the literature (Remington's Pharmaceutical Science, recently, Mack Publishing Company, Easton PA). Carriers, excipients and diluents that may be included include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum acacia, alginates, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxy benzoate, propylhydroxy benzoate, talc, magnesium stearate, mineral oil, and the like. When formulating the pharmaceutical composition, it is prepared using diluents or excipients such as commonly used fillers, extenders, binders, wetting agents, disintegrants, and surfactants. Solid preparations for oral administration include tablets, pills, powders, granules, capsules, etc., and these solid preparations contain at least one excipient such as starch, calcium carbonate, sucrose, and lactose in the composition. , gelatin, etc. are mixed and prepared. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used. Liquid preparations for oral use include suspensions, solutions, emulsions, and syrups. In addition to water and liquid paraffin, which are commonly used simple diluents, various excipients such as wetting agents, sweeteners, aromatics, and preservatives may be included. there is. Preparations for parenteral administration include sterilized aqueous solutions, non-aqueous solvents, suspensions, emulsions, freeze-dried preparations, suppositories, and the like. 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 suspending agents. As a base for the suppository, witepsol, macrogol, tween 61, cacao butter, laurin paper, glycerogeratin and the like may be used.

The pharmaceutical composition of the present invention can be administered in a therapeutically effective amount, which is an amount of an active ingredient or pharmaceutical composition that induces a biological or medical response in a tissue system, animal, or human considered by researchers, veterinarians, doctors, or other clinicians. It is apparent to those skilled in the art that the therapeutically effective dosage and frequency of administration of the pharmaceutical composition of the present invention will vary depending on the desired effect. Therefore, the optimal dose to be administered can be easily determined by those skilled in the art, and depends on the type of disease, the severity of the disease, the content of the active ingredient and other ingredients contained in the composition, the type of formulation, and the age, weight, and general health of the patient. Condition, sex and diet, administration time, administration route and secretion rate of the composition, treatment period, can be adjusted according to various factors including drugs used simultaneously. For desirable effects, the pharmaceutical composition of the present invention may be administered in an amount of 1 to 10,000 mg/kg/day, preferably 1 to 200 mg/kg/day, and may be administered once a day or several times. It can also be administered in divided doses.

The pharmaceutical composition of the present invention can be administered to a subject by various routes. All modes of administration are contemplated, eg oral, rectal or intravenous, intramuscular, subcutaneous, intrauterine intrathecal or intracerebrovascular injection.

In addition, when the composition of the present invention is a food composition, the type of food is not particularly limited, and may include all foods in a conventional sense. Non-limiting examples of foods to which the substance can be added include meat, sausage, bread, chocolate, candy, snacks, confectionery, pizza, ramen, other noodles, gum, dairy products including ice cream, various soups, beverages, tea , drinks, alcoholic beverages, and vitamin complexes. When using the composition as a food additive, the composition may be added as it is or used together with other foods or food ingredients, and may be appropriately used according to conventional methods.

In the present specification, food means a natural product or processed product containing one or more nutrients, preferably means that it can be directly eaten through a certain degree of processing process, and is a conventional meaning, and the food The composition is intended to include all foods, food additives, health functional foods and beverages.

Foods to which the composition of the present invention can be added include, for example, various foods, beverages, gum, candy, tea, vitamin complexes, and functional foods. In addition, in the present invention, the food includes special nutritious food (eg, formula milk, infant food, baby food, etc.), processed meat product, fish meat product, tofu, jelly, noodles (eg, ramen, noodles, etc.), health supplement food, seasoning food ( For example, soy sauce, soybean paste, red pepper paste, mixed soybean paste, etc.), sauces, confectionery (eg, snacks), dairy products (eg, fermented milk, cheese, etc.), other processed foods, kimchi, pickled foods (various types of kimchi, pickles, etc.), beverages (eg, fruit and vegetable beverages, soy milk, fermented beverages, ice cream, etc.), natural seasonings (eg, ramen soup, etc.), vitamin complexes, alcoholic beverages, alcoholic beverages, and other health supplements, but are not limited thereto. The food, beverage or food additive may be prepared by a conventional manufacturing method.

