WO2021040086A1 - Novel lactic acid bacteria, and composition and health functional food, each comprising same - Google Patents

Abstract

The present invention relates to Lactobacillus plantarum L-14 KCTC13497BP strain, and a composition and a health functional food, each comprising same, wherein the strain exhibits various activities such as antibacterial activity, anticancer activity, inhibition of differentiation to adipocytes, antiobesity activity, etc. Accordingly, the strain can be preferably utilized in a medicinal composition, a health functional food, and so on.

Description

Novel lactic acid bacteria, composition and health functional food containing the same

The present invention relates to a novel lactic acid bacteria, a composition comprising the same, and a health functional food.

Lactobacillus is one of the most beneficial microorganisms available to humans, and has a long history and has a direct or indirect relationship with human life. Lactobacillus is widely distributed in nature, such as the oral cavity, digestive tract, and soil of mammals as well as food, and is a beneficial strain for human life that plays an important role related to food preservation and organoleptic properties.

The name lactic acid bacteria is conceptual and not a taxonomic scientific name for bacteria. Bacteria that produce about 50% or more of lactic acid as metabolites with respect to sugar consumed are called lactic acid bacteria. Unlike E. coli, it does not produce decay products. Therefore, bacteria that produce a large amount of lactic acid from sugars, do not produce substances harmful to the human body in the intestines of humans and animals, and prevent decay, are conceptually referred to as lactic acid bacteria. Lactobacillus in a common concept is Gram-positive cocci or bacillus, catalase negative (anaerobic fermentation), does not form spores, and generally has no motility.

Lactic acid bacteria produce various antibacterial substances and organic acids such as lactic acid and acetic acid with antibacterial activity. Lactobacillus produces hydrogen peroxide in the presence of oxygen and does not have catalase activity, so it secretes hydrogen peroxide into the medium and accumulates it to a concentration that has microbial inhibition. In addition, diacetyl, one of the non-proteinaceous antimicrobial substances produced by lactic acid bacteria, is one of the representative flavor components of lactic acid bacteria fermented products, and has a strong inhibitory effect against pathogenic microorganisms by interacting with pH.

In general, bacteriocin is a protein or protein complex material having a relatively small molecular weight and inhibits other microorganisms by disrupting the proton motive force of the bacterial cell membrane. Most of them are stable to heat and are stable even at low pH, but most of them are inactivated by proteolytic enzyme treatment. Nisin, a representative lactic acid bacteria-derived bacteriocin substance, is produced from Lactococcus lactis and has been commercially accepted for a long time because of its broad antimicrobial activity. Lactococcin), Pediocin, and the like are known.

An object of the present invention is to provide a novel lactic acid bacteria.

An object of the present invention is to provide a composition and health functional food utilizing various activities and functions of novel lactic acid bacteria.

The present invention provides Lactobacillus plantarum L-14 (Lactobacillus plantarum L-14) KCTC13497BP.

The present invention provides a composition comprising Lactobacillus plantarum L-14 (Lactobacillus plantarum L-14) KCTC13497BP, a culture solution or an extract thereof.

The present invention provides a health functional food comprising Lactobacillus plantarum L-14 (Lactobacillus plantarum L-14) KCTC13497BP, a culture solution or an extract thereof.

Lactobacillus plantarum L-14 (Lactobacillus plantarum L-14) KCTC13497BP strain of the present invention exhibits various activities such as antibacterial, anti-inflammatory, anti-cancer, adipocyte differentiation inhibition, and anti-obesity. Thus, it can be preferably used as a pharmaceutical composition, health functional food, cosmetic composition, and the like.

Figure 1 is a micrograph of Lactobacillus plantarum L-14 (Lactobacillus plantarum L-14) KCTC13497BP, and the turbidity (O.D.) of the culture, and the number of viable cells (colony forming unit; CFU) was measured.

Figure 2 shows the metabolism of Lactobacillus plantarum L-14 (Lactobacillus plantarum L-14) KCTC13497BP.

3 to 8 show the effect of inhibiting the growth of harmful microorganisms of Lactobacillus plantarum L-14 (Lactobacillus plantarum L-14) KCTC13497BP.

Figure 9 shows the anti-inflammatory effect of Lactobacillus plantarum L-14 (Lactobacillus plantarum L-14) KCTC13497BP.

10 to 13 show the anticancer effect of Lactobacillus plantarum L-14 (Lactobacillus plantarum L-14) KCTC13497BP.

14 to 21 show the effect of inhibiting adipocyte differentiation of Lactobacillus plantarum L-14 (Lactobacillus plantarum L-14) KCTC13497BP.

Figure 22 shows the skin irritation relief effect of Lactobacillus plantarum L-14 (Lactobacillus plantarum L-14) KCTC13497BP.

Hereinafter, the present invention will be described in detail.

The present invention relates to a novel lactic acid bacteria Lactobacillus plantarum L-14 (Lactobacillus plantarum L-14) KCTC13497BP.

The strain was deposited with the Korea Research Institute of Bioscience and Biotechnology Biological Resource Center on March 15, 2018, and the accession number is KCTC13497BP.

The strain may have glycogen metabolism, L-arabinose or α-methyl-D-mannoside metabolism, and may metabolize all of the sugars.

