KR20240079940A - Lactobacillus salivarius HHuMin-U with anti-norovirus activity - Google Patents

Abstract

Lactobacillus salivarius HHuMin-U (KCCM13001P) of the present invention has excellent anti-norovirus efficacy. The composition containing Lactobacillus salivarius HHuMin-U (KCCM13001P), its culture medium, or dried powder of this culture medium of the present invention has excellent efficacy in improving, preventing, or treating norovirus infection.

Description

Lactobacillus salivarius HHuMin-U with anti-norovirus activity {Lactobacillus salivarius HHuMin-U with anti-norovirus activity}

The present invention relates to a composition for improving, preventing or treating norovirus infection, comprising Lactobacillus salivarius HHuMin-U (KCCM13001P), its culture medium, or dried powder of this culture medium.

Norovirus infection is an epidemic viral gastroenteritis caused by norovirus. It is mainly spread through contaminated food or groundwater and is known to be highly infectious. It mainly infects humans through food (fish, shellfish or water), and is so highly contagious that infection is possible even with as few as 100 to 1,000 viral particles in food, accounting for more than 90% of viral food poisoning.

The main symptoms of norovirus infection are nausea, vomiting, diarrhea, headache, and fever. Chills, etc. are confirmed. Fever occurs in half of patients, and diarrhea occurs at least several times a day, at most dozens of times.

Meanwhile, norovirus only proliferates in the human intestine, and cell culture is not possible to date, so related research is insufficient and the development of a vaccine or treatment is still insufficient. However, as murine norovirus has recently become possible to be cultured in RAW 264.7 cells as a surrogate, norovirus research has become active. RAW 264.7 cells are a mouse leukemicmonocyte macrophage cell line obtained from immunocompromised mice lacking STAT1, and are the only cell system in which murine norovirus-1 (MNV-1) is cultured.

Republic of Korea Patent No. 10-1885863 describes a strain with anti-norovirus activity. Republic of Korea Patent Publication No. 10-2021-0043012 describes an anti-norovirus composition.

The present invention seeks to provide a composition for improving, preventing, or treating norovirus infection.

The present invention provides a pharmaceutical composition for preventing or treating norovirus infection, comprising Lactobacillus salivarius HHuMin-U (KCCM13001P), its culture medium, or dried powder of this culture medium.

In addition, the present invention provides a food composition for improving norovirus infection, comprising Lactobacillus salivarius HHuMin-U (KCCM13001P), its culture medium, or dried powder of this culture medium.

In the present invention, it was confirmed that dead cells of Lactobacillus salivarius HHuMin-U (KCCM13001P) have excellent anti-norovirus efficacy.

The composition containing Lactobacillus salivarius HHuMin-U (KCCM13001P), its culture medium, or dried powder of this culture medium of the present invention has excellent efficacy in improving, preventing, or treating norovirus infection.

Figure 1 shows the results of measuring the amount of antiviral cytokine production in an in vitro experiment with Lactobacillus salivarius HHuMin-U (KCCM13001P) of the present invention.
Figure 2 shows the results of measuring the amount of antiviral cytokine production in an in vivo experiment with Lactobacillus salivarius HHuMin-U (KCCM13001P) of the present invention.
Figure 3 shows the results confirming the anti-norovirus efficacy of Lactobacillus salivarius HHuMin-U (KCCM13001P) of the present invention.

The present invention provides a pharmaceutical composition for preventing or treating norovirus infection, comprising Lactobacillus salivarius HHuMin-U (KCCM13001P), its culture medium, or dried powder of this culture medium.

In addition, the present invention provides a food composition for improving norovirus infection, comprising Lactobacillus salivarius HHuMin-U (KCCM13001P), its culture medium, or dried powder of this culture medium.

According to the following experimental example, Lactobacillus salivarius HHuMin-U (KCCM13001P) of the present invention has excellent anti-norovirus efficacy.

In the present invention, norovirus infection refers to epidemic viral gastroenteritis caused by norovirus.

Lactobacillus salivarius HHuMin-U (KCCM13001P) used in the present invention is a strain deposited at the Korea Microorganism Conservation Center on June 1, 2021, and the accession number is KCCM13001P. In addition, it is a strain registered in Korea Patent No. 10-2337950.

Meanwhile, in the present invention, the pharmaceutical composition may further include a pharmaceutically acceptable carrier, diluent, or excipient. Usable carriers, excipients or diluents include lactose, dextrose, sucrose, sorbitol, mannitol, xyritol, erythritol, maltitol, starch, gum acacia, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, Microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and mineral oil, and one or more of these can be used. In addition, when the therapeutic or preventive agent is a drug, fillers, anti-coagulants, lubricants, wetting agents, fragrances, emulsifiers or preservatives may be additionally included.

The formulation of the pharmaceutical composition of the present invention can be prepared in a desired form depending on the method of use, and in particular, methods known in the art can be adopted to provide rapid, sustained or delayed release of the active ingredient after administration to a mammal. It is better to formulate it. Examples of specific dosage forms include PLASTERS, GRANULES, LOTIONS, LINIMENTS, LEMONADES, AROMATIC WATERS, POWDERS, and syrups ( SYRUPS, OPHTALMIC OINTMENTS, LIQUIDS AND SOLUTIONS, AEROSOLS, EXTRACTS, ELIXIRS, OINTMENTS, FLUIDEXTRACTS, EMULSIONS ), SUSPESIONS, DECOCTIONS, INFUSIONS, OPHTHALMIC SOLUTIONS, TABLETS, SUPPOSITIORIES, INJECTIONS, SPIRITS, CATAPLSMA ), CAPSULES, CREAMS, TROCHES, TINCTURES, PASTES, PILLS, soft or hard gelatin capsules. Preferably, it is formulated as a dosage form for oral administration.

