Patent Application
20220151253
The present disclosure relates to a novel Lactobacillus sakei strain and a composition containing the same.
Rheumatoid arthritis is an autoimmune disease and chronic inflammatory disease with a prevalence rate of 1-1.5%. It induces joint swelling and pain due to inflammatory changes in the joint induce systemic and, when aggravated, may cause severe results such as deformity of the joint, difficulty in joint flexion, etc.
The direct cause of arthritis has not been clearly understood yet, and steroids, nonsteroidal anti-inflammatory drugs, anti-gout drugs, immunosuppressants, etc. are used as therapeutic agents for treating arthritis. However, these therapeutic agents do not provide ultimate therapeutic effects and their use is limited due to various side effects. In addition, some chemotherapeutic medications have weaknesses such as insufficient efficacy for arthritis that has occurred already.
Accordingly, development of a therapeutic agent for arthritis which is effective in relieving inflammatory symptoms and pain with few side effects and can be medicated conveniently is necessary. The inventors of the present disclosure have researched on a kimchi-derived lactic acid bacterium which effectively relieves the symptoms of arthritis and inhibits the production of inflammation-related factors in blood.
(Patent document 1) Korean Patent Publication No. 10-2018-0104544.
The present disclosure is directed to providing a novel lactic acid bacterium in the genus Lactobacillus which has superior activity of ameliorating arthritis.
The inventors of the present disclosure have made efforts to find a strain exhibiting an effect of ameliorating arthritis. As a result, they have isolated and identified Lactobacillus sakei WIKIM0109, which is a novel lactic acid bacterium strain in the genus Lactobacillus, and have completed the present disclosure.
The Lactobacillus sakei WIKIM0109 according to the present disclosure is a novel Lactobacillus sakei strain derived from kimchi. Although it is described in the present disclosure that the Lactobacillus sakei WIKIM0109 is isolated and identified from kimchi, it can also be obtained from other sources.
As a result of 16S rRNA base sequencing for identification and classification of the lactic acid bacterium strain isolated from the traditional fermented food in an example of the present disclosure, it has been found to have a nucleic acid sequence of SEQ ID NO: 1.
Therefore, the microorganism of the present disclosure having the 16S rRNA base sequence of SEQ ID NO: 1 was named Lactobacillus sakei WIKIM0109 and was deposited in the Korean Collection for Type Cultures on Mar. 7, 2019 (accession number: KCTC13818BP).
The Lactobacillus sakei WIKIM0109 (accession number: KCTC13818BP) strain of the present disclosure is a Gram-positive bacterium. It is a rod-shaped facultative anaerobe that can grow under both aerobic and anaerobic conditions.
The Lactobacillus sakei WIKIM0109 of the present disclosure has the effect of intestinal regulation and immune enhancement of common lactic acid bacteria. It is well known that the lactic acid bacteria in the genus Lactobacillus have the effect of intestinal regulation and immune enhancement.
In an example which will be described below, it was confirmed that the Lactobacillus sakei WIKIM0109 strain of the present disclosure exhibits the effect of ameliorating arthritis by inhibiting IgG and IgG2a production in blood and inhibiting IFN-γ, IL-17 and TNF-α production. Accordingly, the Lactobacillus sakei WIKIM0109 according to the present disclosure can be used variously for intestinal regulation, amelioration of arthritis, immune enhancement, etc. in human or animals.
In an exemplary embodiment, the present disclosure provides a composition containing the Lactobacillus sakei WIKIM0109, a culture thereof, a lysate thereof or an extract thereof as an active ingredient.
The Lactobacillus sakei WIKIM0109 contained in the composition according to the present disclosure may be in the form of a living bacterium or in the form of a dried or freeze-dried bacterium. The types of lactic acid bacteria suitable for inclusion in various compositions and methods for preparing the same are well known to those skilled in the art.
In a specific exemplary embodiment, the composition may be a composition for oral administration, which contains the Lactobacillus sakei WIKIM0109 strain in the form of a living bacterium.