The strain is excellent in viability, and for example, the number of viable cells may not decrease until 48 hours when cultured in a culture medium.

The strain has a remarkably fast growth rate with reproducible ability. For example, the generation time (doubling time) may be within 20 minutes, specifically within 15 minutes. This is a growth rate similar to that of food poisoning bacteria, and is significantly faster than other conventional lactic acid bacteria (Lactobacillus genus). Thus, when the strain of the present invention is used for antibacterial or the like, various harmful bacteria can be effectively suppressed.

The strain or its culture has several excellent activities. For example, it is an effect of inhibiting the growth of harmful microorganisms, an anti-inflammatory effect, an anti-cancer effect, and an effect of inhibiting differentiation of adipocytes. Thus, the strain, its culture or its extract may be used as an antibacterial composition, a pharmaceutical composition, an anti-inflammatory composition, a health functional food, and the like. The culture may include the strain, or the strain may be an isolated culture.

In addition, the present invention relates to a composition comprising Lactobacillus plantarum L-14 (Lactobacillus plantarum L-14) KCTC13497BP, a culture thereof or an extract thereof.

The culture of the Lactobacillus plantarum L-14 KCTC13497BP strain includes a medium containing the strain (solid, liquid, etc.), and after culturing the strain, a medium from which the strain is isolated (solid, liquid, etc.). I can.

The extract of the strain may be, for example, a lysate of the strain, and specifically, may be a powder obtained by lyophilizing the lysate of the strain or a solution or dispersion thereof.

The composition may be an antibacterial composition.

Lactobacillus plantarum L-14 (Lactobacillus plantarum L-14) KCTC13497BP, its culture or its extract may exhibit antimicrobial activity against various harmful bacteria.

Examples of harmful bacteria may be microorganisms belonging to the genus Staphylococcus, the genus Streptococcus, the genus Enterococcus, the genus Pseudomonas, the genus Escherichia, and the like, and specific examples are Staphylococcus aureus, S. aureus. ), Streptococcus mutans (S.mutans), Enterococcus faecalis (E.faecalis), Pseudomonas aeruginosa; P.aeruginosa, Escherichia coli, Esichia coli (Esichia coli, E. .coli) or the like, but is not limited thereto.

In addition, the antimicrobial composition of the present invention may exhibit excellent antimicrobial activity against harmful bacteria as described above, without having a significant effect on beneficial bacteria such as lactic acid bacteria or other bacteria.

In addition, the composition may be a pharmaceutical composition for preventing or treating diseases caused by the bacteria.

Harmful bacteria such as the genus Staphylococcus, Streptococcus, Enterococcus, Pseudomonas, and Escherichia are known to cause various diseases, and for specific examples, Staphylococcus aureus is food poisoning and sepsis. Etc., and Streptococcus mutans, Enterococcus faecalis, etc. induce tooth decay, oral diseases, etc., and Pseudomonas aruginosa is known to cause endocarditis, pneumonia, meningitis, etc. It can be utilized as a pharmaceutical composition for prophylaxis or treatment. In addition to those exemplified above, it may exhibit a preventive or therapeutic effect on all diseases known to be caused by the above bacteria.

In addition, the composition may be an anti-inflammatory composition.

Lactobacillus plantarum L-14 (Lactobacillus plantarum L-14) KCTC13497BP, its culture or its extract has an effect of inhibiting the secretion of inflammatory cytokines of macrophages, can exhibit excellent anti-inflammatory effect.

In addition, the composition may be a pharmaceutical composition for preventing or treating inflammatory diseases.

The inflammatory diseases include, for example, atopic dermatitis, edema, dermatitis, allergy, asthma, conjunctivitis, periodontitis, rhinitis, otitis media, sore throat, tonsillitis, pneumonia, gastric ulcer, gastritis, Crohn's disease, colitis, hemorrhoids, gout, hepatic spondylitis, Rheumatoid heat lupus, fibromyalgia, psoriatic arthritis, osteoarthritis, rheumatoid arthritis, peri-shoulder joint infection, tendinitis, tendonitis, myositis, hepatitis, cystitis, nephritis, sjogren's syndrome, multiple sclerosis, etc. It is not.

In addition, the composition may be a pharmaceutical composition for preventing or treating cancer.

Lactobacillus plantarum L-14 (Lactobacillus plantarum L-14) KCTC13497BP, its culture or its extract has excellent cancer cell lowering effect, thereby reducing tumor size.

The pharmaceutical composition of the present invention can be applied without limitation to cancer known in the art, for example, brain tumor, gastric cancer, lung cancer, breast cancer, ovarian cancer, liver cancer, bronchial cancer, nasopharyngeal cancer, laryngeal cancer, esophageal cancer, pancreatic cancer, bladder cancer, prostate cancer. Cancer, colon cancer, head and neck cancer, skin cancer, melanoma, colon cancer, and cervical cancer, and the like, but are not limited thereto. For a specific example, it may be a melanoma.

In addition, the composition may be a composition for inhibiting adipocyte differentiation or a composition for anti-obesity.

Lactobacillus plantarum L-14 (Lactobacillus plantarum L-14) KCTC13497BP, its culture or its extract can inhibit the differentiation of adipocytes into adipocytes and stem cells into adipocytes.