The food composition of the present invention may preferably be a health functional food. The health functional food refers to a food manufactured and processed by extracting, concentrating, refining, mixing, etc., a specific ingredient as a raw material or a specific ingredient contained in a food raw material for the purpose of health supplement. It refers to food designed and processed to sufficiently exert biological control functions such as regulation of biorhythms, prevention and recovery of diseases, etc., and functions related to immunity enhancement.

When using the strain according to the present invention as a health functional food, it can be added as it is or used together with other foods or food ingredients, which can be selected and used appropriately as needed.

Cell safety according to the present invention is guaranteed, and since the activity increases in proportion to the concentration, it is obvious that it can be used in an appropriate amount without being limited to a specific range.

In addition, there is no particular limitation on the type of health functional food in which the strain according to the present invention can be used. For example, ramen, other noodles, beverages, tea, drinks, alcoholic beverages, various soups, meat, sausages, bread, chocolate, candy, confectionery, pizza, chewing gum, dairy products including ice cream, or vitamin complexes.

In particular, the strain according to the present invention can be added to various foods for the purpose of enhancing immunity to exhibit functions, and suitable other auxiliary ingredients and known additives that can be commonly contained in health functional foods according to the selection of a person skilled in the art Can be used in combination with The known additives include other microorganisms that can be used together with the strain according to the present invention.

The composition of the present invention for immuno-enhancing feed composition may be a feed additive or a formulated feed.

In the present invention, "feed additive" means a substance added in small amounts to feed for nutritional or specific purposes, and the functional feed additive of the present invention may contain strains as active ingredients. The active ingredient may be blended with known feed additives in a certain ratio. When formulated, the active ingredient of the present invention may be applied in an amount of 0.01 to 99% by weight based on the total weight of the feed additive.

In addition, the feed composition of the present invention may contain a strain based on 100 parts by weight of the formulated feed. As an example of the compound feed, 53.71% by weight of corn, 23.2% by weight of soybean meal, 1.0% by weight of rapeseed meal, 2.0% by weight of corn meal, 3.0% by weight of ginseng, 2.7% by weight of wheat hull, 0.3% by weight of potato starch, 2.8% by weight of oil 9.9% by weight of calcium carbonate, 0.68% by weight of dibasic calcium phosphate, 0.23% by weight of sodium chloride, 0.03% by weight of phosphorus enzyme, 0.05% by weight of choline chloride, 0.2% by weight of methionine, 0.1% by weight of mineral mix, and 0.1% by weight of vitamin mix is formed In addition, it goes without saying that conventional poultry formulated feeds can be used. In addition, it goes without saying that compounded feeds such as cows, pigs, ducks, etc. may be used according to the type of livestock to be raised.

Livestock may be raised by feeding the feed composition, and the strain of the present invention may be diluted in water and provided as drinking water during breeding of livestock. At this time, 100 to 500 ml of the active ingredient (the strain of the present invention, its culture medium, its cell body or its cell-free extract) may be applied to 100 L of water.

The feed composition of the present invention may further include other nutritional components within the range expected by those skilled in the art.

The strain of the present invention can be incorporated into commercially common animal feed compositions, and can be fed, for example, to cattle, swine, sheep, poultry, and the like. To this end, the strain of the present invention and its culture medium can be mixed with common animal feed ingredients and, if necessary, processed into a purified form. Common animal feed ingredients are, for example, corn, barley, oats, soybeans, fishmeal, bran, soybean oil, minerals, trace elements, amino acids and vitamins.

Redundant content is omitted in consideration of the complexity of the present specification, and terms not otherwise defined in the present specification have meanings commonly used in the technical field to which the present invention belongs.

Hereinafter, the present invention will be described in more detail through examples. These examples are only for illustrating the present invention, and it will be apparent to those skilled in the art that the scope of the present invention is not to be construed as being limited by these examples.