In the pharmaceutical composition of the present invention, the dosage should be determined taking into account the administration method, the age, gender and weight of the recipient, and the severity of the disease. For example, based on the active ingredient, it can be administered orally at 0.00001 to 1,000 mg/kg (body weight) more than once per day. However, the above dosage is only an example for illustrative purposes, and may vary depending on the user's condition and doctor's prescription.

Meanwhile, in the food composition of the present invention, the food composition includes, for example, meat, grains, caffeinated beverages, general beverages, chocolate, bread, snacks, confectionery, candy, pizza, jelly, noodles, gum, dairy products, and ice cream. It may be any one selected from the group of foods, alcoholic beverages, alcohol, vitamin complexes, and other health supplements. More preferably, it may be any one selected from lactic acid bacteria fermented milk, soy milk, powdered milk, yogurt, beverages, granules, or health supplements, but is not necessarily limited thereto.

Hereinafter, the contents of the present invention will be described in more detail through the following experimental examples. However, the scope of the present invention is not limited to the following experimental examples and includes modifications of the technical idea equivalent thereto.

[Experimental Example 1: Lactobacillus salivarius ( Lactobacillus salivarius ) Experiment to confirm antiviral cytokine production ability of HHuMin-U (KCCM13001P)]

In this experimental example, we attempted to confirm the antiviral cytokine production ability of Lactobacillus salivarius HHuMin-U (KCCM13001P) in order to confirm its antiviral efficacy.

1) In vitro experiment

To analyze the antiviral effect of HHuMin-U dead cells, immune cell line RAW 264.7 (ATCC) cells were seeded in a 12-well plate and incubated with 10% fetal bovine serum. Serum) and 1% penicillin-streptomycin (DMEM (Dulbecco-modified Eagle medium)) were used and cultured for 24 hours in a 10% CO ₂ incubator (Thermo scientific) at 37°C. After culturing for 24 hours, HHuMin-U dead cells were treated with concentrations of 5, 10, and 20 μg/mL and cultured for 24 hours. After culturing for 24 hours, the medium was recovered and centrifuged at 13000 rpm for 2 minutes at 4°C to obtain the supernatant. As a result of measuring the amount of antiviral cytokine production in the obtained medium using mouse IFN-α, IFN-β Duoset ELISA kit and mouse TNF-α Duoset ELISA kit, HHuMin-U dead cells produced IFN-α and IFN in a concentration-dependent manner. -β, it was confirmed that the amount of TNF-α production was increased (Figure 1).

2) In vivo experiment

To analyze the antiviral effect of HHuMin-U dead cells in vivo, HHuMin-U was orally administered at 3 × ¹⁰ CFU/kg body weight to 8-week-old C57BL/6 mice (source: Oriental Bio) for 5 days. And, distal ileum tissue of the small intestine was isolated from mice administered on the 5th day. The separated tissue was homogenized with lysis buffer, and proteins were extracted and quantified. Afterwards, as a result of visualizing the protein band using an experimental method using ECL equipment, it was confirmed that the amount of IFN-β production was higher in the HHuMin-U administered group compared to the control group (PBS administered group) (Figure 2).

Meanwhile, all experimental animal procedures were performed with approval from the Yonsei University Institutional Animal Care and Use Committee (IACUC).

[Experimental Example 2: Lactobacillus salivarius ( Lactobacillus salivarius ) Measurement of anti-norovirus efficacy of HHuMin-U (KCCM13001P)]

In this experimental example, the anti-norovirus efficacy of Lactobacillus salivarius HHuMin-U (KCCM13001P) strain was measured using RAW 264.7 cells capable of norovirus growth.

Specifically, RAW 264.7 (ATCC) cells were seeded in a 12-well plate, and 10% fetal bovine serum and 1% penicillin-streptomycin were added. This added DMEM (Dulbecco-modified Eagle medium) was used and cultured at 37°C and 10% CO ₂ in an incubator (Thermo scientific) for 24 hours. After culturing for 24 hours, norovirus MNV-1 was infected at an MOI of 0.05 for 1 hour, and then the medium was replaced with medium containing 0, 5, and 20 μg/mL of HHuMin-U dead cells, respectively, and incubated at 37°C and 10% CO. It was cultured for 24 hours in a CO ₂ incubator (Thermo scientific). After 24 hours, the medium was recovered and the supernatant was obtained after centrifugation at 13,000 rpm, 2 minutes, 4°C, and the supernatant was diluted and infected in a 12-well plate seeded with RAW 264.7 cells for 1 hour. Afterwards, a 1:1 mixture of 2% SeaPlaque Agarose and 2X DMEM was added and cultured for 48 hours. Afterwards, it was fixed with 10% formaldehyde, stained with 1% crystal violet, and the number of plaques was counted. The inhibition rate of plaque formation was calculated as {1-(number of plaques tested)/(number of plaques in control group)}×100(%), and the result was 76.0 in the groups treated with 5 and 20 μg/mL of HHuMin-U dead cells, respectively. %, showing an inhibition rate of 99.6% (Figure 3). Through this, the excellent anti-norovirus efficacy of HHuMin-U of the present invention was confirmed.

Name of depository organization: Korea Center for Microbial Conservation (KCCM)

Accession number: KCCM13001P

Trust date: 20210601

Claims (2)

A pharmaceutical composition for preventing or treating norovirus infection, comprising Lactobacillus salivarius HHuMin-U (KCCM13001P), its culture medium, or dried powder of this culture medium.
A food composition for improving norovirus infection, comprising Lactobacillus salivarius HHuMin-U (KCCM13001P), its culture medium, or dried powder of this culture medium.