The stem cells may be, for example, embryonic stem cells, adult stem cells, or induced pluripotent stem cells, and adult stem cells may be mesenchymal stem cells, but are not limited thereto.

The composition of the present invention may be administered orally, parenteral, rectal, topical, transdermal, intravenous, intramuscular, intraperitoneal, subcutaneous, and the like. Formulations for oral administration may be tablets, pills, soft and hard capsules, granules, powders, fine granules, liquids, emulsions, or pellets, but are not limited thereto. Formulations for parenteral administration may be solutions, suspensions, emulsions, gels, injections, drops, suppositories, patches, ointments, or sprays, but are not limited thereto. The formulation can be easily prepared according to a conventional method in the art, and a surfactant, an excipient, a wetting agent, an emulsification accelerator, a suspending agent, a salt or buffer for controlling the osmotic pressure, a colorant, a spice, a stabilizer, a preservative, a preservative or Other commonly used adjuvants may be additionally included.

The applied amount or dosage of the composition of the present invention will vary depending on the age, sex, weight, pathological condition and severity of the subject to be administered, the route of administration, or the judgment of the prescriber. Determination of the applied amount based on these factors is within the level of those skilled in the art, and the daily dose of the active ingredient is, for example, 0.0001 μg/kg/day to 5000 mg/kg/day, more specifically 0.00001 mg/kg/day to 15 mg. It may be /kg/day, but is not limited thereto.

In addition, the present invention relates to a health functional food comprising Lactobacillus plantarum L-14 (Lactobacillus plantarum L-14) KCTC13497BP, its culture or its extract.

The health functional food of the present invention may have health functions (prevention or improvement) such as antibacterial, anti-inflammatory, anti-cancer, adipocyte differentiation inhibition, or anti-obesity.

The formulation of the health functional food of the present invention is not particularly limited, but may be formulated as, for example, a tablet, granule, pill, powder, capsule, liquid such as a drink, caramel, gel, bar, tea bag, or the like. In addition to the active ingredients, the food composition or health food composition of each formulation can be appropriately selected and blended by a person skilled in the art according to the formulation or purpose of use, depending on the formulation or purpose of use. Can happen.

The health functional food of the present invention may be administered by various methods such as simple intake, drinking, injection administration, spray administration, or squeeze administration.

In the health functional food of the present invention, the determination of the dosage of the active ingredient is within the level of those skilled in the art, and the daily dosage thereof may be, for example, 0.00001 mg/kg/day to 15 mg/kg/day, The present invention is not limited thereto, and may vary depending on various factors such as age, health status, and complications of the target to be administered.

The health functional food of the present invention may be, for example, various foods such as chewing gum, caramel products, candies, frozen desserts, sweets, beverage products such as soft drinks, mineral water, alcoholic beverages, and health functional foods including vitamins or minerals. have.

The health functional food of the present invention includes various nutrients, vitamins, minerals (electrolytes), flavoring agents such as synthetic flavors and natural flavoring agents, coloring agents and enhancers (cheese, chocolate, etc.), pectic acid and salts thereof, alginic acid and salts thereof. , Organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohols, carbonates used in carbonated beverages, and the like.

The health functional food of the present invention may include flesh for the manufacture of natural fruit juice and fruit juice beverages and vegetable beverages. These components may be used independently or in combination. The proportion of these additives is not so important, but is generally included in the range of 0 to about 50 parts by weight per 100 parts by weight of the composition according to an aspect of the present invention.

In addition, the present invention relates to a cosmetic composition comprising the Lactobacillus plantarum L-14 (Lactobacillus plantarum L-14) KCTC13497BP, the culture or an extract thereof.

The cosmetic composition of the present invention may have the anti-inflammatory activity described above.

In addition, the cosmetic composition of the present invention may be for relieving skin irritation.

The cosmetic composition of the present invention may include ingredients commonly used in cosmetic compositions in addition to the above as an active ingredient, for example, conventional auxiliary agents such as antioxidants, stabilizers, solubilizers, vitamins, pigments and fragrances, and carriers. can do.

The cosmetic composition of the present invention may be prepared in any formulation conventionally prepared in the art, for example, a solution, a suspension, an emulsion, a paste, a gel, a cream, a lotion, a powder, a soap, a surfactant containing cleansing, It can be formulated as an oil, powder foundation, emulsion foundation, wax foundation and spray. However, it is not necessarily limited thereto. In more detail, it may be prepared in the form of a flexible lotion, astringent lotion, a nutrition lotion, a nutrition cream, a massage cream, an essence, an eye cream, a cleansing cream, a cleansing foam, a cleansing water, a pack, a spray, or a powder.

When the formulation of the cosmetic composition of the present invention is a paste, cream, or gel, as a carrier component, animal oil, vegetable oil, wax, paraffin, starch, tracant, cellulose derivative, polyethylene glycol, silicone, bentonite, silica, talc or zinc oxide, etc. Can be used.

When the formulation of the cosmetic composition of the present invention is a powder or spray, lactose, talc, silica, aluminum hydroxide, calcium silicate or polyamide powder may be used as a carrier component. Propellants such as carbon, propane/butane or dimethyl ether.