[Test method]

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 cultured 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 Bacillus valerensis Kh2-2 in spleen cells was evaluated using the WST-8 cell viability assay kit purchased from BIOMAX (Seoul, Korea). Splenocytes were cultured with concanavalin A (ConA) or various concentrations of Bacillus valergensis Kh2-2 cells (25, 50, 100, 200, 300, 400, 500, 600 μg/mL) at 1x10 ⁶ cells/well. Density was seeded into 96-well plates and cultured 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 No. 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 Kh2-2 Bacillus ballegensis Kh2-2 strain 10 ⁶ CFU/mL treatment group Kh2-2ly Bacillus balegensis Kh2-2 cells 10 ⁶ CFU / mL treatment group LMS CTX-treated group treated with levamisole hydrochloride (40 mg/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 Bacillus valergensis Kh2-2 or its cells, 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 various concentrations of Bacillus valerensis Kh2-2 cells and LPS or ConA, respectively. 100 μl of the culture supernatant was obtained from the cell medium and the secretion of TNF-α, IL-1β, IL-2 and INF-γ was evaluated using ELISA according to the manufacturer's (R & D Systems) instructions.

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, 500 ng 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' IL-10 Forward 5’-TAGAGCTGCGGACTGCCTTCA-3’ Reverse 5'-GGTCTTCAGCTTTCTCACCCAG-3' IL-4 Forward 5'-TGATCACACATTGCATTTCA-3' Reverse 5'-ACACCAGATTGTCAGTCACTTG-3'

Immunoblot analysis

Spleens were lysed for 1 hour using Pierce™ RIPA Buffer (Thermo Fisher Scientific, Rockford, USA). The splenocytes were centrifuged at 12,000 rpm and 4° C. for 20 minutes, and the same amount (50 μg) of total protein was loaded on 10% SDS-PAGE and transferred to a PVDF membrane. 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.

Design of experiments in animals and in vivo

Male ICR mice weighing 28-30*?*g (age, 4 weeks) were purchased from Orient Bio Inc. (Seongnam, Korea). Mice were housed on a 12-h light-dark cycle at 23 ± 2 °C humidity in a controlled room with food and water provided ad libitum. All experiments were performed with the approval of the Animal Care Committee (KHUASP(SE)-14-040) of the Animal Research Institute of Kyung Hee University, and were performed in accordance with the guidelines for the care and use of laboratory animals.

After acclimatization for one week, the mice were randomly divided into 5 groups: control (CON), model control (CTX), positive control (LMS), Kh2-2 and Kh2-2 cell groups. For the next 23 days, all mice had free access to sterile water and feed. An immunosuppressed mouse model was developed by intraperitoneal injection of 60 mg/(kg·d) CTX to all mice except for the control mice from the first day of treatment for 3 consecutive days. Control animals were injected with sterile saline (control). Subsequently, PBS (100 μL/d) was orally administered to the CON and CTX groups. Kh2-2 and Kh2-2 cell supernatant groups were orally administered with Kh2-2 cells (1 × 10 ⁶ CFU/mL) and Kh2-2 cells (1 × 10 ⁶ CFU/mL) at a volume of 10 mL/kg/ . Mice were monitored daily. All animals were sacrificed after strict fasting for 12 hours and complete anesthesia by inhalation of ether. Blood, thymus, spleen and colon of sacrificed mice were immediately collected for further study. The organ index was calculated by the following formula.

Organ weight ratio (%) = Organ weight (g) / Body weight (g) × 100%

Proliferation of splenocytes and cytotoxicity of natural killer (NK) cells

Spleens were collected from mice sacrificed under aseptic conditions for analysis of spleen cell proliferation. After collecting splenocytes, they were plated in 96-well plates. Splenocyte proliferation was assessed using the WST-8 assay with LPS and Con A, respectively. After 30 minutes absorbance was measured in a spectrophotometric microplate reader at 450 nm (SpectraMax ^® ABS plus).

The cytotoxicity of NK cells in spleen cells was measured. Specifically, lymphoblast YAC-1 cells (2×10 ⁵ cells/well), which are target cells, were cultured in a 96-well plate for 5 hours to detect NK cell activity assays (eector cells, 2×10 ⁶ cells/well). well) and co-cultured. The viability of YAC-1 cells was evaluated using the WST-1 assay kit, and the NK cell cytotoxicity was calculated by the following formula.

NK cell cytotoxicity (%) = (T-(S-E))/T, where T is the OD value of the target cell, S is the OD value of the test sample, and E is the OD value of the effector cell.

Delayed-type hypersensitivity (DTH) assay

After shaving the belly hair of the mouse, 20 μL 1% dinitrofluorobenzene (DNFB) was applied for sensitization. The sensitization process was repeated after 24 hours. After 4 days, DNFB was applied to each right ear of the mice for 24 hours. A 5 mm hole was drilled in the corresponding part of both ears of each mouse, 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.

