KR101938801B1 - Lactobacillus sakei WiKim0066 with antioxidant and anti-aging effects - Google Patents

Abstract

The present invention relates to a novel Lactobacillus sakei strain, an antioxidant cosmetic composition containing the same, and an anti-aging cosmetic composition containing the same. The Lactobacillus sakei WiKim0066 strain according to the present invention has excellent UVB resistance through the survival rate of UVB-stressed skin cells, and has excellent antioxidant and anti-aging effects, thus being useful as a cosmetic material.

Description

Antioxidant and anti-aging effects Lactobacillus saccharum Wikiim0066 {Lactobacillus sakei WiKim0066 with antioxidant and anti-aging effects}

The present invention relates to Lactobacillus saccharum WiKim0066 having antioxidant and anti-aging activity.

As the lifespan of humans becomes longer, aging is progressing globally, and it is known that the elderly over 80 years old are the fastest growing. In particular, Korea's aging population is regarded as the highest in the world. Therefore, interest in aging is also increasing. The skin of the human body is physically and biochemically defective, but photoaging is progressed due to chronic light exposure. Deep wrinkles, skin relaxation, capillary vasodilation, increased epidermal and dermal thickness, destruction of collagen structure, and elastic fibrosis. Recently, the intensity and intensity of ultraviolet rays are rapidly increased due to the destruction of the ozone layer caused by environmental pollution, so that skin damage is also increasing. Efforts to prevent skin damage and photoaging and to develop a therapeutic method have been made in various fields.

Cosmetic materials should be balanced in safety, efficacy, and physical properties. In addition, securing economical efficiency is also an important factor. Cosmetic materials are developed by optimizing the permeation of skin, inhibition of aging, physiological activity, safety and physical properties by using various development methods, natural material extraction and fermentation by microorganisms. As the boundaries between cosmetics and pharmaceuticals have collapsed, various cosmetic products have continued to show strong growth due to the combination of functional raw materials and biomaterials that combine the stability of cosmetics and the effectiveness of pharmaceuticals. As the lactic acid bacteria are highlighted as a health functional material and new functions of lactic acid bacteria are studied, the cosmetics containing kimchi fermented lactic acid bacteria metabolites or lactic acid bacteria themselves are called bio cosmetics. As a similar technology, it has been confirmed that the cultured Lactobacillus culture solution has the effect of preventing wound healing and skin infections in Japan and actively researching the development of cosmetics using lactic acid bacteria.

Major components such as lactic acid, lactose, amino acid, and phosphate in the culture medium of lactic acid bacteria are similar to the keratin components of the skin and have been utilized as cosmetic materials having functionalities such as skin moisturizing effect, antioxidative effect and skin microflora controlling effect. Functional cosmetics using probiotic Lactobacillus as well as health functional foods are emerging as blue oceans in the bio-industry market in response to changing consumer desires.

However, no studies have been reported on the lactic acid bacteria of Kimchi for cosmetic materials, which have both UV blocking, antioxidant and anti-aging effects.

Korean Patent Publication No. 2014-0105976

Accordingly, the present inventors have completed the present invention by separating new lactic acid bacteria having both UV-blocking, antioxidant and anti-aging activity simultaneously.

It is an object of the present invention to provide a strain of Lactobacillus sakei WiKim0066 strain of Accession No. KACC92194P and its use.

In order to accomplish the above object, the present invention provides a method for producing Lactobacillus strain Lactobacillus strain KAKC 92194P sakei WiKim0066) strain.

The present invention also relates to an antioxidant cosmetic composition comprising at least one selected from the group consisting of Lactobacillus saccharum WiKim0066 (Accession No. KACC 92194P) strain, a disruption product thereof, a culture thereof, a concentrate of the culture product and a dried product of the culture Lt; / RTI >

The present invention also relates to an anti-aging cosmetic composition comprising at least one member selected from the group consisting of a strain of Lactobacillus saccharum WiKim0066 (Accession No. KACC 92194P), a disruption thereof, a culture thereof, a concentrate of the culture and a dried product of the culture Lt; / RTI >

The present invention also provides a skin external preparation comprising as an active ingredient at least one selected from the group consisting of a strain of Lactobacillus casei KIKC0066 (Accession No. KACC 92194P), a lysate thereof, a culture thereof, a concentrate of the culture and a dried product of the culture to provide.