When the formulation of the cosmetic composition of the present invention is a solution or emulsion, a solvent, a solubilizing agent or an emulsifying agent is used as a carrier component, such as water, ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene Glycol, 1,3-butylglycol oil, glycerol aliphatic ester, polyethylene glycol or fatty acid ester of sorbitan.

When the formulation of the cosmetic composition of the present invention is a suspension, as a carrier component, a liquid diluent such as water, ethanol or propylene glycol, an ethoxylated isostearyl alcohol, a suspending agent such as polyoxyethylene sorbitol ester and polyoxyethylene sorbitan ester , Microcrystalline cellulose, aluminum metahydroxide, bentonite, agar or tracant, and the like may be used.

When the formulation of the cosmetic composition of the present invention is a surfactant containing cleansing, as carrier components, aliphatic alcohol sulfate, aliphatic alcohol ether sulfate, sulfosuccinic acid monoester, isethionate, imidazolinium derivative, methyltaurate, sarcosinate, Fatty acid amide ether sulfates, alkylamidobetaines, fatty alcohols, fatty acid glycerides, fatty acid diethanolamides, vegetable oils, lanolin derivatives or ethoxylated glycerol fatty acid esters may be used.

Hereinafter, examples will be described in detail in order to describe the present invention in detail.

Example

1. Microbiological characteristics

(1) strain selection

A portion of the traditional rice fermentation (flounder sikhae) was resuspended in 20 mL PBS and vortexed. Then, diluted to various concentrations, 2% dextrose, 1% peptic digest of animal tissue, 1% beef extract, 0.5% yeast extract, 0.5% sodium acetate, 0.2% disodium phosphate, 0.2% ammonium citrate, 0.1% polysorbate 80, 0.01 5.5% (w/v) De Man, Rogosa and Sharpe agar (MRS; Hardy Diagnostics, CA, USA) containing% magnesium sulfate, 0.005% manganese sulfate, and 1.5% agar were plated on agar plates at 37°C for 18 hours. . Colonies were randomly collected, re-inoculated in MRS medium, and cultured at 37°C for 18 hours. 98 strains were obtained through the screening process, which were added to glycerol at a final concentration of 15% and frozen at -80°C.

Each L14 microbial isolate was subjected to phylogenetic analysis. The PCR primer used was 27F (5'-AGAGTTTGATCMTGGCTCAG-3', SEQ ID NO: 1)/1492R (5'-TACGGYTACCTTGTTACGACTT-3', SEQ ID NO: 2), and the 16s rRNA sequencing primer was 785F (5'-GGATTAGATACCCTGGTA-3' , SEQ ID NO: 3) / 907R (5'-CCGTCAATTCMTTTRAGTTT -3', SEQ ID NO: 4). PCR sequencing was performed by Cosmo Gentech (Daejeon, Korea).

To calculate the doubling time of L14, it was inoculated into MRS medium and cultured at 37°C. The absorbance was measured at 600 nm using a microplate reader (Molecular Devices, LLC, CA, USA) and a colony forming unit (CFU/mL), spreading L14 on the MRS agar plate at regular intervals, and counting the number of colonies.

The morphology of L14 was observed through a scanning electron microscope (SEM). L14 cells were straight, rounded at the end, 0.6-0.9 μm in width, and 1.2-2.4 μm in length, in the form of a rod (Fig. 1).

(2) Check doubling time

In order to analyze the doubling time of L14, the turbidity (O.D.) of the culture medium and the change in the number of viable cells (colony forming unit; CFU) according to time were measured (FIG. 1).

The turbidity and viable cell count change graph showed consistent improvement, and it was confirmed that the viable cell count did not decrease until about 48 hours.

As a result of analyzing the doubling time of L14, it was found to be about 14.9 minutes, which is a significantly faster growth rate than the previously known lactic acid bacteria L. acidophilus for about 70 minutes, E. coli 20 minutes, Pseudomonas aeruginosa 35 minutes, and B. megaterium 25 minutes. As such, it can have a positive effect on production efficiency, so it can be considered to be more marketable than other lactic acid bacteria. The doubling times of microorganisms in the genus Lactobacillus known for reference are shown in Table 1 below.

Cell strains Doubling time (min) Lactobacillus plantarum ATCC 14917 34.4 Lactobacillus plantarum spp. 79.8 Lactobacillus fermentum spp. 36.6 Lactobacillus reuteri (DSM 20016, DSM 17938, ATCC 53608) 51-83.4 Lactobacillus paracasei LFBM 01 104.4 Lactobacillus paracasei LFBM 05 114.6 Lactobacillus paracasei LFBM 06 100.8 Lactobacillus paracasei LFBM 10 106.2 Lactobacillus rhamnosus ATCC 9595 82.8 Lactobacillus plantarum LFBM 02 103.78 Lactobacillus plantarum LFBM 04 112.2 Lactobacillus plantarum LFBM 07 108 Lactobacillus plantarum LFBM 08 90 Lactobacillus plantarum LFBM 09 117

(3) metabolism

The metabolic properties of L14 were confirmed using an API identification kit. When compared with the experimental results of the API 50 CHL kit used for the identification of lactic acid bacteria and the metabolic ability table of standard L. plantarum written in the kit manual, it was confirmed that L14 has the metabolic ability closest to L. plantarum (Fig. 2). Reference).