High throughput sequencing and bioinformatics analysis

Fresh fecal samples from 5 mice randomly selected from each group were rapidly frozen in liquid nitrogen. Fecal samples were stored at -80 °C for long-term preservation. Genomic DNA was extracted from fecal samples using the FastDNA Spin Kit for Soil (MP Biomedicals, CA, USA). The V3 and V4 regions of 16S rDNA were amplified from genomic DNA using PCR using universal primers according to a previous study and purified by ChunLab, Inc. (Seoul, Korea) on an Illumina MiSeq Sequencing system (Illumina, USA) according to the manufacturer's instructions. ) was sequenced. The generated high-quality sequences were classified into operational taxonomic units (OTUs) using the EzBioCloud 16S-based MTP (Cheonlab's bioinformatics cloud platform) for further bioinformatics analysis. Finally, species level was clustered according to OTU homology.

statistical analysis

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

[Example]

Example 1. Separation and purification of Bacillus valergensis Kh2-2 and cell preparation

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 Bacillus valerensis, and it was confirmed that it was a new strain having a 16S rRNA sequence different from previously known strains. It was named Kh2-2. The Bacillus balegensis Kh2-2 was deposited at the Center for Biological Resources on July 22, 2021 and assigned a KCTC14642BP number. The 16S rRNA sequence subjected to genetic analysis of the strain is represented by SEQ ID NO: 1.

Cells of Bacillus valergensis Kh2-2 were used in the experiments described below. In order to prepare the Bacillus valerensis Kh2-2 cells, the strain was cultured in 100 mL LB medium at 36 ° C for 24 hours until it reached an optical density of 1.2 Å at 600 nm, then the medium was removed, and then washed with PBS. was repeated three times and then suspended as pellets for freeze-drying.

In addition, Kh2-1, Kh8-3 and Kh3-1 strains were used as control groups for experiments described later. Kh2-1, Kh8-3, and Kh3-1 strains were separated together when strain Kh2-2 was isolated.

Example 2. Characterization of Bacillus balegensis Kh2-2

Acid resistance, bile resistance and beta-glucosidase activity of Bacillus valergensis Kh2-2 were confirmed.

2.1 acid resistance

After culturing the Bacillus valergensis Kh2-2 strain in MRS medium at pH 2.0 for 3, 6 and 12 hours, the survival rate was confirmed. The results of confirming the survival rate of Bacillus valerensis Kh2-2 in acidic conditions are shown in FIG. 1A.

As shown in Figure 1A, it was confirmed that the survival rate of Bacillus valerensis Kh2-2 was high in acidic conditions. This means that Bacillus valerensis Kh2-2 has excellent acid resistance.

2.2 Biliary resistance

After culturing the Bacillus valergensis Kh2-2 strain in MRS medium containing 0.5% bile salt for 3, 6 and 12 hours, the survival rate was confirmed. The results of confirming the survival rate of Bacillus valerensis Kh2-2 under the above conditions are shown in FIG. 1B.

As shown in Figure 1B, it was confirmed that the survival rate of Bacillus valerensis Kh2-2 was high even in the presence of bile salts. This means that Bacillus valerensis Kh2-2 has excellent bile resistance.

2.3 beta-glucosidase activity

Beta-glucosidase activity of the Bacillus ballegensis Kh2-2 strain was analyzed. Bacillus valerensis Kh2-2 strain hydrolyzes bile esculin and releases esculetin into the medium using free sugar as energy. It then reacts with ferric citrate to form a phenolic iron complex, which changes the color of the colonies to dark brown. The results of analyzing beta-glucosidase activity are shown in FIG. 1C.

As shown in Fig. 1C, strains Kh2-2 and Kh3-1 exhibited beta-glucosidase activity, which was confirmed by the colonies turning black. The above results suggest that strains Kh2-2 and Kh3-1 were grown on bile esculin medium. The ability to hydrolyze bile salts can help microbes maintain the balance of the gut microbiota.