Hereinafter, the configuration of the present invention will be described in detail.

The present invention relates to Lactobacillus strain < RTI ID = 0.0 > sakei WiKim0066) strain, Accession No. KACC 92194P).

Lactobacillus saccharum WiKim0066 (Accession No. KACC 92194P) according to the present invention is a novel strain of Lactobacillus saca derived from kimchi. Although the lactobacillus sacrifice strain WiKim0066 in the present invention is isolated and identified in kimchi, the supply route is not limited thereto.

As a result of 16S rRNA sequencing for microbial identification and classification, the lactic acid bacteria strain having excellent antioxidative and / or anti-aging effect isolated from kimchi through the following examples was found to have the nucleotide sequence of SEQ ID NO: 1. The homology of the nucleic acid sequence was confirmed by a blast search program provided by NCBI GenBank (http://www.ncbi.nin.gov). As a result, it was confirmed that the NBRC15893 strain of the Lactobacillus casei strain 99%.

Thus, the microorganism of the present invention having the 16S rRNA nucleotide sequence of SEQ ID NO: 1 was incubated with Lactobacillus < RTI ID = 0.0 > sakei WiKim0066) and deposited on October 16, 2017 (Accession No. KACC 92194P) at the National Institute of Agricultural Science and Technology.

Lactobacillus saccharum WiKim0066 of the present invention is a gram-positive bacterium and is a facultive anaerobe capable of growing under both aerobic and anaerobic conditions, taking the form of bacillus. In addition, it was confirmed that the skin cell survival rate against UVB stress was excellent, and the ultraviolet screening, antioxidative and anti-aging effects were excellent.

As used herein, the term 'antioxidant' means an application for preventing oxidation through an action of inhibiting or eliminating active oxygen in an unstable state of oxygen.

As used herein, the term " anti-aging "means an application for preventing, delaying or improving the aging phenomena caused by internal factors including genetic factors and external factors including ultraviolet rays. For example, it means preventing, delaying, improving or alleviating the phenomenon of skin changes due to aging such as reduction of skin elasticity, wrinkle formation, increase in depth or number of wrinkles, and dryness.

The present invention also provides an antioxidant composition comprising at least one selected from the group consisting of a strain of Lactobacillus saccharum WiKim0066 (Accession No. KACC 92194P), a disruption thereof, a culture thereof, a concentrate of the culture and a dried product of the culture, / ≪ / RTI > anti-aging cosmetic composition.

The Lactobacillus casei WiKim0066 (Accession No. KACC 92194P) strain may be derived from kimchi.

In addition, the Lactobacillus casei WiKim0066 (Accession No. KACC 92194P) strain may contain the 16s rRNA of SEQ ID NO: 1.

The cosmetic composition may be provided in all formulations suitable for topical application. Lotion such as soft lotion or nutrition lotion; Lotion such as facial lotion or body lotion; Creams such as nutritional creams, moisture creams or eye creams; essence; Makeup ointment; spray; Gel; pack; Sunscreen; Makeup base; A foundation such as a liquid type, a solid type or a spray type; powder; Makeup removers such as cleansing creams, cleansing lotions, cleansing oils; Detergents such as cleansing foams, soaps, body wash, and the like.

The cosmetic composition may contain, in addition to the above-mentioned substances, other ingredients which can give synergistic effects to the main effect, so long as they do not impair the main effect. The cosmetic composition according to the present invention may include a material selected from the group consisting of vitamins, high molecular weight peptides, high molecular weight polysaccharides and sphingo lipids. The cosmetic composition according to the present invention may also contain a moisturizing agent, an emollient agent, a surfactant, an ultraviolet absorber, an antiseptic, a bactericide, an antioxidant, a pH adjuster, an organic and inorganic pigment, a fragrance, . The compounding amount of the above components can be easily selected by a person skilled in the art within the range not impairing the object and effect of the present invention, and the amount thereof may be 0.01 to 10% by weight, specifically 0.01 to 5% by weight based on the total weight of the composition have.

The present invention also includes an external preparation for skin comprising, as an active ingredient, at least one selected from the group consisting of a strain of Lactobacillus saccharum WiKim0066 (Accession No. KACC92194P), a disruption thereof, a culture thereof, a concentrate of the culture and a dried product of the culture .