The principle of the API kit is to grow bacteria in a kit strip containing 50 kinds of sugar to see if it can metabolize sugar. If it can metabolize a specific sugar, the color changes from purple to yellow due to pH change.

2, L14 has Glycogen metabolism ability differently from Company A's L. plantarum ML PRIMETM (LALLEMAND, South Australia, Australia, hereinafter LS), and Company B's L. plantarum lactofy kimchi lactobacillus (Probiotic Co., Ltd., Jeollabuk-do , Jeonju, LP), it can be seen that it has the ability to metabolize L-arabinose.

2. Inhibition of the growth of harmful microorganisms

(1) Disk diffusion analysis

Two types of modified MRS (i.e., ML and MT) agar were prepared to perform disk diffusion analysis. ML contained 4.5% (w/v) MRS, 1.25% (w/v) LB and 1.5% (w/v) agar. MT contained 4.5% (w/v) of MRS, 1.5% (w/v) of TSB and 1.5% (w/v) of agar. E. coli was pre-cultured in LB medium, and the other four pathogens (MRSA, P. aeruginosa, S. mutans, and E. faecalis) were cultured in TSB medium. The surface of the culture dish containing the MRS medium was wiped with LB or TSB as an indicator strain of the pathogen. Then, a commercially available paper disc (7 mm in diameter, ADVANTEC, Taipei, Taiwan) was placed in 20 μL of 7 log CFU/μL of L14, 20 μL of 7 log CFU/μL of commercial L. reuteri or 20 μL of ampicillin (1.0 mg/μL). mL) was added. Paper discs were gently placed on the surface of each media and incubated at 37° C. for 1-2 days before assessing the formation of a microbial growth inhibition zone around the disc.

(2) Comparison with L. reuteri

To investigate whether L14 exhibits antimicrobial properties similar to those of commercially available antimicrobial L. reuteri, disc diffusion analysis using 3 groups (negative control, L14 and L. reuteri treatment groups) was performed. L14 significantly inhibited the growth of E. coli (p <0.01), MRSA (p <0.05), and E. faecalis (p <0.01) compared to L. reuteri, which is known to have an antibacterial effect (Fig. 3). These data show that the antimicrobial properties of L14 against three types of oral pathogens are superior to that of commercial L. reuteri.

(3) Escherichia coli (E. coli), Staphylococcus aureus (S. aureus), Pseudomonas aeruginosa; P. aeruginosa)

Live L14 bacteria have the ability to inhibit the growth of Escherichia coli (E. coli), Staphylococcus aureus (S. aureus), and Pseudomonas aeruginosa (P. aeruginosa), which can cause food poisoning and sepsis. The antibacterial effect was confirmed by the following method.

L14 was inoculated in 5 mL of MRS liquid medium, E. coli was inoculated in 5 mL of LB liquid medium, and S. aureus and P. aeruginosa were inoculated in 5 mL of TSB liquid medium, respectively, and cultured at 37°C for 18 hours. To 100 mL of distilled water, 3.66 mg of commercially available MRS medium powder, 1 mg of LB medium powder, 1 mg of TSB medium powder, and 1.5 mg of agar were added and sterilized with an autoclave. Thereafter, when the medium was sufficiently cooled, 1 mL of E. coli, S. aureus, and P. aeruginosa cultured in the above LB and TSB liquid medium was added each, and after sufficiently mixing, poured into a petri dish to make a solid medium. . After centrifuging the cultured L14 bacteria at 12,000 g for 3 min, only about 500 μL of the medium was left, and the supernatant was removed, and then resuspended with the remaining liquid medium (1/10 concentration). 20 μL of PBS (Negative control; NC), antibiotic ampicillin (1 mg/mL), and concentrated L14 live bacteria were inoculated on sterilized disk paper, placed on a solid medium, and cultured at 37°C for 18 hours.

Referring to Figure 4, compared to the group inoculated with PBS (negative control), it can be seen that L14 live bacteria inhibit the growth of surrounding E. coli and S. aureus bacteria. In addition, it can be confirmed that it has the effect of inhibiting harmful bacteria such as ampicillin at a concentration 10 times higher than the concentration used in the existing laboratory (0.1 mg/mL). In addition, it can be confirmed that L14 has an antibacterial effect against Pseudomonas aeruginosa, which has no effect on the antibiotic ampicillin.

(4) Streptococcus mutans (S.mutans) and Enterococcus faecalis (E.faecalis)

L14 live bacteria were confirmed to have the ability to inhibit growth against the harmful microorganisms Streptococcus mutans (S. mutans) and Enterococcus faecalis (E. faecalis), which are known to cause tooth decay and oral disease.

20 μL of distilled water and concentrated L14 live bacteria were inoculated on sterilized disk paper and placed on a solid medium coated with S. mutans and E. faecalis bacteria, and the effect of inhibiting the growth of S. mutans and E. faecalis was confirmed.

