KR102424598B1 - Lactobacillus pentosus OKBL-L.PE 1 strain having antioxidant activity, anti-inflammatory activity, skin whitening activity, antimicrobial activity against pathogenic microorganism and anti-thrombotic activity and uses thereof - Google Patents

Abstract

The present invention relates to Lactobacillus pentosus OKBL-L.PE 1 strain having an accession number KCCM12791P having antioxidant activity, anti-inflammatory activity, skin whitening activity, antibacterial activity and antithrombotic activity, and to a use thereof.

Description

Lactobacillus pentosus OKBL-L.PE 1 strain having antioxidant activity, anti-inflammatory activity, skin whitening activity, antibacterial activity against harmful microorganisms and antithrombotic activity and uses thereof {Lactobacillus pentosus OKBL-L.PE 1 strain having antioxidant activity, anti-inflammatory activity, skin whitening activity, antimicrobial activity against pathogenic microorganism and anti-thrombotic activity and uses thereof}

The present invention relates to a Lactobacillus pentosus OKBL-L.PE 1 strain having antioxidant activity, anti-inflammatory activity, skin whitening activity, antibacterial activity against harmful microorganisms and antithrombotic activity, and uses thereof.

Lactic acid bacteria are involved in the fermentation of various foods and not only impart a unique flavor, but also produce antibacterial substances to increase the shelf life and stability of foods. Examples of the antimicrobial substances produced by these lactic acid bacteria include diacetyl and bacteriocin in addition to organic acids such as lactic acid.

Probiotics refer to strains that have a beneficial effect on the intestinal environment when ingested and reach the intestine, and are supplied to humans or animals in the form of dry cells or fermented products to improve the intestinal flora. Most probiotics known to date are lactic acid bacteria and are mainly used in the form of fermented milk, granules, or powders made using Lactobacillus genus strains. In order for strains including lactic acid bacteria to be used as probiotics, they must survive gastric and bile acids, reach the intestines, multiply and settle, and must be non-toxic and non-pathogenic. Recently, new strains having various useful activities as well as basic characteristics of lactic acid bacteria have been isolated, identified and reported.

On the other hand, Korean Patent No. 1088685 discloses 'a composition having an immunomodulatory action containing Lactobacillus pentosus', and Korean Patent No. 1073791 discloses 'New lactobacillus with excellent intestinal enzyme activity, acid resistance and bile resistance. Although the Bacillus pentosus PL-11 strain and probiotics for fish using the same have been disclosed, Lactobacillus pentosus OKBL having 'antioxidant activity, anti-inflammatory activity, skin whitening activity, antibacterial activity against harmful microorganisms and antithrombotic activity' of the present invention -L.PE 1 strain and its use' is not described.

The present invention was derived by the above requirements, and the present inventors isolated Lactobacillus pentosus OKBL-L.PE strain (Accession No.: KCCM12791P) from Kkakdugi Kimchi, and excellent antioxidant activity and anti-inflammatory activity of the isolated strain , skin whitening activity, antibacterial activity against harmful microorganisms, and antithrombotic activity were confirmed, thereby completing the present invention.

In order to solve the above problems, the present invention provides an Lactobacillus pentosus ( Lactobacillus pentosus ) OKBL-L.PE 1 strain whose deposit number is KCCM12791P having antioxidant activity, anti-inflammatory activity, skin whitening activity, antibacterial activity and antithrombotic activity do.

In addition, the present invention provides an antibacterial composition for Gram-negative bacteria containing the strain, its culture solution, the concentrate of the culture solution, or a dried product thereof as an active ingredient.

In addition, the present invention provides a starter composition for producing a fermented food containing the strain, its culture, the concentrate of the culture or its dried product as an active ingredient.

In addition, the present invention provides a probiotic composition containing the strain, its culture solution, the concentrate of the culture solution, or a dried product thereof as an active ingredient.

In addition, the present invention provides a health functional food composition for preventing or improving thrombosis containing the strain, its culture solution, the concentrate of the culture solution, or a dried product thereof as an active ingredient.

In addition, the present invention provides a cosmetic composition for skin whitening containing the strain, its culture solution, the concentrate of the culture solution, or a dried product thereof as an active ingredient.

In addition, the present invention provides a feed additive composition containing the strain, its culture, the concentrate of the culture or its dried product as an active ingredient.