The Lactobacillus casei WiKim0066 (Accession No. KACC 92194P) strain may be derived from kimchi.

In addition, the Lactobacillus casei WiKim0066 (Accession No. KACC 92194P) strain may contain the 16s rRNA of SEQ ID NO: 1.

When the composition is used as the external preparation for skin, it may further contain at least one selected from the group consisting of fatty substances, organic solvents, solubilizers, thickening and gelling agents, softening agents, antioxidants, suspending agents, stabilizing agents, foaming agents, Or any of those commonly used in nonionic emulsifiers, fillers, sequestering and chelating agents, preservatives, vitamins, barrier agents, wetting agents, essential oils, dyes, pigments, hydrophilic or lipophilic active agents, lipid vesicles or dermatological topical agents And may contain adjuvants conventionally used in the field of dermatology, such as other ingredients. The components can also be introduced in amounts commonly used in the field of dermatology.

When the active ingredient is provided as an external preparation for skin, it may have a formulation such as, but not limited to, ointments, patches, gels, creams or sprays.

In one embodiment of the present invention, the external preparation for skin may contain the active ingredient in an amount of 0.01 wt% to 10 wt% (preferably 0.001 wt% to 5 wt%) based on the total weight of the external preparation for skin.

The new strain according to the present invention is excellent in UVB resistance through the survival rate of skin cells against UVB stress and is useful as a cosmetic material to be applied to skin using Lactobacillus skeei WiKim0066 strain having excellent antioxidative and anti- Lt; / RTI >

Figure 1 shows the 16S rRNA gene sequence of Lactobacillus sacrifice strain WiKim0066.
Fig. 2 shows a phylogenetic analysis result of Lactobacillus sacrifice strain WiKim0066.
Figure 3 shows the cytotoxicity of HaCaT skin cells on the treatment of lactic acid bacteria culture.
Fig. 4 shows the cytotoxicity of HaCaT skin cells on the treatment of lactic acid bacterium disruption.
Fig. 5 shows skin cell survival rate of lactic acid bacteria derived from Kimchi against UV stress.
6 shows the results of measurement of DPPH free radical scavenging activity (antioxidative effect) by the culture broth and disintegration treatment of Kimchi lactic acid bacteria.
Fig. 7 shows MMP-1 activation as an anti-aging index by the treatment of disruption of Kimchi lactic acid bacteria.
Fig. 8 shows the measurement results of UVB-induced MMP-9 and MMP-2 activation inhibition (anti-aging effect).
Fig. 9 shows the ability of Kimchi lactic acid bacteria to protect the skin cells by the culture broth and disruption treatment.

Hereinafter, the present invention will be described in detail with reference to examples. The following examples illustrate the invention and are not intended to limit the scope of the invention.

Example  One: Lactobacillus Sakai WiKim0066  Isolation and Identification of Strain

The cabbage kimchi samples were taken aseptically and grinded with a blender, diluted with 0.85% NaCl in a sterile filter bag, and stomached for 1 minute. After dilution, 100 μl of the diluted suspension was plated on MRS agar plate and cultured in an anaerobic incubator using anaerobic system gaspack at 30 ° C for 48 hours. After culture, different strains of lactic acid bacteria were used depending on the type of colonies.

The isolated lactic acid bacteria were identified through 16S rRNA sequencing [FIGS. 1 and 2].

The isolated strain, WiKim0066, was purified and purified by MRS plate, and then subjected to 16S rRNA sequencing using a universal primer set of 27f and 1492r as a primer.

As a result, a total sequence of 1,430 bp was determined using 16S rRNA of the isolated strain WiKim0066, and the results are shown in SEQ ID NO: 1.

Based on the nucleotide sequence, the homology of the nucleotide sequence was checked by the Blast program (http://www.ncbi.nlm.nhi.gov) for the information registered in the GeneBank database. As a result, Lactobacillus sacrifice strain NBRC15893 ( Lactobacillus sakei NBRC15893) and 99% homology to the Lactobacillus sake.

The selected strain, Lactobacillus sake, was named Lactobacillus saccharum WiKim0066 and deposited with the National Institute of Agricultural Science and Technology on October 16, 2017 and granted accession number KACC 92194P.

