KR102678449B1 - Composition for anti-inflammatory comprising the Lactiplantibacillus plantarum WiKim0145 - Google Patents

Abstract

The present invention relates to a novel Lactiplantibacillus plantarum WiKim0145 isolated from kimchi and a composition containing the same. Lactiplantibacillus plantarum WiKim0145 according to the present invention exhibits an inhibitory effect on inflammatory cytokines, and can be used in a variety of ways for anti-inflammatory purposes, prevention, improvement, and treatment of inflammatory diseases.

Description

Anti-inflammatory composition comprising the Lactiplantibacillus plantarum WiKim0145 {Composition for anti-inflammatory comprising the Lactiplantibacillus plantarum WiKim0145}

The present invention relates to a novel Lactiplantibacillus plantarum strain isolated from kimchi and an anti-inflammatory composition containing the same.

Inflammation is a local or systemic defense mechanism against cell and tissue damage or infection. It is mainly caused by the humoral mediators that make up the immune system react directly or by stimulating the local or systemic effector system. It is caused by a chain of biological reactions that occur. The inflammatory response removes damaged and necrotic cells and aids recovery, but excessive secretion of inflammatory substances can actually cause necrosis of surrounding tissues and cause various diseases such as rheumatic diseases and various cancers. Treatment is needed. Recently, as interest in improving immunity has increased, various studies have been conducted on immunomodulation methods using lactic acid bacteria. Lactobacillus not only modulates immunity but also produces various metabolites to provide beneficial effects to the human body, so the discovery of functional lactic acid bacteria It is expected that it can be of great help in improving human health.

Major inflammatory diseases include digestive diseases such as gastritis and inflammatory enteritis, oral diseases such as periodontitis, respiratory diseases such as asthma, chronic obstructive pulmonary disease (COPD), and rhinitis, skin diseases such as atopic dermatitis, hair loss, and psoriasis, degenerative arthritis, and other inflammatory diseases. Arthritis such as rheumatoid arthritis; and metabolic diseases such as obesity, diabetes, and liver cirrhosis (Patent Document 0001).

Inflammatory mediators expressed at the site of inflammation, i.e., cytokines such as IL-1α, IL-1β, IL-6, and IL-8, chemokines, reactive oxygen intermediates, and cyclooxygenase-2 ( Cycoxygenase-2, COX-2), 5-lipoxygenase (5-LOX), and matrix metalloproteinase (MMP) play an important role in the development and maintenance of inflammatory responses. The expression of these inflammatory mediators is regulated by transcription factors such as NF-κB (nuclear factor κB), STAT3 (signal transducer and activator of transcription 3), AP-1 (activator protein 1), and HIF-1a (hypoxiainducible factor 1a). It is known to be (patent document 0001).

Korean Patent Publication No. 10-2021-0031301

The present invention seeks to provide novel Lactiplantibacillus plantarum lactic acid bacteria with excellent anti-inflammatory efficacy.

Accordingly, the present inventors tried to find lactic acid bacteria strains that exhibit anti-inflammatory effects from traditional fermented foods, and as a result, they isolated and identified Lactiplantibacillus plantarum WiKim0145 ( Lactiplantibacillus plantarum WiKim0145), a novel Lactiplantibacillus lactic acid bacteria strain. The invention was completed.

The present invention provides Lactiplantibacillus plantarum WiKim0145 ( Lactiplantibacillus plantarum WiKim0145).

Lactiplantibacillus plantarum WiKim0145 of the present invention is a new Lactiplantibacillus plantarum strain derived from kimchi.

The lactic acid bacteria strain isolated through the examples of the present invention was found to have a nucleic acid sequence of SEQ ID NO: 1 as a result of 16S rDNA base sequence analysis for identification and classification of microorganisms.

Therefore, the microorganism of the present invention having the 16S rDNA base sequence of SEQ ID NO: 1 was named Lactiplantibacillus plantarum WiKim0145 ( Lactiplantibacillus plantarum WiKim0145), and was registered at the Korea Research Institute of Bioscience and Biotechnology Biological Resources Center on December 20, 2023. It was deposited (accession number KCTC 15742BP).