Lactobacillus pentosus of the present invention ( Lactobacillus pentosus ) OKBL-L.PE 1 strain has excellent antioxidant activity, anti-inflammatory activity, skin whitening activity, antibacterial activity and antithrombotic activity, so antibacterial composition, fermented food manufacturing starter, probiotic , a composition for preventing thrombosis, a composition for skin whitening, and a feed additive, etc., can be very usefully used in related industrial fields.

1 is a result of measuring the DPPH free radical scavenging activity of Lactobacillus pentosus isolated in the present invention ( Lactobacillus pentosus ) OKBL-L.PE 1 strain. 0.05% BHT is butylated hydroxytoluene, a synthetic antioxidant used as a positive control, MRSB is MRS medium used as a negative control, and L.FE_O and L.PL_O are L. FE_O and L.PL_O, respectively, Lactobacillus fermentum and It is a culture medium of Lactobacillus plantarum, L.PE_O means the culture medium of the strain of the present invention, L.PE_I means the cells of the strain of the present invention, and L.PE_A means the culture medium of the strain of the present invention + cells .
Figure 2 is a result of measuring the lipoxygenase inhibitory activity of Lactobacillus pentosus ( Lactobacillus pentosus ) OKBL-L.PE 1 strain isolated in the present invention. 0.1% IDM is indomethacin used as a positive control, MRSB is MRS medium used as a negative control, and L.FE_O and L.PL_O are L.FE_O and L.PL_O of Lactobacillus fermentum and Lactobacillus plantarum, respectively, used as a control group. It is a culture medium, L.PE_O means the culture medium of the strain of the present invention, L.PE_I means the cells of the strain of the present invention, and L.PE_A means the culture medium of the strain of the present invention + cells.
3 is a result of measuring the NO production inhibitory ability of Lactobacillus pentosus isolated in the present invention ( Lactobacillus pentosus ) OKBL-L.PE 1 strain. C is distilled water, LPS is lipopolysaccharide, L.FE_O and L.PL_O are the cultures of Lactobacillus fermentum and Lactobacillus plantarum used as comparative groups, respectively, and L.PE_O means the culture medium of the strain of the present invention.
Figure 4 is a result of analyzing the tyrosinase inhibitory activity of the Lactobacillus pentosus isolated in the present invention ( Lactobacillus pentosus ) OKBL-L.PE 1 strain. 0.05% Arbutin is a tyrosinase inhibitor used as a positive control, L.FE_O and L.PL_O are cultures of Lactobacillus fermentum and Lactobacillus plantarum, respectively, used as a comparison group, and L.PE_O is the culture medium of the strain of the present invention means
Figure 5 is a result of measuring the antibacterial activity of Lactobacillus pentosus ( Lactobacillus pentosus ) OKBL-L.PE 1 strain against Escherichia coli ( Escherichia coli ) isolated in the present invention. MRSB is the MRS medium used as a negative control, L.FE_O and L.PL_O are the cultures of Lactobacillus fermentum and Lactobacillus plantarum used as comparative groups, respectively, and L.PE_O means the culture medium of the strain of the present invention.
6 is a result of measuring the antithrombotic activity of Lactobacillus pentosus isolated in the present invention ( Lactobacillus pentosus ) OKBL-L.PE 1 strain. MRSB is the MRS medium used as a negative control, L.FE_O and L.PL_O are the cultures of Lactobacillus fermentum and Lactobacillus plantarum used as comparative groups, respectively, and L.PE_O means the culture medium of the strain of the present invention.

In order to achieve the object of the present invention, the present invention is Lactobacillus pentosus ( Lactobacillus pentosus ) OKBL-L.PE 1 strain whose accession number is KCCM12791P having antioxidant activity, anti-inflammatory activity, skin whitening activity, antibacterial activity and antithrombotic activity provides

In the present invention, a strain having antioxidant activity, anti-inflammatory activity, skin whitening activity, antibacterial activity and antithrombotic activity was isolated from kkakdugi kimchi, and as a result of identification through 16S rRNA sequencing of the finally selected strain, Lactobacillus pentosus ( Lactobacillus pentosus ) was confirmed to be a strain. The present inventor named the final selected strain Lactobacillus pentosus ( Lactobacillus pentosus ) OKBL-L.PE 1 strain and deposited in Korea Center for Microbiology (KCCM) on September 18, 2020 (Accession No.: KCCM12791P) .

The present invention also provides an antibacterial composition for Gram-negative bacteria containing, as an active ingredient, the Lactobacillus pentosus OKBL-L.PE 1 strain, the culture solution thereof, the concentrate of the culture solution, or the dried product thereof, the deposit number of which is KCCM12791P. provides

In the antibacterial composition according to an embodiment of the present invention, the Gram-negative bacteria may be Escherichia coli , but is not limited thereto.