Example  2: Preparation of lactic acid culture broth and disruption liquid sample

Kimchi lactic acid bacteria culture solution, and lactic acid bacteria-derived lysate of the six seed culture in MRS agar medium to prepare lactic acid bacteria, Lactobacillus brevis WiKim47 (Lactobacillus brevis , Pediococcus penisusiusius WiKim20 ( Pediococcus pentosaceus ) Lactobacillus plantarum WiKim18 ( Lactobacillus plantarum , Lactococcus lactis , Lactobacillus casei WKim0066 ( Lactobacillus sakei WIKIM0066), Leukonostomycesenteroids WiKim19 ( Leuconostoc mesenteroides ) were inoculated in a 15 ml tube containing 5 ml of MRS broth as a growth medium. 2% (v / v) was inoculated in the same sterilization medium and incubated under the same conditions as the previous step. Finally, to obtain a large amount of culture medium, 20 ml of MRS liquid medium was placed in a 50 ml tube, sterilized in the same conditions as described above, and inoculated in the medium at 2% (v / v) And cultured until 600 at 600 nm to obtain a culture. The number of lactic acid bacteria was measured by 3M Petrifilm by decidual dilution method. As a result, 6 kinds of lactic acid bacteria were similarly 2 to 3 × 10 ⁹ CFU / ml, and 10 ml of the culture was centrifuged at 4,000 rpm for 20 minutes at 4 ° C.

In the case of the lactic acid bacteria culture solution, the centrifuged supernatant was removed with a 0.22 μm syringe filter (sartorius stedim biotech), and the polar substances present in the sample were removed using a Sep-Pak C18 cartridge (Waters) Me-OH < / RTI > After the methanol was blown off at 60 ° C for 2 hours using an Integrated Speed-Vac (SPD2010, Thermo Fisher), the cells were dissolved in 1 ml of serum-free DMEM medium and diluted to various concentrations to determine appropriate dilution factors. And samples were stored at -20 ° C.

After centrifugation, the cells were washed twice with sterilized water, and 1 ml of distilled water was added. The beads were added to the beads and subjected to beating on 20 sec, off 5 min for 5 replicates (FastPrep-24, MP) Lt; / RTI > The cell debris was removed by centrifugation at 4,000 rpm for 20 minutes at 4 ° C and the supernatant was collected in a sample using a 0.22 μm syringe filter (sartorius stedim biotech) and a Sep-Pak C18 cartridge (Waters) Existing polar material was removed and eluted with 1 ml of 100% Me-OH. After the methanol was blown off at 60 ° C for 2 hours using an Integrated Speed-Vac (SPD2010, Thermo Fisher), the cells were dissolved in 1 ml of serum-free DMEM medium and diluted to various concentrations to determine appropriate dilution factors. And samples were stored at -20 ° C.

The value can be cultured lactic acid bacteria until a 1.0 in OD 600nm at 30 ℃ thermostat of kimchi-derived lactic acid bacteria under anaerobic conditions in decimal dilution method to confirm and plated on 3M Petrifilm a result, 6 kinds of lactic acid bacteria similar to 2 ~ 3 X 10 ⁹ CFU / ml.

Measurement of the number of lactic acid bacteria living bacteria using 3M Petrifilm (CFU / ml) LABs L. b P. p L. p Lac . l WiKim0066 Leu . m 10 ^-8 21 26 25 24 22 32 CFU / ml 2 X 10 ⁹ 2 X 10 ⁹ 2 X 10 ⁹ 2 X 10 ⁹ 2 X 10 ⁹ 3 X 10 ⁹

Example  3: Cytotoxicity measurement of culture and broth of lactic acid bacteria MTT Assay )

The cell lines used for the experiments were 10% (v / v) of Dulbecco's modified Eagle medium (DMEM, ATCC), a HaCaT cell line (purchased from AddexBio, T0020001), a human keratinocyte skin, and a B16F10 cell line derived from mouse melanoma cells / v) fetal bovine serum (FBS, Gibco BRL) and 100 U / ml penicillin / streptomycin were added and incubated at 37 ° C in a 5% CO ₂ incubator (MCO-18AC, SANYO). Cells were washed lightly with PBS for cell desorption when 80% of a 100 × 20 mm cell culture dish (eppendorf) grew, then treated with 0.25% typsin-EDTA (Gibco) and left at 37 ° C for 2 minutes Cells were harvested. Subculture was performed every 2 to 3 days.