Example 3. Confirmation of cytotoxicity of Bacillus balegensis Kh2-2

The cytotoxicity of Bacillus valerensis Kh2-2 cells in RAW264.7 cells was investigated. Specifically, RAW264.7 cells were treated with Bacillus valergensis Kh2-2 cells using a LIVE/DEAD staining kit (L-3224, Invitrogen, Carlsbad, CA, USA), and 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 Bacillus valerensis Kh2-2 cells were treated with 25 μg/mL, 50 μg/mL and 100 μg/mL, and LPS (1 μg/mL), and then cultured. 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). The result of confirming cytotoxicity is shown in FIG. 1D.

As shown in Fig. 1D, it was confirmed that the Bacillus valerensis Kh2-2 cell had almost no cytotoxicity.

After cytotoxicity analysis, the effect of Bacillus ballegensis Kh2-2 cell treatment on NO production and cytokine (iNOS) secretion was confirmed. The results of confirming NO production and cytokine secretion are shown in FIGS. 1E and F, respectively.

As shown in FIG. 1E, it was confirmed that the Bacillus valergensis Kh2-2 cell treatment group induced NO production in a concentration-dependent manner. This is a result showing that Bacillus balegensis Kh2-2 can enhance cytokine secretion in macrophages of RAW264.7 cells.

As shown in FIG. 1F, it was confirmed that iNOS expression significantly increased in the group treated with Bacillus valergensis Kh2-2 cells. The above results support that the secretion of cytokines is increased according to the treatment of Bacillus valerensis Kh2-2 cells.

Example 4. Confirmation of induction of RAW264.7 cell differentiation of Bacillus valerensis Kh2-2 cells

Bacillus valerensis Kh2-2 cells were treated with RAW264.7 cells at various concentrations (25, 50, 100 μg/ml), and then whether or not cell differentiation was induced was confirmed through fluorescence images, and the results are shown in FIG. 2A. .

As shown in FIG. 2A, it was confirmed that the Bacillus valerensis Kh2-2 cells increased cell size in RAW264.7 cells and elongated pseudopods (yellow arrows in FIG. 2A) to induce differentiation. The above result means that the Bacillus ballegensis Kh2-2 strain can induce cell differentiation without cytotoxicity.

Example 5. Cytokine expression analysis according to Bacillus ballegensis Kh2-2 cell treatment

Bacillus valerensis Kh2-2 cells were treated with RAW264.7 cells at various concentrations (25, 50, 100 μg/ml), and then mRNA expression of cytokines IL-6, TNF-α and IL-1β was analyzed. . The results of cytokine mRNA analysis are shown in FIG. 2B.

As shown in Fig. 2B, it was confirmed that the Bacillus valerensis Kh2-2 cells reduced the mRNA expression of IL-6, TNF-α and IL-1β, which were increased by LPS treatment.

Based on the cytokine mRNA expression analysis results, the protein expression of each target cytokine was measured by ELISA. The results of analyzing the protein expression of the cytokine proteins IL-6, TNF-α and IL-1β are shown in FIG. 2C.

As shown in Figure 2C, it was confirmed that the immune enhancement effect was excellent in a concentration-dependent manner even when only the cells were treated.

Example 5. Effects of Bacillus valergensis Kh2-2 cells on NF-κB and MAPKs signaling pathways in macrophages

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 the Bacillus balegensis Kh2-2 strain on the signaling pathway in regulating cytokine expression, the signaling pathway-related proteins (ERK, p38, JNK, p65 and IκBα) and their phosphorylated protein amounts were analyzed did The results of analyzing protein expression are shown in Figures 3A and B.

As shown in FIGS. 3A and B, it was confirmed that NF-κB signaling and activation of MAPK protein were significantly increased (ie, immune enhancement) in the LPS-treated group. It was confirmed that the protein expression of phosphorylated NF-κB and IκBα and phosphorylated JNK, ERK, and p38 were increased in a concentration-dependent manner in the group treated with Bacillus valergensis Kh2-2 cells. The above result means that even when only the Bacillus valerensis Kh2-2 cells are treated, the immune enhancing effect is excellent like that of LPS.

Example 6. Immunostimulatory effect of Bacillus balegensis Kh2-2 in mouse splenocytes

The cytotoxicity of Bacillus valergensis Kh2-2 cells in mouse splenocytes was investigated, and the results are shown in FIG. 4A.

As shown in FIG. 4A, it was confirmed that the Bacillus bellegensis Kh2-2 cell did not exhibit cytotoxicity in mouse splenocytes.