-Sequence List-

KCTC 15742BP

SEQ ID NO: 1

TGCAGTCGAACGAACTCTGGTATTGATTGGTGCTTGCATCATGATTTACATTTGAGTGAGTGGCGAACTGGTGAGTAACACGTGGGAAACCTGCCCAGAAGCGGGGGATAACACCTGGAAACAGATGCTAATACCGCATAACAACTTGGACCGCATGGTCCGAGTTTGAAAGATGGCTTCGGCTATCACTTTTGGATGGTCCCGCGGCGTATTAGCTAGATGGTGGGGTAACGGCTCACCATGGCAATGATACGTAGCCGACCTGAGAGGGTAATCGGCCACATTGGGACTGAGACACGGCCCAAACTCCTACGGGAGGCAGCAGTAGGGAATCTTCCACAATGGACGAAAGTCTGATGGAGCAACGCCGCGTGAGTGAAGAAGGGTTTCGGCTCGTAAAACTCTGTTGTTAAAGAAGAACATATCTGAGAGTAACTGTTCAGGTATTGACGGTATTTAACCAGAAAGCCACGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTGTCCGGATTTATTGGGCGTAAAGCGAGCGCAGGCGGTTTTTTAAGTCTGATGTGAAAGCCTTCGGCTCAACCGAAGAAGTGCATCGGAAACTGGGAAACTTGAGTGCAGAAGAGGACAGTGGAACTCCATGTGTAGCGGTGAAATGCGTAGATATATGGAAGAACACCAGTGGCGAAGGCGGCTGTCTGGTCTGTAACTGACGCTGAGGCTCGAAAGTATGGGTAGCAAACAGGATTAGATACCCTGGTAGTCCATACCGTAAACGATGAATGCTAAGTGTTGGAGGGTTTCCGCCCTTCAGTGCTGCAGCTAACGCATTAAGCATTCCGCCTGGGGAGTACGGCCGCAAGGCTGAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGAAGCTACGCGAAGAACCTTACCAGGTCTTGACATACTATGCAAATCTAAGAGATTAGACGTTCCCTTCGGGGACATGGATACAGGTGGTGCATGGTTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTATTATCAGTTGCCAGCATTAAGTTGGGCACTCTGGTGAGACTGCCGGTGACAAACCGGAGGAAGGTGGGGATGACGTCAAATCATCATGCCCCTTATGACCTGGGCTACACACGTGCTACAATGGATGGTACAACGAGTTGCGAACTCGCGAGAGTAAGCTAATCTCTTAAAGCCATTCTCAGTTCGGATTGTAGGCTGCAACTCGCCTACATGAAGTCGGAATCGCTAGTAATCGCGGATCAGCATGCCGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCATGAGAGTTTGTAACACCCAAAGTCGGTGGGGTAACCTTTTAGGAACCAGCCGCCTA

Lactiplantibacillus plantarum WiKim0145 of the present invention is a Gram-positive bacterium and a facultative anaerobe capable of growing under both aerobic and anaerobic conditions. It does not form spores, is non-motile, and its cells take the form of bacilli. there is.

In the following examples, Lactiplantibacillus plantarum WiKim0145 of the present invention produces anti-inflammatory cytokines (IL-4, IL-10, and IFN-γ), which are indicators of immune response, and inflammatory cytokines (IL-1β). , IL-6, TNF-α) expression level inhibition activity and probiotic properties were confirmed to be excellent. Therefore, Lactiplantibacillus plantarum WiKim0145 according to the present invention can be used for anti-inflammation and prevention, treatment or improvement of inflammatory diseases by expression of IL-1β, IL-6 and TNF-α. In addition, it has excellent acid resistance, bile resistance, and heat resistance, and does not produce harmful substances including β-glucuronidase, so it can be applied to foods as a probiotic.

The present invention provides an anti-inflammatory food composition containing Lactiplantibacillus plantarum WiKim0145, its culture, its lysate, its fermentation product, or its extract as an active ingredient.

The inflammation may be due to the expression of one or more cytokines selected from the group consisting of IL-1β, TNF-α, and IL-6.