In another exemplary embodiment, the present disclosure provides a composition for intestinal regulation, which contains the Lactobacillus sakei WIKIM0109, a culture thereof, a lysate thereof or an extract thereof as an active ingredient. The composition according to the present disclosure for intestinal regulation can be used for preventing, treating or ameliorating gastrointestinal disease of animals including human. Specifically, the animals include livestock such as cow, horse and pig. The ‘gastrointestinal disease’ includes infection by gastrointestinal harmful bacteria and inflammatory bowel disease. For example, it includes infectious diarrhea caused by pathogenic microorganisms (E. coli, Salmonella, Clostridium, etc.), gastroenteritis, inflammatory bowel disease, neurological enteritis syndrome, microorganism overgrowth in the small intestine, intestinal acute diarrhea, etc., although not being limited thereto.
Specifically, the composition according to the present disclosure for intestinal regulation may be administered orally. The administration dosage may vary depending on the type of the gastrointestinal disease, the severity of the disease, age, sex, race, purpose such as treatment or prevention, etc. In general, 10 million to 100 billion bacteria may be administered per day for an adult.
The present disclosure provides a composition for enhancing immunity, which contains the Lactobacillus sakei WIKIM0109, a culture thereof, a lysate thereof or an extract thereof as an active ingredient. It is well known that the lactic acid bacteria in the genus Lactobacillus have the effect of enhancing immunity.
The present disclosure provides a composition for preventing and ameliorating arthritis, which contains the Lactobacillus sakei WIKIM0109, a culture thereof, a lysate thereof or an extract thereof as an active ingredient. The arthritis may be one or more disease selected from a group consisting of rheumatoid arthritis, ankylosing spondylitis, osteoarthritis or psoriatic arthritis, although not being limited thereto.
In a specific exemplary embodiment, the arthritis may be rheumatoid arthritis.
Because of these advantageous activities, the Lactobacillus sakei WIKIM0109 according to the present disclosure may be included in drugs, functional health foods, foods, feeds, feed additives or lactic acid bacterium starters for fermentation of foods or feeds.
In a specific exemplary embodiment, the composition of the present disclosure is used as a pharmaceutical composition, the pharmaceutical composition of the present disclosure may be prepared using a pharmaceutically suitable and physiologically acceptable adjuvant in addition to the active ingredient. As the adjuvant, an excipient, a disintegrant, a sweetener, a binder, a coating agent, a swelling agent, a lubricant, a glidant, a flavorant, etc. may be used.
Specifically, the pharmaceutical composition may be formulated by including one or more pharmaceutically acceptable carrier in addition to the active ingredient described above.
For example, for formulation into a tablet or a capsule, the active ingredient may be combined with an oral, nontoxic, pharmaceutically acceptable inert carrier such as ethanol, glycerol, water, etc. In addition, if necessary or desired, a suitable binder, lubricant, disintegrant or colorant or a mixture thereof may also be included. Suitable binders include, but are not limited to, starch, gelatin, natural sugars such as glucose or β-lactose, corn sweetener, natural or synthetic gums such as acacia, tragacanth or sodium oleate, sodium stearate, magnesium stearate, sodium benzoate, sodium acetate, sodium chloride, etc. Disintegrants include, but are not limited to, starch, methyl cellulose, agar, bentonite, xanthan gum, etc. For a composition formulated into a liquid solution, as a pharmaceutically acceptable carrier, one or more of saline, sterile water, Ringer's solution, buffered saline, albumin injection, dextrose solution, maltodextrin solution, glycerol and ethanol, which are biocompatible and suitable for sterilization, may be used. If necessary, other common additives such as an antioxidant, a buffer, a bacteriostat, etc. may be added. In addition, a diluent, a dispersant, a surfactant, a binder and a lubricant may be further added to formulate an injectable formulation such as an aqueous solution, a suspension, an emulsion, etc., a pill, a capsule, a granule or a tablet.
Specifically, the formulation may be prepared depending on the particular disease or ingredients using the methods described in Remington's Pharmaceutical Science (Mack Publishing Company, Easton, Pa.).