By "antimicrobial" in the present invention is meant the ability to reduce, prevent, inhibit, or eliminate the growth or survival of microorganisms at certain concentrations.

The "antibacterial composition" of the present invention is a composition having an activity to inhibit the growth of microorganisms such as bacteria, and may have the same meaning as antibiotics, which is a generic term for antimicrobial agents, and is used in various fields requiring an antibacterial effect. The form may be included, for example, it may be in the form of pharmaceuticals, quasi-drugs, food additives or feed additives.

The antibacterial composition of the present invention may be the strain or the culture solution of the strain or the concentrate of the culture solution of the strain itself, and may further include commonly used additives such as excipients or diluents necessary for formulation, and of the present invention It may further include an antibacterial substance other than the strain or its culture solution. The mixing amount of the active ingredient may be suitably determined according to the purpose of its use.

The content of the strain of the present invention or the culture solution of the strain or the concentrate of the culture solution of the strain included in the antibacterial composition may be appropriately selected in consideration of the purpose and method of use, for example, based on the entire composition 1 to 100% by weight, preferably 10 to 100% by weight, more preferably 50 to 100% by weight.

The "culture solution" of the present invention includes processed products derived from the culture solution itself, such as the culture solution itself cultured in a liquid medium, and the filtrate (centrifuged supernatant) from which the strain is removed by filtration or centrifugation of the culture solution. In order to remove the culture medium in the culture medium and recover only the concentrated cells, centrifugation or filtration may be performed, and these steps may be performed by those skilled in the art as needed. Concentrated cells can be preserved so as not to lose their activity by freezing or lyophilizing according to a conventional method.

The method of culturing the strain of the present invention may be cultured according to a method commonly used in the art, and is not limited to a particular method.

The present invention also provides a Lactobacillus pentosus ( Lactobacillus pentosus ) OKBL-L.PE 1 strain whose accession number is KCCM12791P, a culture solution thereof, a concentrate of the culture solution, or a starter for manufacturing fermented food containing the dried product as an active ingredient. A composition is provided.

In the present invention, a fermented food production starter (starter) means a preparation or composition containing microorganisms involved in fermentation for the production of fermented food. It is used to provide microorganisms that can grow in fermented foods or microorganisms that can grow as dominant species by adding them during the manufacture of fermented foods. When food is manufactured using the starter for manufacturing fermented food, the quality of the food may be uniformly controlled or a specific purpose may be achieved by the microorganisms included in the starter for manufacturing the fermented food. In the present invention, by using the Lactobacillus pentosus OKBL-L.PE 1 strain or a culture medium thereof as a starter strain, a fermented food having antioxidant activity, anti-inflammatory activity, skin whitening activity, antibacterial activity and antithrombotic activity can be prepared.

In the starter composition for manufacturing fermented food according to an embodiment of the present invention, the fermented food may be traditional kimchi, but is not limited thereto.

The present invention also provides the Lactobacillus pentosus ( Lactobacillus pentosus ) OKBL-L.PE 1 strain of which the accession number is KCCM12791P, a culture solution thereof, a concentrate of the culture solution or a dry product thereof as an active ingredient. A probiotic composition containing as an active ingredient provides

The probiotic composition of the present invention may further include conventional pharmaceutical carriers and excipients in addition to the above active ingredients, and these compositions are heat-dried or freeze-dried according to a conventional probiotic composition preparation method to form a probiotic. can be manufactured and used.

Since the strain of the present invention has various activities as described above, it can be used for preparing probiotic compositions such as food, medicine, cosmetics, and health food.

The present invention also provides the Lactobacillus pentosus ( Lactobacillus pentosus ) OKBL-L.PE 1 strain whose accession number is KCCM12791P, a culture solution thereof, a concentrate of the culture solution or a dried product thereof as an active ingredient for preventing or improving health for thrombosis Provided is a nutraceutical composition.

As used herein, the term “thrombosis” refers to a disease caused by a thrombus, also called thromboembolism, and in particular, refers to a disease in which blood vessels are blocked by a thrombus.

The term "thrombus" refers to the aggregation of blood components consisting mainly of platelets and fibrin surrounding cellular components, intermingled with other formed blood components of the blood, and mainly composed of aggregates of platelets and fibrin.