Lactiplantibacillus plantarum WiKim0145 included in the composition according to the present invention may exist as live or dead cells, and may also exist in dried or freeze-dried form. Forms and formulation methods of lactic acid bacteria suitable for inclusion in various compositions are well known to those skilled in the art. For example, Lactiplantibacillus Plantarum WiKim0145 is a culture obtained by culturing in a known liquid medium or solid medium, a fermentation product obtained by culturing the strain and additional components together, or the strain in an organic solvent. It can be formulated in the form of an extract extracted, a lysate (or lysate) obtained by dissolving the cell membrane of the strain, or crushed or homogenized, but is not limited thereto.

In one embodiment, the composition may be a composition containing Lactiplantibacillus plantarum WiKim0145 strain that exists as live or dead bacteria.

The food composition according to the present invention can be used as a health functional food or added to various foods. Foods to which the composition of the present invention can be added include, for example, beverages, vitamin complexes, health supplements, etc.

The food composition of the present invention may include ingredients commonly added during food production, and includes, for example, proteins, carbohydrates, fats, nutrients, seasonings, and flavoring agents. Examples of the above-mentioned carbohydrates include monosaccharides such as glucose, fructose, etc.; Disaccharides such as maltose, sucrose, oligosaccharides, etc.; and polysaccharides, such as common sugars such as dextrin and cyclodextrin, and sugar alcohols such as xylitol, sorbitol, and erythritol. As flavoring agents, natural flavoring agents (thaumatin, stevia extract (e.g., rebaudioside A, glycyrrhizin, etc.)) and synthetic flavoring agents (saccharin, aspartame, etc.) can be used. For example, when the food composition of the present invention is manufactured as a drink or beverage, citric acid, high fructose corn syrup, sugar, glucose, acetic acid, malic acid, fruit juice, and various plant extracts may be additionally included.

The composition according to the present invention can be used as a feed additive or feed.

When used as a feed additive, the composition can be a 20 to 90% highly concentrated solution or prepared in powder or granule form. The feed additives include organic acids such as citric acid, malic acid, adipic acid, lactic acid, and malic acid, phosphates such as sodium phosphate, potassium phosphate, acid pyrophosphate, and polyphosphate (polymerized phosphate), polyphenol, catechin, alpha-tocopherol, and rosemary. It may additionally contain one or more of natural antioxidants such as extract, vitamin C, green tea extract, licorice extract, chitosan, tannic acid, and phytic acid. When used as feed, the composition may be formulated in the form of a normal feed and may include common feed ingredients.

The feed additives and feeds include grains such as ground or crushed wheat, oats, barley, corn and rice; Vegetable protein feeds, such as those based on rape, soybean, and sunflower; Animal protein feeds such as blood meal, meat meal, bone meal and fish meal; It may further include dry ingredients such as sugar and dairy products, such as various powdered milk and whey powder, and may further include nutritional supplements, digestion and absorption enhancers, growth promoters, etc.

The feed additive may be administered to animals alone or in combination with other feed additives in an edible carrier. Additionally, the feed additives can be easily administered to animals as top dressing, by mixing them directly into animal feed, or in an oral dosage form separate from the feed. When the feed additive is administered separately from animal feed, it can be prepared into an immediate-release or sustained-release formulation by combining it with a pharmaceutically acceptable edible carrier, as is well known in the art. These edible carriers may be solid or liquid, such as corn starch, lactose, sucrose, soy flakes, peanut oil, olive oil, sesame oil and propylene glycol. When a solid carrier is used, the feed additive may be a tablet, capsule, powder, troche or sugar-containing tablet, or a top dressing in microdisperse form. When a liquid carrier is used, the feed additive may be in the form of gelatin soft capsules, syrup, suspension, emulsion, or solution.

In addition, the feed additives and feeds may contain auxiliaries such as preservatives, stabilizers, wetting or emulsifying agents, solution accelerators, etc. The feed additive can be used by adding it to animal feed by injecting, spraying, or mixing it.

The feed or feed additive of the present invention can be applied to a number of animal diets, including mammals, poultry, and fish.

In the present invention, animals include mammals and can be used for pigs, cows, sheep, goats, laboratory rodents, and laboratory rodents as well as pets (e.g., dogs, cats), etc., but are not limited thereto. no.

In addition, the present invention provides a lactic acid bacteria starter for fermentation consisting of Lactiplantibacillus plantarum WiKim0145 or a culture thereof.