In another specific exemplary embodiment, the present disclosure may provide a food composition for preventing and ameliorating arthritis, which contains the Lactobacillus sakei WIKIM0109, a culture thereof, a lysate thereof or an extract thereof as an active ingredient. The food composition may be in the form of a functional health food, a beverage, a bar, etc.
In the present disclosure, the food composition containing the strain as an active ingredient may be in the form of a beverage such as fermented milk, etc. Therefore, the present disclosure provides a lactic acid bacterium starter for fermenting foods or feeds, which contains the Lactobacillus sakei WIKIM0109, a culture thereof, a lysate thereof or an extract thereof as an active ingredient.
The food composition according to the present disclosure may be formulated in the same manner as the pharmaceutical composition and may be used as a functional food or may be added to various foods. Examples of the food to which the composition of the present disclosure may be added include beverages, vitamin complexes, dietary health supplements, etc.
The food composition of the present disclosure may contain ingredients commonly added when preparing foods. Examples include a protein, a carbohydrate, a fat, a nutrient, a seasoning agent and a flavorant. Examples of the carbohydrate include common sugars such as monosaccharides, e.g., glucose, fructose, etc., disaccharides, e.g., maltose, sucrose, etc., oligosaccharides and polysaccharides, e.g., dextrin, cyclodextrin, etc. and sugar alcohols such as xylitol, sorbitol, erythritol, etc. As the flavorants, natural flavorants [thaumatin, stevia extract (e.g., rebaudioside A, glycyrrhizin, etc.)] or synthetic flavorants (saccharin, aspartame, etc.) may be used. For example, when the food composition of the present disclosure is prepared into a drink or a beverage, it may further contain citric acid, fructose syrup, sugar, glucose, acetic acid, malic acid, fruit juice, various plant extracts, etc.
The composition according to the present disclosure may be used as a feed additive or a feed.
When used as a feed additive, the composition may be prepared into a solution with a high concentration of 20-90%, a powder or a granule. The feed additive may further contain one or more of an organic acid such as citric acid, fumaric acid, adipic acid, lactic acid, malic acid, etc., a phosphate such as sodium phosphate, potassium phosphate, acidic pyrophosphate, polyphosphate, etc. or a natural antioxidant such as polyphenol, catechin, α-tocopherol, rosemary extract, vitamin C, green tea extract, licorice extract, chitosan, tannic acid, phytic acid, etc. When used as a feed, the composition may be prepared into an ordinary feed and may further contain common feed ingredients.
The feed additive and the feed may further contain a grain, e.g., ground or crushed wheat, oats, barley, maize and rice, a vegetable protein feed, e.g., a feed containing rape, bean and sunflower as main ingredients, an animal protein feed, e.g., blood meal, meat meal, bone meal and fish meal, a sugar or dairy product, e.g., various powder milk and whey powder. In addition, a nutritional supplement, a digestion- and absorption-enhancing agent, a growth-promoting agent, etc. may be further contained.
The feed additive may be administered to an animal either or in combination with another feed additive in an edible carrier. Further, the feed additive may be mixed directly into an animal feed as a top dressing or may be administered easily to an animal as an oral dosage form separately from a feed. When the feed additive is administered separately from an animal feed, it can be prepared into an immediate-release or sustained-release formulation in combination with a pharmaceutically acceptable edible carrier well known in the art. The edible carrier may be either solid or liquid, e.g., corn starch, lactose, sucrose, soybean flake, peanut oil, olive oil, sesame oil and propylene glycol. When a solid carrier is used, the feed additive may be in the form of a tablet, a capsule, a powder, a troche, a sugar-coated tablet or a non-dispersed top dressing. When a liquid carrier is used, the feed additive may be in the form of a soft gelatin capsule, a syrup, a suspension, an emulsion or a solution.
Further, the feed additive and the feed may contain an adjuvant, e.g., a preservative, a stabilizer, a wetting agent, an emulsifying agent, a solubilization promoter, etc. The feed additive may be added to an animal feed by dipping, spraying or mixing.
The feed or feed additive of the present disclosure can be provided to various animals, including mammals, poultry and fish.
The mammal may include pig, cow, sheep, goat, laboratory rodents, pets (e.g., dog or cat), etc. The poultry may include chicken, turkey, duck, goose, pheasant, quail, etc., and the fish may include trout, etc., although not being limited thereto.