Specifically, L14 bacteria were inoculated into 5 mL of MRS liquid medium, and S.mutans and E.faecalis were inoculated into 5 mL of TSB liquid medium, respectively, and cultured at 37°C for 18 hours. To 100 mL of distilled water, 3.66 mg of commercially available MRS medium powder, 1 mg of TSB medium powder, and 1.5 mg of agar were added and sterilized with an autoclave. When the medium was sufficiently cooled, 1 mL each of S. mutans and E. faecalis cultivated in the above TSB liquid medium was added, thoroughly mixed, and poured into a petri dish to make a solid medium. After centrifuging the cultured L14 bacteria at 12,000 g for 3 min, only about 500 μL of the medium was left, and the supernatant was removed, and then resuspended with the remaining liquid medium (1/10 concentration). 20 μL of PBS (Negative control; NC), antibiotic ampicillin (1 mg/mL), and concentrated L14 live bacteria were inoculated on sterilized disk paper, placed on a solid medium, and cultured at 37°C for 18 hours.

Referring to Figure 5, compared to the group inoculated with PBS (negative control), it can be seen that L14 live bacteria inhibit the growth of S. mutans and E. faecalis bacteria around. In addition, it can be confirmed that it has the effect of inhibiting harmful bacteria such as ampicillin at a concentration 10 times higher than the concentration used in the existing laboratory (0.1 mg/mL).

The results of FIGS. 4 and 5 are shown in a graph in FIG. 6.

(5) Staphylococcus aureus (S.aureus) and Pseudomonas aeruginosa (P.aeruginosa)

The growth inhibitory ability of the L14 culture medium against Staphylococcus aureus (S. aureus) and Pseudomonas aeruginosa (P. aeruginosa), which can cause food poisoning and sepsis, was confirmed.

After centrifuging the cultured L14 bacteria, the culture solution was separated, and 20 μL of distilled water and L14 culture solution were inoculated on sterilized disk paper and placed on a solid medium in which S.aureus and P.aeruginosa were already inoculated to grow S.aureus and P.aeruginosa. The inhibitory ability was confirmed.

Specifically, L14 bacteria were inoculated into 5 mL of MRS liquid medium and S.aureus and P.aeruginosa were inoculated into 5 mL of TSB liquid medium, respectively, and cultured at 37°C for 18 hours. To 100 mL of distilled water, 3.66 mg of commercially available MRS medium powder, 1 mg of TSB medium powder, and 1.5 mg of agar were added and sterilized with an autoclave. When the medium was sufficiently cooled, 1 mL each of S.aureus and P.aeruginosa cultured in the above TSB liquid medium was added, mixed sufficiently, and poured into a petri dish to make a solid medium. After the cultured L14 bacteria were centrifuged at 12,000 g for 3 min, only the supernatant (culture solution) was separated. Distilled water and 20 μL of the L14 culture solution were inoculated on sterilized disk paper, placed on a solid medium, and cultured at 37°C for 18 hours.

Referring to FIG. 7, it can be seen that the L14 culture medium inhibits the growth of S. aureus and P. aeruginosa bacteria in comparison with the disk paper inoculated with distilled water (control).

(6) Inhibitory effect of Escherichia coli and Staphylococcus aureus in real environment

The two were co-cultured in a liquid medium in which L14 and harmful bacteria (E. coli and S. aureus) could grow together in order to confirm the antibacterial effect in a practical environment, not a laboratory environment.

L14 was inoculated in 5 mL of MRS liquid medium, E. coli was inoculated in 5 mL of LB liquid medium, and S. aureus was inoculated in 5 mL of TSB liquid medium, followed by incubation at 37°C for 18 hours. To 100 mL of distilled water, 3.66 mg of commercially available MRS medium powder, 1 mg of LB medium powder, and 1 mg of TSB medium powder were added and sterilized with an autoclave. When the medium was sufficiently cooled, the previously cultured E. coli, S. aureus, and L14 bacteria were inoculated with various CFU, and cultured at 37° C. for 18 hours. Thereafter, only E. coli and S. aureus were grown and inoculated with 1 mL of film media and 100 uL of MRS agar plate, and cultured at 37°C for 18 hours.

Referring to FIG. 8, when L14 is inoculated together, it has the effect of inhibiting the growth of E. coli and S. aureus irrespective of CFU. In particular, L14 is inoculated with 1/100 of E. coli and 1/10 of S. aureus. It can be seen that it also inhibits growth when done.

And, regardless of the initial inoculation amount, a similar number of L14 colonies were formed in the MRS medium, and it was confirmed that even a small number of L14 could suppress the growth of harmful bacteria and become dominant.

3. Anti-inflammatory effect

It was confirmed that L14 directly acts on mouse macrophages known as immune cells (ATCC RAW 264.7) to inhibit the secretion of inflammation-inducing cytokines.

RAW264.7 was inoculated into a 12 well plate at 1.0 × 10 ⁶ cells/well and stabilized for one day. The next day, L14, previously cultured, ^{was inoculated at 1.0 × 10 6} CFU/mL and incubated for 6 hours. While replacing with a new medium, 10-100 ug/mL of endotoxin lipopolysaccharide (LPS), which is an inflammation inducing substance, was inoculated. After incubation for 6 hours, the culture solution was separated, and the inflammatory cytokines contained in the separated culture solution were quantified through enzyme immunoassay (ELISA).