In addition, the present invention provides a pharmaceutical composition for preventing, improving, or treating inflammatory diseases containing Lactiplantibacillus plantarum WiKim0145, its culture, lysate, or extract thereof as an active ingredient.

In the present invention, the inflammatory disease includes gastritis, stomatitis, atopic dermatitis, acne, psoriasis, sinusitis, rhinitis, conjunctivitis, asthma, dermatitis, inflammatory collagen vascular disease, glomerulonephritis, encephalitis, inflammatory enteritis, chronic obstructive pulmonary disease, sepsis, Septic shock, pulmonary fibrosis, undifferentiated spondyloarthrosis, undifferentiated arthropathy, arthritis, inflammatory osteolysis, chronic inflammatory disease caused by viral or bacterial infection, ulcerative colitis, inflammatory bowel disease, rheumatoid arthritis, reactive arthritis, osteoarthritis, scleroderma , osteoporosis, atherosclerosis, myocarditis, endocarditis, pericarditis, cystic fibrosis, Hashimoto's thyroiditis, Graves' disease, leprosy, syphilis, Lyme disease, Borreliosis, neurogenic-borreliosis, tuberculosis, sarcoidosis ( Sarcoidosis), lupus, lupus chilblains, lupus tuberculosis, lupus nephritis, systemic lupus erythematosus, macular degeneration, uveitis, irritable bowel syndrome, Crohn's disease, Sjögren's syndrome, fibromyalgia, chronic fatigue syndrome, chronic fatigue immune dysfunction syndrome, myalgic encephalomyelitis. , amyotrophic lateral sclerosis, Parkinson's disease, and multiple sclerosis, and may preferably be an inflammatory disease caused by the expression of one or more cytokines selected from the group consisting of IL-1β, TNF-α, and IL-6. there is.

When the composition according to the present invention is used as a pharmaceutical composition, the pharmaceutical composition of the present invention can be prepared using pharmaceutically suitable and physiologically acceptable adjuvants in addition to the Lactiplantibacillus plantarum WiKim0145 strain. , The auxiliary agent may be an excipient, disintegrant, sweetener, binder, coating agent, swelling agent, lubricant, lubricant, or flavoring agent.

For administration, the pharmaceutical composition may be preferably formulated as a pharmaceutical composition containing one or more pharmaceutically acceptable carriers in addition to the active ingredients described above.

For example, for formulation in the form of tablets or capsules, the active ingredient may be combined with an oral, non-toxic, pharmaceutically acceptable inert carrier such as ethanol, glycerol, water, etc. Additionally, if desired or necessary, suitable binders, lubricants, disintegrants and coloring agents may also be included in the mixture. Suitable binders include, but are not limited to, starch, gelatin, natural sugars such as glucose or beta-lactose, corn sweeteners, natural and synthetic gums such as acacia, tracacance or sodium oleate, sodium stearate, magnesium stearate, sodium benzoate. Includes sodium acetate, sodium chloride, etc. Disintegrants include, but are not limited to, starch, methylcellulose, agar, bentonite, xanthan gum, etc. Acceptable pharmaceutical carriers in compositions formulated as liquid solutions include those that are sterile and biocompatible, such as saline solution, sterile water, Ringer's solution, buffered saline solution, albumin injection solution, dextrose solution, maltodextrin solution, glycerol, ethanol, and One or more of these ingredients can be mixed and used, and other common additives such as antioxidants, buffers, and bacteriostatic agents can be added as needed. Additionally, diluents, dispersants, surfactants, binders, and lubricants can be additionally added to formulate injectable formulations such as aqueous solutions, suspensions, emulsions, etc., pills, capsules, granules, or tablets.

Furthermore, it can be preferably formulated according to each disease or ingredient using a method disclosed by Remington's Pharmaceutical Science, Mack Publishing Company, Easton PA, as an appropriate method in the field.

In addition, the present invention provides an anti-inflammatory cosmetic composition containing Lactiplantibacillus plantarum WiKim0145, its culture, its lysate, its fermentation product, or its extract as an active ingredient.

The inflammation may be due to the expression of one or more cytokines selected from the group consisting of IL-1β, TNF-α, and IL-6.