The amount of the Lactobacillus sakei WIKIM0109 strain contained in the composition according to the present disclosure may be about 106-1012 CFU/g, e.g., 107-1011 CFU/g or 108-1010 CFU/g, based on a single dose. Specifically, the strain may be administered as living bacteria, and may be killed or attenuated before intake. Further, a sterilization process through heat treatment may be performed additionally when the composition is prepared using a culture supernatant, etc. The amount of the strain required to achieve a minimal effect and the daily dose thereof may vary depending on the physical or health condition of a patient. Generally, the daily dose can be about 106-1012 CFU/g, e.g., 107-1011 CFU/g or 108-1010 CFU/g.
The advantages and features of the present disclosure and the methods of accomplishing the same will become apparent by the following examples described in detail. However, the present disclosure is not limited to the examples set forth below but may be embodied in many different forms. The examples are provided such that the disclosure of the present disclosure is complete and are provided to fully inform the scope of the present disclosure to those having ordinary knowledge in the art to which the present disclosure belongs. The scope of the present disclosure will only be defined by the appended claims.
Because the Lactobacillus sakei WIKIM0109 according to the present disclosure exhibits an activity of inhibiting IgG and IgG2a production in blood and inhibiting IFN-γ, IL-17 and TNF-αproduction, it can be utilized variously as a lactic acid bacterium for intestinal regulation, immune enhancement, prevention or amelioration of gastrointestinal disease and arthritis, etc. in human or animals. Furthermore, it can be usefully used as a starter for fermentation.
Because the Lactobacillus sakei WIKIM0109 according to the present disclosure, which is a lactic acid bacterium isolated form kimchi, exhibits an activity of inhibiting IgG and IgG2a production in blood and inhibiting IFN-γ, IL-17 and TNF-α production, it can be utilized variously for prevention or amelioration of arthritis. Furthermore, it can be usefully used as a starter for fermentation.
Hereinafter, the present disclosure is described in detail through examples. The following examples are for illustrative purposes only and the scope of the present disclosure is not limited by the examples.
A bacterial single colony obtained by smearing a crude liquid extract of kimchi in MRS culture medium was collected and cultured in MRS broth. DNA was extracted using the QIAamp DNA Mini Kit (Qiagen, Germany). The extracted DNA was identified using 1% agarose gel. PCR was conducted using the extracted genomic DNA as a template to amplify the 16S rRNA gene. The PCR condition was 30 cycles of denaturation at 95° C. for 1 minute, annealing at 45° C. for 1 minute and extension at 72° C. for 1 minute 30 seconds. The sequence of the obtained PCR product was analyzed by Macrogen (Seoul, Korea). Bacterial identification was performed by similarity analysis of the 16S rRNA sequence using the Basic Local Alignment Search Tool (BLAST) search engine of the National Center for Biotechnology Information (NCBI, www.ncbi.nlm.nih.gov).
As a result of the 16S rRNA base sequence analysis for identification of the microorganism, the strain isolated in this example was found to have a nucleic acid sequence of SEQ ID NO: 1.
The microorganism of the present disclosure was named Lactobacillus sakei WIKIM0109 and deposited on Mar. 7, 2019 in the Korean Collection for Type Cultures (accession number: KCTC13818BP).
The Lactobacillus sakei WIKIM0109 strain isolated in Example 1 was cultured in MRS medium at 30° C. for 24 hours, and the cultured bacteria were centrifuged at 8,000 rpm for 5 minutes and then washed with PBS to remove the remaining medium components. Then, the number of the bacteria was quantified to be 1×1010 CFU/mL using PBS, and 0.1 mL (1×109 CFU) was orally administered to a collagen-induced arthritis mouse model using a sonde, five times a week. Sterile PBS was administered to negative and positive control groups.