Lactobacillus pentosus of the present invention ( Lactobacillus pentosus ) OKBL-L.PE 1 strain, the antithrombotic health functional food composition containing the culture medium thereof as an active ingredient has a preventive or ameliorating effect on various diseases, for example, hypertension, Heart disease, stroke, myocardial infarction, etc., but are not limited thereto, and diseases caused by blood clots and platelet aggregation may all be included therein.

The health functional food composition of the present invention may be prepared in any one formulation selected from powder, granule, pill, tablet, capsule, candy, syrup, effervescent tablet and beverage, but is not limited thereto.

When the health functional food composition of the present invention is used as a food additive, the health functional food composition may be added as it is or used together with other foods or food ingredients, and may be appropriately used according to a conventional method. The active ingredient may be used appropriately depending on the purpose of its use (prevention or improvement). However, in the case of long-term intake for the purpose of health control, it can be used in an amount within a range that does not cause any problems in terms of safety. There is no particular limitation on the type of the food. Examples of foods to which the health functional food composition can be added include meat, sausage, bread, chocolate, candy, snacks, confectionery, pizza, ramen, other noodles, gums, dairy products including ice cream, various soups, beverages, tea There are drinks, alcoholic beverages, vitamin complexes, and the like, and includes all health foods in the ordinary sense.

In addition, the health functional food composition of the present invention may be prepared as a food, particularly a functional food. The functional food of the present invention includes ingredients commonly added during food production, for example, proteins, carbohydrates, fats, nutrients and seasonings. For example, when manufactured as a drink, natural carbohydrates or flavoring agents may be included as additional ingredients in addition to the active ingredient. The natural carbohydrates include monosaccharides (eg, glucose, fructose, etc.), disaccharides (eg, maltose, sucrose, etc.), oligosaccharides, polysaccharides (eg, dextrin, cyclodextrin, etc.) or sugar alcohols (eg, , xylitol, sorbitol, erythritol, etc.). As the flavoring agent, natural flavoring agents (eg, taumatine, stevia extract, etc.) and synthetic flavoring agents (eg, saccharin, aspartame, etc.) may be used. In addition to the health functional food composition, various nutrients, vitamins, electrolytes, flavoring agents, coloring agents, pectic acid and its salts, alginic acid and its salts, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohol, carbonic acid It may further contain a carbonation agent and the like used in beverages.

The present invention also provides a cosmetic composition for skin whitening containing, as an active ingredient, the Lactobacillus pentosus OKBL-L.PE 1 strain, the culture solution thereof, the concentrate of the culture solution, or the dried product thereof, the deposit number of which is KCCM12791P. do.

In the cosmetic composition for skin whitening according to an embodiment of the present invention, the Lactobacillus pentosus ( Lactobacillus pentosus ) OKBL-L.PE 1 strain whose accession number is KCCM12791P is excellent in tyrosinase (tyrosinase) inhibitory activity, so the skin It has a whitening effect.

The skin whitening may be characterized by inhibiting tyrosinase, but is not limited thereto.

In the cosmetic composition according to an embodiment of the present invention, the cosmetic composition for skin whitening is a skin, a skin softener, a skin toner, a lotion, a milk lotion, a moisture lotion, a nourishing lotion, a massage cream, a nourishing cream, an eye cream, and a moisture. It may have any one formulation selected from cream, hand cream, essence, nutritional essence, pack, cleansing foam, cleansing water, cleansing cream, body lotion, body cleanser, soap and powder, but is not limited thereto. The cosmetic composition consisting of each of these formulations may contain various bases and additives necessary and appropriate for the formulation of the formulation, and the types and amounts of these components can be easily selected by those skilled in the art.

The cosmetic composition of the present invention may contain at least one active ingredient for skin whitening that exhibits the same or similar function in addition to the active ingredient. Skin lightening active ingredients include kojic acid and its derivatives, arbutin, ascorbic acid and its derivatives, hydroquinone and its derivatives, resorcinol, cycloalkanone, methylenedioxyphenyl alkanol, 2,7-dinitroindazole or plant extracts such as tendril extract, rice extract, and licorice extract, but are not limited thereto.

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

When the formulation of the cosmetic composition of the present invention is a powder or a spray, lactose, talc, silica, aluminum hydroxide, calcium silicate or polyamide powder may be used as a carrier component, and in particular, in the case of a spray, additional chlorofluorohydro It may contain a propellant 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, solvating agent or emulsifying agent is used as a carrier component, for example, water, ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene fatty acid esters of glycol, 1,3-butylglycol oil, glycerol aliphatic esters, polyethylene glycol or 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, ethoxylated isostearyl alcohol, a suspending agent such as polyoxyethylene sorbitol ester and polyoxyethylene sorbitan ester; Microcrystalline cellulose, aluminum metahydroxide, bentonite, agar, or tracanth may be used.