Referring to FIG. 9, it can be seen that when L14 and RAW 264.7 are co-cultured, the secretion of inflammatory cytokines IL-6, TNFa, and MCP1 by LPS is reduced.

4. Anti-cancer effect

The inhibitory effect of L14 lactic acid bacteria extract on human malignant melanomas (A375P & A375SP) in vitro and in vivo was confirmed.

L14 lactic acid bacteria extract was prepared as follows. First, L14 lactic acid bacteria were inoculated into 10 mL of sterilized MRS liquid medium and cultured at 35° C. for 18 hours (pre-culture). The cultured L14 lactic acid bacteria were inoculated into 1 L of sterilized MRS liquid medium and cultured at 35° C. for 18 hours (main culture). The main cultured L14 bacteria were centrifuged at 4°C, 12,000g, 15 min, and then the supernatant was removed and only L14 bacteria were obtained. After resuspending with PBS, centrifugation at 4° C., 12,000 g, 15 min, and then removing the supernatant was repeated twice. After resuspending with sterile distilled water, centrifugation at 4° C., 12,000 g, 15 min, removing the supernatant, and resuspending with 100 mL distilled water. Using an ultrasonic crusher, the L14 lactic acid bacteria resuspended with distilled water were sufficiently ultrasonicated. After centrifugation at 4° C., 12,000 g, 15 min, only the supernatant was obtained and filtered through a 0.45 μm filter, and the resulting crushed extract was lyophilized for 3 days. The lyophilized L14 lactic acid bacteria extract powder was dissolved in PBS at an appropriate concentration, and then filtered through a 0.45 μm filter to obtain an extract.

(1) in vitro

The anticancer effect of L14 lactic acid bacteria extract against melanoma cell lines in vitro was confirmed as follows. Human dermal fibroblast (HDF) and human melanoma cell lines A375P and A375SM were inoculated at 10,000 cells/well in 12 well plates and 1,000 cells/well in 96 well plates and cultured for 24 hours. At the same time as the medium was changed, the L14 lactic acid bacteria extract was treated with different concentrations. After 3 days, the anticancer effect of the L14 lactobacillus extract was observed through a microscope in a 12 well plate, and confirmed by WST-1 viability assay in a 96 well plate.

Referring to Figure 10, when the treatment concentration of the L14 lactic acid bacteria extract is increased, there is no effect on human skin fibroblasts (HDF), whereas it can be confirmed that a distinct anticancer effect appears in A375P and A375SM, and referring to Figure 11, L14 lactic acid bacteria When the treatment concentration of the extract was increased, it can be seen that A375P and A375SM exhibit a distinct anticancer effect.

(2) in vivo

The anticancer activity of the human melanoma cell line A375SM of L14 lactic acid bacteria extract was confirmed in vivo.

Tumors were artificially generated by subcutaneous inoculation of the A375SM cell line in immune-deficient nude mice, and the groups were randomly divided and intraperitoneally injected with PBS and L14 lactobacillus extract at 2-day intervals for 22 days. Referring to Figure 12, it can be seen that the tumor size of the group nude mice treated with L14 lactic acid bacteria extract is smaller than that of the control group.

In order to quantitatively show the anticancer effect of L14 lactic acid bacteria extract in vivo, the weight and volume of the resected tumor were measured and analyzed. Referring to FIG. 13, it can be seen that the weight and volume of the tumor are significantly reduced by the L14 lactic acid bacteria extract. (Control group n;6, L14-treated group n;9)

5. Adipocyte differentiation inhibitory effect

L14 lactobacillus extract was confirmed to inhibit the differentiation of mouse adipocyte progenitor cell lines (3T3-L1) and human mesenchymal stem cells (hMSCs) into adipocytes.

(1) The effect of L14 lactobacillus extract on inhibiting adipocyte differentiation of the mouse adipocyte progenitor cell line (3T3-L1) was confirmed as follows.

3T3-L1 was inoculated into 24 well plates at 100,000 cells/well and cultured for several days. When the confluency reached 95-100%, the medium was changed with an adipocyte differentiation induction medium (MDI medium) and L14 lactic acid bacterium extract was treated with each concentration at the same time (Day 0). After 2 days (Day 2), the medium was changed with the adipocyte differentiation induction medium, and the L14 lactic acid bacteria extract was treated again. From Day 4 to L14 lactobacillus extract, the medium was changed to adipocyte differentiation maintenance medium, and the medium was changed every 2 days until Day 12. Using the cells of Day 12, which were differentiated into adipocytes, the effect of inhibiting adipocyte differentiation was confirmed through Oil Red O staining, gene analysis, and Triacylglycerol (TAG) assay.

Referring to FIG. 14, it can be seen that the inhibitory effect increases as the treatment concentration of the L14 lactic acid bacteria extract increases.

In addition, it was confirmed through the WST-1 viability assay to determine whether the adipocyte inhibitory effect of the L14 lactic acid bacteria extract is due to toxicity to cells, but referring to FIG. 15, it can be confirmed that there is no negative effect on 3T3-L1 even at high concentration.