1) Measurement of Incidence of Arthritis and Arthritic Score in Collagen-Induced Arthritis Mouse Model
Arthritic score and incidence of arthritis for 12 weeks during which the Lactobacillus sakei WIKIM0109 strain was orally administered to the collagen-induced arthritis mouse model are shown in
As a result, the collagen-induced arthritis control group (CIA-No LAB) showed the highest incidence of arthritis and arthritic score, and the incidence of arthritis and arthritic score were decreased significantly in the Lactobacillus sakei WIKIM0109 treatment group (WIKIM0109).
2) Sensory Evaluation and Measurement of Paw Thickness
After orally administering the Lactobacillus sakei WIKIM0109 strain to experimental animals for 4 weeks, the condition of arthritis was evaluated by clinical visual evaluation and paw thickness was measured.
As shown in
3) Measurement of Concentrations of IgG and IgG2a in Blood
After taking blood from the experimental animals, the concentrations of IgG, collagen antigen-specific IgG, IgG2a and collagen antigen-specific IgG2a in blood were measured.
As seen from
4) Measurement of Production of Collagen Antigen-Specific IFN-γ, IL-17 and TNF-α
After obtaining single cells from the lymph nodes around the joint of the experimental animals, 5×105 cells were dispensed onto a 96-well plate. After treating with collagen (50 μg/mL) for 48 hours, the concentrations of antigen-specific IFN-γ, IL-17 and TNF-α were measured from the supernatant. As seen from
5) Comparative Experiments of WIKIM0109 with Other Strains of Lactobacillus sakei
In order to investigate whether the effect of the Lactobacillus sakei WIKIM0109 is common to Lactobacillus sakei, (1) the increase in the production of the immune-suppressing cytokine IL-10 and (2) the decrease in the production of the immune-enhancing cytokine IL-17 were compared with Lactobacillus sakei LS35 and LS41 acquired from the World Institute of Kimchi, an annex of the Korea Food Research Institute.
Lactobacillus sakei LS35 was isolated from watery kimchi and Lactobacillus sakei LS41 were isolated from pa-gat kimchi. They were statically cultured in a 30° C. incubator for 24 hours using Lactobacilli MRS (BD Difco) liquid medium. After sequencing of the gene encoding 16S rRNA, they were identified as Lactobacillus sakei through homology analysis using the Basic Local Alignment Search Tool (BLAST) of the National Center for Biotechnology Information (NCBI).
OTII mouse having OVA-specific T cells were purchased from Jackson Laboratory and used after breeding. 5×105 single splenocytes of the OTII mouse were dispensed onto a 96-well plate and cultured at 37° C. for 72 hours after treating with OVA peptide (OVA323-339) and kimchi lactic acid bacteria at MOI 1. The amount of IL-17 and IL-10 present in the culture medium was measured using the Cytometric Bead Assay (CBA) mouse Th1/Th2/Th17 kits purchased from BD Bioscience. The production of the cytokines was analyzed based on standard curves by using FACSCanto II (BD Bioscience).
As a result, it was confirmed that the WIKIM0109 strain resulted in the highest production of IL-10, the lowest production of IL-17 and the highest IL-10/IL-17 ratio among the different strains of Lactobacillus sakei (
Although the LS41 strain also increased the production of the immune-suppressing cytokine IL-10, the WIKIM0109 strain showed about 40% or more production of IL-10. The production by the LS35 strain was only about ⅓ of that of the WIKIM0109 strain.
For the immune-enhancing cytokine IL-17, although the LS41 strain did not show significant decrease in production, the WIKIM0109 strain significantly decreased the production of IL-17. On the contrary, the LS35 strain increased the production of IL-17.
Although the specific exemplary embodiments of the present disclosure have been described in detail, it will be obvious to those having ordinary knowledge in the art that they are merely preferred exemplary embodiments and the scope of the present disclosure is not limited thereto. Accordingly, it is to be understood that the substantial scope of the present disclosure is defined by the appended claims and their equivalents.
[Deposition Number]
Depository agency: Korean Collection for Type Cultures (international).
Accession number: KCTC13818BP.
Date of accession: Mar. 7, 2019.
| Number | Date | Country | Kind |
|---|---|---|---|
| 10-2019-0034211 | Mar 2019 | KR | national |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/KR2020/003883 | 3/20/2020 | WO | 00 |