After the cytotoxicity test, the amount of NO production according to the treatment of Bacillus bellegensis Kh2-2 cells was analyzed. The results of analyzing the amount of NO production are shown in FIG. 4B.

As shown in FIG. 4B, it was confirmed that NO production increased in a concentration-dependent manner in the Bacillus bellegensis Kh2-2 cell treatment group.

Experiments were performed to confirm whether Bacillus balegensis Kh2-2, as a paraprobiotic, could also induce immune-related cytokine production in spleen cells. Specifically, after treating Bacillus valergensis Kh2-2 cells with various concentrations (25, 50, 100 μg/ml) in mouse spleen cells, the cytokines IFN-γ, IL-2, TNF-α, IL-4 , mRNA expression of IL-6 and IL-10 was analyzed. The results of cytokine mRNA analysis are shown in Figure 4C.

As shown in Fig. 4C, it was confirmed that the Bacillus valerensis Kh2-2 cells increased the mRNA expression of IFN-γ, IL-2, TNF-α, IL-4, IL-6 and IL-10.

Based on the cytokine mRNA expression analysis results, the protein expression of each target cytokine was measured by ELISA. The results of analyzing the protein expression of the cytokine proteins TNF-α, IFN-γ and IL-2 are shown in FIG. 4D.

As shown in Fig. 4D, it was confirmed that the Bacillus balegensis Kh2-2 cells increased the protein expression of the cytokine proteins TNF-α, IFN-γ and IL-2 compared to the control group (CON). In particular, it was confirmed that the expression of IFN-γ increased in a concentration-dependent manner in Bacillus ballegensis Kh2-2 cells.

Example 6. Confirmation of immune enhancing effect of Bacillus balegensis Kh2-2 strain and its cells in immunosuppressed mice

With the control group and the experimental group described in FIG. 5A, an in vivo immune test described later was performed. Specifically, the experimental group was a group treated with Bacillus balegensis Kh2-2 strain (10 ⁶ CFU/mL treatment group); and a group treated with Bacillus valerensis Kh2-2 strain cells (10 ⁶ CFU/mL treatment group). Cyclophosphoamine (CTX)-induced immunosuppressed mice were used, and LMS 50 mg/kg treated group was used as a positive control group.

6.1 Organ index and confirmation of innate immunity effect

Based on the results showing that the Bacillus valergensis Kh2-2 strain can exhibit an immunomodulatory effect, its immunomodulatory effect was additionally confirmed in vivo. 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 Bacillus ballegensis Kh2-2 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. 5B.

As shown in Fig. 5B, organ indices of spleen and thymus, which can be considered indirect indicators of CTX-induced immunological disorders, decreased with CTX treatment. It was confirmed that it was recovered according to the treatment of Bacillus valerensis Kh2-2 cells, and it was confirmed that the organ index recovery effect was better than that of the LMS-treated group used as a positive control group. The above result means that the Bacillus valerensis Kh2-2 strain can exhibit an effect on the atrophic immune system induced by CTX in mice.

6.2 Confirmation of NK cell activity enhancement effect

Experiments were performed to determine whether the Bacillus valergensis Kh2-2 strain could also affect the activity of NK cells, which are effector lymphocytes of the innate immune system that control infection and play an immune regulatory role. To prepare a spleen cell suspension, they were suspended in a complete RPMI-1640 medium solution to a final density of 1 x 10 ⁶ cells/mL and placed on a 96-well plate. For the NK cell activity assay, YAC-1 cells were used as target cells and cultured with spleen cells (effector cells). 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.

NK cytotoxicity (%) = (ODT- (ODS-ODE)) / ODT

* ODT is the absorbance of the target cell control, ODS is the absorbance of the test sample, and ODE is the absorbance of the effector cell control.

The results of analyzing toxicity to NK cells are shown in FIG. 5C.

As shown in Figure 5C, compared to the control (Con) mice, CTX-treated mice had significantly reduced NK cell activity, and in mice treated with Bacillus valergensis Kh2-2 cells, the reduced NK cell activity was significantly recovered confirmed that it is. The above result means that the Bacillus valergensis Kh2-2 strain or its cells can exhibit an effect on CTX-induced suppression of innate immunity.