Then, the oil red o of the stained adipocytes was dissolved again with isopropanol, and the content was dissolved in O.D. The values were measured and analyzed. Referring to FIG. 16, it can be seen that the degree of staining with oil red o significantly decreased as the treatment concentration of the L14 lactic acid bacteria extract increased.

And, by accurately quantifying the amount of fat accumulated in adipocytes through TAG assay, the effect of inhibiting adipocyte differentiation of L14 lactic acid bacteria extract was reconfirmed. Referring to FIG. 17, it can be seen that the inhibitory effect of adipocyte differentiation increases as the treatment concentration of the L14 lactic acid bacterium extract increases.

And, the effect of inhibiting the differentiation of mouse adipocytes of L14 lactic acid bacteria extract was confirmed at the gene level. Expression of C/EBPα, PPARγ, and FABP4, which are key gene markers for adipocyte differentiation, were confirmed. Referring to FIG. 18, it can be seen that as the concentration of L14 lactic acid bacteria extract treatment increases, the amount of expression of the three genes significantly decreases compared to the control. NC: negative control (normal 3T3-L1 cells not differentiated into adipocytes).

In addition, in order to compare the inhibitory effect on mouse adipocyte differentiation of L. plantarum (LS, LP) and L14 extract from other companies at the gene level, the expression of PPARγ, C/EBPα, and FABP4, which are key gene markers for adipocyte differentiation, was confirmed. Referring to FIG. 19, it can be seen that the L14 extract effectively suppresses the expression of adipocyte differentiation-related genes at a molecular level than the LS and LP extracts (compared with the results of FIG. 14).

(2) The effect of L14 lactic acid bacteria extract on inhibiting adipocyte differentiation of human mesenchymal stem cells (hMSCs) was confirmed as follows.

hMSCs were inoculated into 24 well plates at 100,000 cells/well and cultured for several days. When the confluency reached 95-100%, the medium was changed with an adipocyte differentiation induction medium (MDI medium) and L14 lactic acid bacterium extract was treated with each concentration at the same time (Day 0). After 2 days (Day 2), the medium was changed with the adipocyte differentiation induction medium, and the L14 lactic acid bacteria extract was treated again. From Day 4 to L14 lactobacillus extract, the medium was changed to adipocyte differentiation maintenance medium, and the medium was changed every 2 days until Day 7. The effect of inhibiting adipocyte differentiation was confirmed through the oil red o staining method using the cells of Day 7 that were differentiated into adipocytes.

Referring to Figure 20, it can be seen that the higher the concentration of L14 lactic acid bacteria extract treatment, the effect of inhibiting differentiation of adipocytes increases.

In addition, AICAR (AMPK activator) and Compound C (AMPK inhibitor, Dorsomorphin) were treated with L14 extract in order to confirm whether the effect of L14 extract inhibiting adipocyte differentiation of mouse adipocyte 3T3-L1 is achieved through the AMPK pathway. After 4 days of culture, the protein was extracted and the AMPK pathway marker was confirmed through Western blot. As a result, when the L14 extract was treated, it was confirmed that phosphorylation of AMPK was increased, and SREBP1, SCD1, FAS, PPARγ, and FABP4 were decreased (FIG. 21).

6. Relieve skin irritation

A sample containing 70% of 1,3-Butylene Glycol, 29% of purified water, and 1% of lactic acid bacteria (L-14) crushed extract filtrate was prepared.

Twenty-two healthy adult women aged 20 to 60 years old were selected as test subjects.

The skin barrier was artificially damaged by designating the sample use and unused area on the test site (left forearm) and applying 1% SLS (Sodium Lauryl Sulfate) for 24 hours. The amount of transepidermal water evaporation on the used and unused samples was measured with a Vapometer (Delfin, Finland) according to the measurement schedule. Each measurement was performed once, and the unit was expressed in g/m2/h. In addition, it was taken with Antera 3D CS (Miravex, Ireland) for photo data. As the measured value decreases, it means improvement.

Referring to FIG. 22, when the amount of transepidermal water evaporation in the sample used group was compared with the unused portion, it can be seen that the stimulation was statistically significantly relieved on the 7th and 14th days.

Claims (10)

Lactobacillus plantarum L-14 KCTC13497BP. The strain according to claim 1, which has glycogen metabolism and L-arabinose metabolism. The antibacterial composition comprising the strain of claim 1, the culture or strain extract. The composition for antibacterial according to claim 3, which has antibacterial activity against microorganisms of the genus Staphylococcus, genus Streptococcus, genus Enterococcus, genus Pseudomonas or genus Escherichia. As a pharmaceutical composition for the prevention or treatment of infectious diseases comprising the strain of claim 1, the culture or strain extract, The infectious disease is at least one composition selected from the group consisting of food poisoning, sepsis, tooth decay, endocarditis, pneumonia, and meningitis. Anti-inflammatory composition comprising the strain of claim 1, the culture or strain extract. A pharmaceutical composition for the prevention or treatment of cancer comprising the strain of claim 1, its culture or strain extract. A cosmetic composition comprising the strain of claim 1, the culture or strain extract. The composition of claim 8, wherein the cosmetic composition is for anti-inflammatory or skin irritation relief. The composition for anti-obesity comprising the strain of claim 1, the culture or strain extract.

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