When the composition according to the present invention is used as a cosmetic composition, it can be prepared in any formulation commonly prepared in the art. For example, the cosmetic composition may have the formulation of lotion, essence, lotion, cream, ampoule, pack, gel, powder, foundation, soap, or detergent, but is not limited thereto.

The cosmetic composition of the present invention can be used according to conventional usage methods, and the number of times of use can be varied depending on the user's skin condition or preference.

In addition, the cosmetic composition of the present invention may further include all kinds of ingredients that can be used in conventional cosmetics, such as moisturizers, sunscreens, neutralizers, thickeners, fragrances, preservatives, antioxidants, or colorants.

The amount of Lactiplantibacillus plantarum WiKim0145 strain included in the composition according to the present invention may be about 10 ⁶ to 10 ¹² cfu/g, for example, 10 ⁷ to 10 ¹¹ cfu/g, 10 8 to 10 ⁸ It may be 10 ¹⁰ cfu/g. When administering a strain, it is preferable to administer it in a live state, and it can be killed before ingestion or administered in an attenuated state. In addition, when manufacturing using culture supernatant, etc., it may additionally undergo a sterilization process through a heat treatment process. The amount of strain and daily intake required to have minimal efficacy may vary depending on the body or health status of the ingestor, but may generally be about 10 ⁶ to 10 ¹² cfu/g, for example, 10 ⁷ to 10 ¹¹ cfu/g. , it may be 10 ⁸ to 10 ¹⁰ cfu/g.

The advantages and features of the present invention and methods for achieving them will become clear with reference to the embodiments described in detail below. However, the present invention is not limited to the embodiments disclosed below and will be implemented in various different forms. The present embodiments are merely provided to ensure that the disclosure of the present invention is complete and to provide common knowledge in the technical field to which the present invention pertains. It is provided to fully inform those who have the scope of the invention, and the present invention is only defined by the scope of the claims.

Lactiplantibacillus plantarum WiKim0145 according to the present invention is a lactic acid bacterium isolated from kimchi and exhibits an excellent inflammatory cytokine inhibition effect, so it can be used in various ways for anti-inflammatory purposes, prevention, improvement, and treatment of inflammatory diseases. there is.

Figure 1 is a graph measuring the anti-inflammatory activity of various kimchi lactic acid bacteria isolated from kimchi ((A) Nitric oxide, (B) IL-1β, (C) IL-6, and (D) TNF-α)
Figure 2 shows the 16S rRNA identification results of Kimchi lactic acid bacterium WiKim0145.
Figure 3 is a graph confirming the probiotic properties of Kimchi lactic acid bacterium Wikim0145 ((A) acid resistance, (B) bile resistance, and (C) heat resistance).
Figure 4 is a graph analyzing the expression level of inflammation-related cytokines of Kimchi lactic acid bacterium WiKim0145 ((A) IL-1β, (B) IL-6, (C) TNF-α, (D) IL-4, (E) IL- 10, and (F) IFN-γ).
Figure 5 is a photo of the results of a hemolytic investigation of Kimchi lactic acid bacteria Wikim0145.
Figure 6 is a photograph of the enzyme activity measurement results of kimchi lactic acid bacterium WiKim0145 (the number of samples in the photograph corresponds to Table 2).

Hereinafter, the present invention will be described in detail through examples. The following examples are merely illustrative of the present invention and the scope of the present invention is not limited to the following examples.

[Example]

Example 1: Isolation and anti-inflammatory activity investigation of Lactiplantibacillus plantarum WiKim0145

To isolate various kimchi lactic acid bacteria, kimchi such as cabbage kimchi, radish kimchi, mustard kimchi, and bachelor kimchi were collected, and the kimchi crushed liquid was smeared and cultured on LBS (Lactobacillus selective medium) solid medium, a lactic acid bacteria selection medium, to purify and isolate lactic acid bacteria colonies. . 100 types of kimchi lactic acid bacteria were isolated from various kimchi, and after culturing the isolated kimchi lactic acid bacteria, the culture medium was taken and kimchi lactic acid bacteria with anti-inflammatory activity were selected through nitric oxide scavenging ability analysis.