When the formulation of the cosmetic composition of the present invention is surfactant-containing cleansing, aliphatic alcohol sulfate, aliphatic alcohol ether sulfate, sulfosuccinic acid monoester, acetionate, imidazolinium derivative, methyl taurate, sarcosinate as carrier components , fatty acid amide ether sulfate, alkylamidobetaine, fatty alcohol, fatty acid glyceride, fatty acid diethanolamide, vegetable oil, linolin derivative or ethoxylated glycerol fatty acid ester, and the like can be used.

The present invention also provides a feed additive composition containing, as an active ingredient, the Lactobacillus pentosus OKBL-L.PE 1 strain, the culture solution thereof, the concentrate of the culture solution, or the dried product thereof, the accession number of which is KCCM12791P.

The strain is as described above, and may be added as a feed additive composition for the purpose of antioxidant, anti-inflammatory, antibacterial and antithrombotic properties. The feed additive of the present invention corresponds to an auxiliary feed under the Feed Management Act.

The feed additive composition of the present invention may be added to a feed, and "feed" means any natural or artificial diet, one meal, etc., or a component of the one meal diet, suitable for or for the animal to eat, ingest, and digest. can do. The type of feed is not particularly limited, and feed commonly used in the art may be used. Non-limiting examples of the feed include plant feeds such as grains, root fruits, food processing by-products, algae, fibers, pharmaceutical by-products, oils and fats, starches, gourds or grain by-products; and animal feeds such as proteins, inorganic materials, oils and fats, minerals, oils and fats, single cell proteins, zooplankton, or food.

Hereinafter, the present invention will be described in detail by way of Examples. However, the following examples are merely illustrative of the present invention, and the content of the present invention is not limited thereto.

Example 1. Isolation and identification of strains

After the kkakdugi sample was diluted in physiological saline, it was inoculated in LB medium (1% sucrose, 1% yeast extract and 0.15% NaH ₂ PO ₄ ), and the flask containing the medium was incubated at 30° C. for 24 hours. Thereafter, the colonies formed by smearing the culture medium on a solid medium were selected and pure separation was performed. As a result of analyzing the nucleotide sequence (SEQ ID NO: 1) of the 16S rRNA gene of the selected strain, Lactobacillus It was found to be pentosus ( Lactobacillus pentosus ), named Lactobacillus pentosus OKBL-L.PE 1 strain, and deposited at the Korea Center for Conservation of Microorganisms (KCCM) on September 18, 2020 (Accession No.: KCCM12791P).

Example 2. Characterization of selected strains

Selected strains were inoculated into MRS liquid medium, incubated at 37°C for 18 hours, centrifuged at 3,000 rpm for 10 minutes, and the supernatant was taken and named strain culture solution (L.PE_O), and the pellet obtained after centrifugation was ultrasonicated After decomposition (sonication) and centrifugation at 3,000 rpm for 10 minutes, the supernatant was taken and named as a cell (L.PE_I), and after culturing the strain, sonication was performed and centrifuged at 3,000 rpm for 10 minutes, followed by centrifugation at 3,000 rpm for 10 minutes. The solution was taken and named as strain culture solution + cell (L.PE_A). In order to analyze the characteristics of the strain of the present invention, the sugar content and pH of the strain culture medium (L.PE_O), the cell body (L.PE_I), and the strain culture medium + the cell body (L.PE_A) were measured, respectively. Sugar content was expressed by measuring the total soluble solids (Brix) content with a saccharometer (Hand refractormeter, Germany). The pH was expressed as an average value measured three times using a pH meter (SevenCompact™ pH/Ion S220, Switzerland).

As a result, the pH and sugar content of the Lactobacillus pentosus OKBL-L.PE 1 strain of the present invention showed lower values compared to the control, and the strain culture solution (L.PE_O), the cells (L.PE_I) and the culture medium + cells (L) .PE_A) showed different characteristics (Table 1).

pH and sugar content of Lactobacillus pentosus OKBL-L.PE 1 strain Sample pH sugar content control MRS medium 6.69 5.7 control group Lactobacillus Fermentum (L.FE) 4.31 4.3 Lactobacillus plantarum (L.PL) 3.77 4.9 experimental group Lactobacillus pentosus OKBL-L.PE 1 Culture (L.PE_O) 3.83 4.9 Cells (L.PE_I) 3.95 0.2 Culture medium + cells (L.PE_A) 3.75 4.6