6.3 Confirmation of splenocyte proliferation effect

After treating the Bacillus valergensis Kh2-2 cells in the CTX-treated mice, it was confirmed whether they could induce spleen proliferation. The results confirming the splenocyte proliferation effect are shown in FIG. 5D.

As shown in Figure 5D, it was confirmed that the proliferation of spleen cells was significantly inhibited in the CTX-treated group, but the proliferation of spleen cells in the Bacillus valerensis Kh2-2 strain or cell-treated group was significantly increased compared to the CTX-treated group.

6.3 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. To evaluate the adaptive immune effect through delayed hypersensitivity response, the abdominal hair of the mouse was removed and the mouse was 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, and the weight of each ear was recorded. The degree of ear swelling was expressed as the difference in weight between the left and right ears of each mouse. To evaluate the adaptive immune effect, changes in ear edema were confirmed in each experimental group, and the results are shown in FIG. 5E.

As shown in Fig. 5E, 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 the Bacillus valerensis Kh2-2 strain or cell treated group, which was confirmed to exhibit a better effect than the LMS treated group.

The above result means that the Bacillus valerensis Kh2-2 strain or strain enhances the DTH response and enhances the immune response.

6.4 Comparison of serum IgA and IgG content

The immune response is regulated by B-lymphocytes, which are known to differentiate into plasma cells, produce immunoglobulins (IgA and IgG) and bind to 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. 5F.

As shown in Fig. 5F, it was confirmed that serum IgA and IgG levels were greatly reduced in CTX-treated mice compared to control mice (CON). It was confirmed that the serum IgA and IgG levels of the Bacillus valergensis Kh2-2 treated group were increased. The above result means that humoral immunity can be improved in Bacillus valergensis Kh2-2 strain or CTX-treated mice.

6.5 Confirmation of serum cytokine secretion enhancement effect

The effect of Bacillus ballegensis Kh2-2 cell treatment on serum cytokine levels was confirmed. Specifically, the Bacillus balegensis Kh2-2 strain or cell was administered to immunosuppressed mice by CTX treatment, and the levels of IL-2, IL-6, and IFN-γ in serum were examined. The results of confirming the levels of IL-2, IL-6 and IFN-γ in the serum are shown in FIGS. 6A to C, respectively.

As shown in Figures 6A to C, serum cytokines were significantly suppressed due to the immunosuppressive reaction by CTX injection, but the Bacillus valergensis Kh2-2 cell treatment group recovered serum cytokine levels compared to the CTX treatment group Confirmed. These results indicate that the Bacillus valergensis Kh2-2 strain can restore cytokine gene expression in the serum of immunosuppressed mice.

Example 7. Confirmation of the effect of Bacillus balegensis Kh2-2 cells on NF-κB and MAPKs signaling pathways in immunosuppressed mice

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 the Bacillus balegensis Kh2-2 strain on the signaling pathway in regulating cytokine expression, the signaling pathway-related proteins (ERK, p38, JNK, p65 and IκBα) and their phosphorylated protein amounts were analyzed did The results of analyzing protein expression are shown in Figures 7A and B.

As shown in Figures 7A and B, NF-κB signaling and activation of MAPK protein were greatly suppressed in the CTX-treated group, but phosphorylated NF-κB (p-p65) in the group treated with Bacillus valerensis Kh2-2 cells. , IκBα and phosphorylated JNK, ERK, and p38 protein expression was confirmed to be restored. These results indicate that the Bacillus valerensis Kh2-2 strain can activate the NF-κB and MAPK signaling pathways to restore immune suppression induced by CTX.

Example 8. Investigation of the effects of Bacillus balegensis Kh2-2 strains on CTX-induced colon damage and intestinal microbial composition in immunosuppressed mice

CTX treatment may cause colon damage leading to intestinal immunosuppression; and that bowel length reflects the colon damage index; is known in the art. Accordingly, the colon length according to the Kh2-2 cell treatment was analyzed, and the results are shown in FIG. 8A.

As shown in Fig. 8A, it was confirmed that the colon length of the CTX-treated group was shorter than that of the control group. However, it was confirmed that the colon length of the mice treated with the Kh2-2 strain was restored to the CTX-treated mice.