Nitric oxide scavenging ability was tested using RAW264.7 cells, a mouse macrophage cell line. To cultivate RAW264.7 cells, 10% FBS and 1% P/S were added to DMEM medium and used as a culture medium, and the experiment was conducted by culturing at 37°C under CO ₂ 5% conditions. Anti-inflammatory activity was analyzed using an ELISA kit for nitric oxide scavenging ability in RAW264.7 cell culture. In addition, the expression level of inflammatory cytokines was also measured simultaneously with the analysis of NO expression level. Cytokine expression levels were measured using a Quantikine ELISA kit, and the measured cytokines were interleukin-1β (IL-1β), interleukin-6 (IL-6), and TNF-α. The results are shown in Figure 1. It was.

After inducing NO production in RAW264.7 cells by treatment with LPS (1 μg/mL), the anti-inflammatory effect of the lactic acid bacteria culture was measured. As a result, it was confirmed that all selected kimchi lactic acid bacteria cultures inhibited NO production, and among them, anti-inflammatory Kimchi lactic acid bacteria WiKim0145 strain with excellent inflammatory activity was selected.

It was confirmed that there was no cytotoxicity in all lactic acid bacteria cultures. To perform an ELISA assay according to the above results, a sample (10 ㎕) was added to the RAW264.7 cell line and cultured in a 37°C 5% CO ₂ incubator for 48 hours. As a result of checking the expression level of the cytokine IL-1β, an excellent reduction effect was observed from 121.3 pg/mL to 53.8 pg/mL, and in the case of TNF-α, a reduction effect was confirmed from 1773.7 pg/mL to 1157.4 pg/mL. did. In the case of IL-6, the expression level decreased from 1638.4 pg/mL to 1100.1 pg/mL, confirming that the WiKim0145 strain had the best anti-inflammatory activity among the selected kimchi lactic acid bacteria.

As a result of analyzing the phylogenetic tree of the Kimchi Lactobacillus WiKim0145 strain, which was selected as the strain with the best anti-inflammatory activity through the above experiment, using 16S rRNA base sequence analysis, a genetic identification method, it was 99% identical to Lactiplantybacillus plantarum. was confirmed, and the microorganism of the present invention was named Lactiplantibacillus plantarum WiKim0145 ( Lactiplantibacillus plantarum WiKim0145), and was deposited at the Korea Research Institute of Bioscience and Biotechnology Biological Resources Center on December 20, 2023 (accession number KCTC 15742BP).

The results of analysis of Lactiplantibacillus plantarum WiKim0145 using the API 50 CHL kit (Biomerieux) are shown in Table 1 below.

test number reaction substrate WiKim0145 test number reaction substrate WiKim0145 24h 48h 24h 48h 0 control - - 25 Esculine - - One glycerol - - 26 salicin - - 2 Erythritol - - 27 cellobiose - - 3 D-arabinose - - 28 maltose + + 4 L-arabinose - - 29 lactose - - 5 Ribose + + 30 melibius + + 6 D-xylose - - 31 sucrose + + 7 L-xylose - - 32 trehalose - - 8 adonitol - - 33 Inulin - - 9 ß Methyl-D-xyloside - - 34 Melezitos - - 10 galactose - - 35 Raffinose + + 11 glucose + + 36 starch - - 12 fructose + + 37 glycogen - - 13 Mannose - - 38 xylitol - - 14 Sorbos - - 39 ß Gentiobios - - 15 rhamnose - - 40 D-Turanos - - 16 Dulcitol - - 41 D-License - - 17 inositol - - 42 D-Tagatose - - 18 Mannitol - - 43 D-fucose - - 19 Sorbitol - - 44 L-fucose - - 20 Methyl-D-mannoside - - 45 D-arabitol - - 21 Methyl-D-glucoside - - 46 L-arabitol - - 22 N-acetyl-glucosamine - - 47 gluconate + + 23 Amygdalin - - 48 2-keto-gluconate - - 24 arbutin - - 49 5-keto-gluconate + +

Example 2: Results of confirming acid resistance, bile resistance, and heat resistance of Lactiplantibacillus plantarum WiKim0145

The probiotic properties of acid resistance and bile resistance of the selected Lactiplantibacillus plantarum WiKim0145 strain were confirmed, and are shown in Figure 3.