Example 3. Antioxidant Activity Assay

The antioxidant activity of Lactobacillus pentosus OKBL-L.PE 1 strain was analyzed according to the method of Blois et al. After dissolving 16 mg of DPPH (2,2-diphenyl-1-picryl-hydrazyl) in 100 ml of ethanol, 100 ml of distilled water was mixed and filtered through Whatman filter paper NO.2 to prepare a reaction solution. DPPH free radical scavenging activity was measured by adding 4.6 ml of the DPPH reaction solution to 0.4 ml of OKBL-L.PE 1 strain culture medium, cells or culture medium + cells, mixing well, and reacting in the dark for 30 minutes with a spectrophotometer (Hitachi U-2900, Japan). ), the absorbance was measured at 528 nm and then substituted into the equation below. At this time, for activity comparison, BHT (Butylated hydroxytoluene), a synthetic antioxidant mainly used as an antioxidant, was added at a concentration of 0.05%, and absorbance was measured in the same manner as in the sample, and used as a positive control.

DPPH free radical scavenging activity (%)=[1-(A/B)x100]

A: Absorbance of sample-added DPPH solution at 528 nm

B: Absorbance of sample-free DPPH solution at 528 nm

The results obtained from the experiment were expressed as the mean and standard error (mean±SE) by a one-way ANOVA test, and the significance test between each experimental group was analyzed through Duncan's multiple range tes.

As a result, it was confirmed that the Lactobacillus pentosus OKBL-L.PE 1 strain of the present invention had excellent antioxidant activity compared to the negative control (MRS medium), and the culture medium (L.PE_O) and the culture medium + cells (L.PE_A). ) was confirmed to have superior antioxidant activity than the bacterial cells of the strain (L.PE_I) (FIG. 1).

Example 4. Anti-inflammatory activity assay

(1) Measurement of lipoxygenase inhibitory activity

Lipoxygenase (lipoxygenase) has insufficient activity in normal cells, but increases in activity due to an increase in leukotrienes when inflammation is induced. After adding 1 ml of 200 mM borate buffer (pH 9.0) to 100 μl of the strain culture medium with OD ₆₀₀ = 1.0, 2 ml of linoleic acid (0.6 mM/ml in ethanol) as a substrate was added and mixed, and then 234 Calibration values were measured in nm. Thereafter, 100 μl of 10,000 unit/ml of lipoxygenase was added to the mixture and reacted at room temperature for 10 minutes, and then absorbance was measured at 234 nm. The positive control was used by dissolving indomethacin (IDM) in ethanol at 0.1% (w/v). Lipoxygenase inhibitory activity (%) was calculated by the following formula.

Lipoxygenase inhibitory activity (%) = {1-(b'-b)/(a'-a)}x100

a: enzyme-free group

a': enzyme addition group

b: enzyme-free and sample-added group

b': enzyme and sample addition group

As a result, it was confirmed that the Lactobacillus pentosus OKBL-L.PE 1 strain of the present invention had significant lipoxygenase inhibitory activity compared to the negative control (MRS medium), and the comparative groups Lactobacillus fermentum and Lactobacillus plantarum. It was confirmed that it was superior to the inhibitory activity of the culture medium (FIG. 2).

(2) NO production inhibition rate measurement

In order to analyze the anti-inflammatory activity of the Lactobacillus pentosus OKBL-L.PE 1 strain, the inhibition rate of NO production in RAW 264.7 cells induced by lipopolysaccharide (LPS) was measured. RAW 264.7 cells were purchased from Korea Cell Line Bank (KCLB, Korea) and cultured in DMEM medium supplemented with 10% FBS and penicillin-streptomycin under conditions of 37° C. and 5% CO ₂ and used for subsequent experiments. After culturing RAW 264.7 cells in a 24-well plate at 1x10 ⁴ cells/well for 24 hours, the strain culture solution of the present invention (L.PE_O) was pretreated with 10% and then treated with LPS (1 μg/ml) and for 24 hours. reacted. Then, 100 μl of the cell culture solution was taken and reacted with a grease reagent (1% sulfonic acid, 0.1% N-1-naphthyl ethylene diamine dihydrochloride, 2.5% phosphoric acid), and absorbance was measured at 546 nm with a microplate reader.

As a result, it was confirmed that the Lactobacillus pentosus OKBL-L.PE 1 strain culture solution (L.PE_O) of the present invention has an effect of inhibiting NO production by LPS treatment (FIG. 3).