In addition, the expression of TNF-α, IL-1β and IL-6 according to Kh2-2 cell treatment was analyzed in the colon tissues of CTX-treated mice. The analysis results are shown in Figure 8B.

As shown in Figure 8B, it was confirmed that the expression of TNF-α, IL-1β and IL-6 was suppressed in the CTX-treated group. However, it was confirmed that the expression of TNF-α, IL-1β, and IL-6 was increased in mice treated with Kh2-2 cells in CTX-treated mice.

These results indicate that the Kh2-2 strain exhibits immunostimulatory activity in CTX-treated mice by improving systemic immunity including thymus/spleen index, colon length, and cytokine expression. As previous studies have shown that host immunity has a symbiotic relationship with the gut microbiota, certain bacterial species can modulate the gut immune response, including T and B cell activation. Therefore, the immune enhancing effect of Kh2-2 strains may be positively correlated with the regulation of intestinal microflora.

Example 9. Intestinal bacterial composition analysis

The shared abundance of bacteria was analyzed by Venn diagram in the control, CTX, and Kh2-2 cell groups. The Venn diagram analysis results are shown in FIG. 8C.

As shown in Figure 8C, the control group identified 39 unique operational taxonomic units (OTUs). On the other hand, it was confirmed that the CTX, Kh2-2, and Kh2-2 cell supernatant groups had 28, 8, and 6 unique OTUs, respectively.

In order to further confirm the change in intestinal bacterial composition after treatment with Kh2-2 and Kh2-2 cell supernatants, the bacterial composition of the control group, CTX, Kh2-2 and Kh2-2 cell supernatant groups were compared at the phylum and genus level, and the results were It is shown in Figures 8D and E.

As shown in Fig. 8D and E, Bacteroidetes and Firmicutes were confirmed to be the dominant bacterial communities in all groups at the phylum level (> 95%). CTX increased the relative abundance of Firmicutes and Proteobacteria with a corresponding decrease in Bacteroidetes. Compared with the CTX group, Kh2-2 treatment partially reversed the intestinal bacterial imbalance. This is consistent with previous studies suggesting that probiotics can enhance the immune response by regulating CTX-induced gut bacterial imbalance. Interestingly, treatment of Kh2-2 cells did not seem to affect the increase in the ratio of Firmicutes/Bacteroidetes induced by CTX, but it was confirmed that the relative abundance of Tenericutes was increased.

The above results suggest that Kh2-2 and the supernatant of Kh2-2 can control the intestinal bacterial imbalance induced by CTX through various patterns of action.

Based on Fig. 8D and E above, the intestinal microflora was further analyzed. The analysis results are shown in Figure 8F.

As shown in FIG. 8F, it was confirmed that the CTX group had higher abundances of Helicobacter, Barnesiella, Alistipes, and Ruminococcus than the control group, and the Kh2-2 or Kh2-2 supernatant treatment group had low abundances of the strains.

These results suggest that Kh2-2 effectively promotes the growth of some beneficial bacteria and inhibits the growth of several harmful bacteria in CTX-induced immunodeficient mice.

In the above, specific parts of the present invention have been described in detail, and for those skilled in the art, it is clear that these specific descriptions are only preferred embodiments, and the scope of the present invention is not limited thereby. something to do. Accordingly, the substantial scope of the present invention will be defined by the appended claims and their equivalents.

Biological Resource Center KCTC14642BP 20210722

Claims (9)

Bacillus velenzensis containing the nucleotide sequence of SEQ ID NO: 1 ( Bacillus velenzensis ) Kh2-2 strain (accession number: KCTC14642BP). delete The strain according to claim 1, wherein the strain has acid resistance or bile resistance. The strain according to claim 1, wherein the strain has an immune enhancing activity. The strain according to claim 4, wherein the immunity is innate immunity or adaptive immunity. A pharmaceutical composition for enhancing immunity, comprising the strain of any one of claims 1, 3 to 5, its culture medium, and its cells. A food composition for enhancing immunity, comprising the strain according to any one of claims 1, 3 to 5, its culture medium, and its cells. Claims 1, 3 to 5 of any one of the strain, a culture medium thereof, a health functional food composition for immunity enhancement, including its cells. Claims 1, 3 to 5 of any one of the strain, its culture medium, comprising a cell body, immune enhancement feed composition.

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