Since only lactic acid bacteria that can survive under conditions of gastric acid (pH 3 or pH 2 or lower) can exhibit probiotic functionality, when treated for 4 hours in MRS medium adjusted to pH 2.0, Lactiplantibacillus plantarum WiKim0145 strain produced 7.71 log CFU in pH 2.0 medium. The number of viable bacteria was shown at /mL, and it was confirmed that it had excellent acid resistance with a 99% survival rate when compared to the control group's 7.75 log CFU/mL.

In order for probiotic lactic acid bacteria to reach the intestinal tract, it is important not only to have acid resistance to gastric juice, but also to have resistance to bile acids secreted from the pancreas to the duodenum. Bile resistance was tested in MRS liquid medium containing 0.3% oxgall for 12 hours, and the viable cell count in the 0.3% oxgall medium was confirmed to be 7.44 log CFU/mL. It was confirmed to have excellent bile resistance when compared to the live bacterial count of 8.14 log CFU/mL in the control group.

Exposure to a high-temperature environment during the probiotic formulation process may reduce probiotic activity, so the thermal stability of lactic acid bacteria is one of the important criteria for selecting probiotics. The heat resistance of the selected strains was tested at 50℃ and 60℃ for 1 hour each. The control group showed 7.58(log CFU/mL) at 30℃, 5.87(log CFU/mL) at 50℃, and 5.17(log CFU/mL) at 60℃. /mL) of live bacteria (p<0.05). When heat treated at 50℃, the survival rate gradually decreased to 78%, and the Lactiplantibacillus plantarum WiKim0145 strain had a very high survival rate even in harsh environments such as acidity, bile, and high temperature, and showed excellent probiotic properties.

Example 3: Results of confirming the expression level of inflammation-related cytokines in Lactiplantibacillus plantarum WiKim0145

Analysis of inflammation-related cytokine expression levels was additionally conducted. In additional experiments, Caco-2 cells (1×10 ⁵ cells/well) were used. The level of expression of inflammation-related cytokines was measured through PCR analysis, and inflammatory cytokines (IL-1β, IL-6, TNF-α) and anti-inflammatory cytokines (IL-4, IL-10, IFN) were measured. -γ) was confirmed and shown in Figure 4.

The Control treatment group was treated with DMEM medium (2 mL/well) for 8 hours, and the LPS treatment group was treated with DMEM medium (2 mL/well) for 6 hours and then LPS (1 μl/mL) for 2 hours. The LPS+10^7 treatment group and the LPS+10^8 treatment group were treated with DMEM medium (2 mL/well) to which Wikim0145 was added at 10 ⁷ CFU/mL and 10 ⁸ CFU/mL, respectively, for 6 hours, followed by LPS (1 μl/well). mL) was treated for 2 hours.

The expression level of inflammatory cytokines (IL-1β, IL-6, TNF-α) was significantly reduced in the LPS+10^8 treatment group compared to the LPS treatment group (IL-1β 77.9%, IL-6 67.0%, TNF- α 88.6%), showing excellent anti-inflammatory activity. The expression level of anti-inflammatory cytokines (IL-4, IL-10, and IFN-γ) significantly increased in the LPS+10^8 treatment group (IL-4 1102.3%, IL-10 797.1%, IFN-γ 918.2% ), it was confirmed that treatment with 10 ⁸ CFU/mL of Lactiplantibacillus plantarum WiKim 0145 had an excellent anti-inflammatory effect in suppressing inflammation.

Example 4: Safety test results of Lactiplantibacillus plantarum WiKim0145

In order to apply probiotic lactic acid bacteria to food, substances harmful to the human body must not be produced, so the production of hemolytic and harmful enzymes was measured.

In order to ensure the stability of the selected Lactiplantibacillus plantarum WiKim0145 strain, 5% sheep blood was added to MRS and hemolysis was measured using a solid medium supplemented with 1.5% agar, and the results are shown in Figure 5. After streak smearing on 5% sheep blood agar solid medium and culturing at 30°C for 48 hours, the presence or absence of hemolysis was determined based on the shape of the ring around the strain. Hemolysis is divided into α-hemolysis, which produces a green, opaque ring due to incomplete lysis of red blood cells, β-hemolysis, which completely dissolves red blood cells and creates a yellow ring, and γ-hemolysis, which does not cause hemolysis. As a result of confirming the presence or absence of hemolysis by culturing on 5% sheep blood agar solid medium, Lactiplantibacillus Plantarum WiKim0145 did not show hemolysis, confirming that it is safe for application to food.