Example 5. Skin whitening activity assay

In order to analyze the skin whitening activity of the Lactobacillus pentosus OKBL-L.PE 1 strain, the mushroom tyrosinase inhibitory activity was measured in vitro . In a 1.5 ml plastic cuvette, 0.3 ml of 2.5 mM L-DOPA (3,4-dihydroxyphenylalanine), 0.1 ml of the strain culture solution of the present invention (L,PE_O), and 0.1 M phosphate buffer solution (pH 6.8) were mixed to 25 The reaction was carried out at ℃. After that, 0.05 ml of 1,380 units/ml mushroom tyrosinase (Sigma, USA) was added, reacted at 25° C. for 2 minutes, absorbance was measured at 470 nm using a spectrophotometer, and then tyrosinase inhibitory activity was obtained by substituting the following formula calculated. As a control, a sample dissolution solution containing no sample was used, and as a positive control, 0.05% arbutin, known as a tyrosinase inhibitor, was used.

Tyrosinase inhibitory activity (%)=100-[(A-B)/A]x100

A: The difference in absorbance between 0.5 and 1 minute of the reaction solution without the sample

B: The difference in absorbance between 0.5 and 1 minute of the reaction solution containing the sample

As a result, it was confirmed that the Lactobacillus pentosus OKBL-L.PE 1 strain culture solution (L.PE_O) of the present invention had a superior tyrosinase inhibitory activity than the positive control (0.05% arbutin), and the comparative group, Lactobacillus fur It was confirmed that the Lactobacillus pentosus OKBL-L.PE 1 strain culture (L.PE_O) had skin whitening activity by confirming that the tyrosinase inhibitory activity was superior to that of the culture medium of Mentum and Lactobacillus plantarum (Fig. 4). ).

Example 6. Antimicrobial Activity Assay

To analyze the antibacterial activity of the Lactobacillus pentosus OKBL-L.PE 1 strain of the present invention, Escherichia coli, E. coli O157:H7 (ATCC 43894) from the American Type Culture Collection Antibacterial activity was measured using the disc diffusion method. After culturing E. coli in LB (Luria-Bertani) liquid medium at 37° C. for 24 hours, 200 μl of the culture solution was spread on LB solid medium and a sterilized 10 mm paper disk was placed on the solid medium. Thereafter, the Lactobacillus pentosus OKBL-L.PE 1 strain culture solution (L.PE_O) was treated on the paper disk and cultured at 37° C. for 24 hours, and then a clear zone around the paper disk was observed.

As a result, it was confirmed that the Lactobacillus pentosus OKBL-L.PE 1 strain culture solution (L.PE_O) of the present invention has excellent antibacterial activity against E. (FIG. 5).

Example 7. Antithrombotic Activity Assay

In order to analyze the antithrombotic activity of the Lactobacillus pentosus OKBL-L.PE 1 strain of the present invention, the fibrinogen dissolving ability to coagulate blood was measured. 0.6% fibrinogen (Sigma, USA) was added to 0.05 M sodium borate buffer (pH 7.5) and dissolved at 37° C. for 1 hour, then dispensed 10 ml each in a Petri dish, and 1,000 units of thrombin (Sigma, USA) was diluted to a concentration of 40 units/ml, treated in a Petri dish to make a total of 20 units, and shaken well to mix evenly. Thereafter, it was hardened for about 15 minutes to form a fibrin clot and used in the experiment. A paper disk is placed on a Petri dish in which the fibrin clot is formed, and 50 μl of a strain culture solution (L.PE_O) having an OD ₆₀₀ = 1.0 is added dropwise and reacted in an incubator at 37° C., followed by dissolution of fibrin (clear zone) observed.

As a result, it was confirmed that the culture medium of Lactobacillus pentosus OKBL-L.PE 1 strain (L.PE_O) of the present invention had superior antithrombotic activity than the culture medium of Lactobacillus fermentum and Lactobacillus plantarum, which are comparative groups (FIG. 6). ).