Additionally, in order to confirm the safety of probiotic lactic acid bacteria, it has been reported that they should not produce β-glucuronidase, a carcinogenic enzyme that converts benzopyrene into a carcinogen. The presence or absence of β-glucuronidase activity, a carcinogenic enzyme, was tested using the API zym kit, and the results are shown in Table 2 and Figure 6. Colonies cultured on MRS agar for 24 hours were taken and mixed with suspension medium, dispensed into the API zym kit, and the mixture was cultured at 37°C for 4 hours. Color development reagents zym A and zym B were added to the mixture, respectively, and color was developed for 5 minutes. Enzyme activity was confirmed through color intensity. As a result, the kimchi lactic acid bacterium Lactiplantibacillus plantarum Wikim0145 was tested for β-glucuronidase, a carcinogenic enzyme. Since it does not show any activity, its safety against carcinogenesis was confirmed, and it was judged that it could be used in various fields as a safe probiotic lactic acid bacteria in the future.

No enzyme WiKim
0145 No enzyme WiKim
0145 One Control - 11 Acid phosphatase - 2 Alkaline phosphatase - 12 Naphthol-AS-BI-phosphohydrolase + 3 Esterase - 13 α-Galactosidase - 4 Esterase lipase - 14 β-Galactosidase - 5 lipase - 15 β-Glucuronidase - 6 Leucine arylamidase + 16 α-Glucosidase - 7 Valine arylamidase + 17 β-Glucosidase + 8 Cystine arylamidase - 18 N-Acetyl-β-glucosaminidase + 9 Trypsin - 19 α-Mannosidase - 10 α-Chymotrypsin - 20 α-Fucosidase -

Korea Research Institute of Bioscience and Biotechnology Biological Resources Center (KCTC) KCTC15742BP 20231220

Sequence list electronic file attached

Claims (13)

Lactiplantibacillus plantarum WiKim0145 { Lactiplantibacillus plantarum WiKim0145 } strain with accession number KCTC 15742BP. According to claim 1,
The strain is a strain that has acid resistance, heat resistance, and bile resistance. According to claim 1,
The strain is a strain that does not produce β-glucuronidase. An anti-inflammatory food composition comprising Lactiplantibacillus plantarum WiKim0145 ( Lactiplantibacillus plantarum WiKim0145 ; accession number KCTC 15742BP), its culture, its lysate, or its extract as an active ingredient. According to claim 4,
The food composition, wherein the inflammation is caused by the expression of one or more cytokines selected from the group consisting of IL-1β, TNF-α, and IL-6. According to claim 4,
The food is a food composition that is a health functional food. A lactic acid bacteria starter for fermentation consisting of Lactiplantibacillus plantarum WiKim0145 (Accession number KCTC 15742BP) or a culture thereof. A feed or feed additive composition containing Lactiplantibacillus plantarum WiKim0145 ( Lactiplantibacillus plantarum WiKim0145 ; accession number KCTC 15742BP), its culture, its lysate, or its extract as an active ingredient. An anti-inflammatory pharmaceutical composition comprising Lactiplantibacillus plantarum WiKim0145 ( Lactiplantibacillus plantarum WiKim0145 ; accession number KCTC 15742BP), its culture, its lysate, or its extract as an active ingredient. According to clause 9,
A pharmaceutical composition, wherein the inflammation is caused by the expression of one or more cytokines selected from the group consisting of IL-1β, TNF-α, and IL-6. An anti-inflammatory cosmetic composition comprising Lactiplantibacillus plantarum WiKim0145 ( Lactiplantibacillus plantarum WiKim0145 ; accession number KCTC 15742BP), its culture, its lysate, or its extract as an active ingredient. According to claim 11,
The cosmetic composition wherein the inflammation is caused by the expression of one or more cytokines selected from the group consisting of IL-1β, TNF-α, and IL-6. delete