Korea Microorganism Center (Overseas) KCCM12791P 20200918

<110> OKBIOLAND Co., Ltd. <120> Lactobacillus pentosus OKBL-L.PE 1 strain having antioxidant activity, anti-inflammatory activity, skin whitening activity, antimicrobial activity against pathogenic microorganisms and anti-thrombotic activity and uses thereof <130> PN20310 <160> 1 <170> KoPatentIn 3.0 <210> 1 <211> 1452 <212> DNA <213> Lactobacillus pentosus <400> 1 ccagcgctgc taactgcagt cgacgaactc tggtattgat tggtgcttgc tcatgattta 60 catttgagtg agtggcgaac tggtgagtaa cacgtgggaa acctgcccag aagcggggga 120 taacacctgg aaacagatgc taataccgca taacaacttg gaccgcatgg tccgagtttg 180 aaagatggct tcggctatca cttttggatg gtcccgcggc gtattagcta gatggtgggg 240 taacggctca ccatggcaat gatacgtagc cgacctgaga gggtaatcgg ccacattggg 300 actgagacac ggcccaaact cctacgggag gcagcagtag ggaatcttcc acaatggacg 360 aaagtctgat ggagcaacgc cgcgtgagtg aagaagggtt tcggctcgta aaactctgtt 420 gttaaagaag aacatatctg agagtaactg ttcaggtatt gacggtattt aaccagaaag 480 ccacggctaa ctacgtgcca gcagccgcgg taatacgtag gtggcaagcg ttgtccggat 540 ttattgggcg taaagcgagc gcaggcggtt ttttaagtct gatgtgaaag ccttcggctc 600 aaccgaagaa gtgcatcgga aactgggaaa cttgagtgca gaagaggaca gtggaactcc 660 atgtgtagcg gtgaaatgcg tagatatatg gaagaacacc agtggcgaag gcggctgtct 720 ggtctgtaac tgacgctgag gctcgaaagt atgggtagca aacaggatta gataccctgg 780 tagtccatac cgtaaacgat gaatgctaag tgttggaggg tttccgccct tcagtgctgc 840 agctaacgca ttaagcattc cgcctgggga gtacggccgc aaggctgaaa ctcaaaggaa 900 ttgacggggg cccgcacaag cggtggagca tgtggtttaa ttcgaagcta cgcgaagaac 960 cttaccaggt cttgacatac tatgcaaatc taagagatta gacgttccct tcggggacat 1020 ggatacaggt ggtgcatggt tgtcgtcagc tcgtgtcgtg agatgttggg ttaagtcccg 1080 caacgagcgc aacccttatt atcagttgcc agcattaagt tgggcactct ggtgagactg 1140 ccggtgacaa accggaggaa ggtggggatg acgtcaaatc atcatgcccc ttatgacctg 1200 ggctacacac gtgctacaat ggatggtaca acgagttgcg aactcgcgag agtaagctaa 1260 tctcttaaag ccattctcag ttcggattgt aggctgcaac tcgcctacat gaagtcggaa 1320 tcgctagtaa tcgcggatca gcatgccgcg gtgaatacgt tcccgggcct tgtacacacc 1380 gcccgtcaca ccatgagagt ttgaacaccc aaagtcgggg ggaaccttta gaaccaccgc 1440 tagggacgag gg 1452

Claims (8)

Antioxidative activity, anti-inflammatory activity, skin whitening activity, Escherichia coli ( Escherichia coli ) Lactobacillus pentosus ( Lactobacillus pentosus ) OKBL-L.PE 1 strain with an accession number KCCM12791P having antibacterial activity and antithrombotic activity. The Lactobacillus pentosus of claim 1 ( Lactobacillus pentosus ) OKBL-L.PE 1 strain, a culture solution thereof, a concentrate of the culture solution or a dried product thereof as an active ingredient. An antibacterial composition against Escherichia coli . delete The Lactobacillus pentosus of claim 1 ( Lactobacillus pentosus ) OKBL-L.PE 1 strain, a culture solution thereof, a concentrate of the culture solution, or a starter composition for manufacturing fermented food containing the dried product as an active ingredient. The Lactobacillus pentosus of claim 1 ( Lactobacillus pentosus ) OKBL-L.PE 1 strain, a culture solution thereof, a concentrate of the culture solution, or a probiotic composition for producing a fermented food containing a dried product thereof as an active ingredient. Claim 1 Lactobacillus pentosus ( Lactobacillus pentosus ) OKBL-L.PE 1 strain, a culture solution thereof, a concentrate of the culture solution, or a health functional food composition for preventing or improving thrombosis containing the dried product as an active ingredient. The Lactobacillus pentosus of claim 1 ( Lactobacillus pentosus ) OKBL-L.PE 1 strain, a culture solution thereof, a concentrated solution of the culture solution or a dry product thereof as an active ingredient. A cosmetic composition for skin whitening. The Lactobacillus pentosus of claim 1 ( Lactobacillus pentosus ) OKBL-L.PE 1 strain, a culture solution thereof, a concentrate of the culture solution, or a feed additive composition containing a dried product thereof as an active ingredient.

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