To determine the degree of cytotoxicity of the samples, we used the in vitro toxicology assay kit (Sigma) using Mosmann (1983) and Kotnik (1990). HaCaT cells were seeded at a density of 2 × 10 ⁴ cells / well in 96-well plates with 100 μl of medium. After incubation at 37 ° C in a 5% CO ₂ incubator for 24 hours, the medium was discarded and washed with PBS. free DMEM medium was treated with 100 μl of each sample at various concentrations ranging from 0.5X to 2X for 24 hours. After cultivation, the culture broth and disruption solution of lactic acid bacteria were removed, and 100 μl of serum-free DMEM medium and 10 μl of 5 mg / ml MTT dissolved in serum-free DMEM medium were added to each well and shaded with aluminum foil during time 37 ℃, and cultured at 5% CO _2. After removing the culture medium and adding 100 μl of MTT Solubilization Solution, the reaction was carried out for 20 min at room temperature for 20 min until MTT formazan crystals were dissolved and then absorbance was measured at 570 nm using an ELISA reader.

This study was conducted to investigate the cytotoxicity of lactic acid bacteria derived from kimchi on the culture medium and disruption treatment. As shown in FIGS. 3 and 4, the cell viability was slightly inhibited at the concentration of 2X in both the culture medium and the disruption solution of the lactic acid bacteria in HaCaT skin cells, and the cell survival rate was increased with almost no cytotoxicity at 1X concentration. Based on these results, skin efficacy tests were conducted using culture broth and disintegration liquid samples of 1X concentration of lactic acid bacteria.

Example  4: To skin cells  About UV  Establishment of treatment conditions and UVB  Measurement of skin cell survival rate for stress

To establish treatment conditions for UV stress (UVB, 312 nm) on skin cells (HaCaT cells), HaCaT cells were first seeded at a density of 2 × 10 ⁴ cells / well in a 96-well plate and incubated at 37 ° C in a 5% After incubation for 24 hours, the medium was discarded, and the cells were washed with PBS. Then, 20 μL of PBS was added and UVB 10, 20, 30 mJ / cm ² (UV-6-LM, 312 nm, VILBER LOURMAT) was irradiated in a CN- PBS was discarded, washed twice with fresh PBS, and incubated for 0-24 hours. After the culture was completed, the culture medium was removed and cell viability was measured using an in vitro Toxicology Assay kit (Sigma).

For 24 hours in a first, dispensing the HaCaT cells in a 96 well plate 2 density of × 10 ⁴ cell / well and then, the incubator _{(37 ℃, 5% CO 2} ) in order to measure the skin cell viability of kimchi-derived lactic acid bacteria for the UVB stress After incubation, the medium was discarded and the cells were washed with PBS. After 20 μl of PBS was added, the cells were irradiated with UVB 30 mJ / cm ² in a CN-6 cabinet. PBS was discarded and washed twice with fresh PBS. Liquid 1X samples were treated with 100 ㎕ each well and cultured for 18 hours. Subsequently, the medium was discarded and washed with PBS. Cell viability was measured using an in vitro toxicology assay kit (Sigma).

The survival rate of HaCaT skin cells was measured by UVB ultraviolet rays (30 mJ / cm ² ) and cultured for 18 hours to induce UV stress. The survival rate of HaCaT skin cells after treatment with Lactobacillus sakei The cell viability and disruption of WiKim0066 increased cell viability by 195% and 219%, respectively (Fig. 5).

Example  6: DPPH  Measurement of free radical scavenging activity (antioxidant effect)

Six Kimchi microorganisms, Lactobacillus brevis WiKim47 ( Lactobacillus brevis , Pediococcus penisusiusius WiKim20 ( Pediococcus pentosaceus ) Lactobacillus plantarum WiKim18 ( Lactobacillus plantarum ), Lactococcus lactis WiKim48 ( Lactococcus lactis ), Lactobacillus casei WiKim0066 ( Lactobacillus sakei ), Leuconostoc mesenteroids WiKim 19 ( Leuconostoc mesenteroides ) were cultured at 2 to 3 x 10 ⁹ CFU / ml, and 10 ml of the culture was centrifuged at 4,000 rpm for 20 minutes at 4 ° C. After centrifugation, the culture supernatant was prepared by 0.22 μm syringe filter (sartorius stedim biotech). DPPH (Sigma) was dissolved in methanol at a concentration of 0.2 mM. 1 ml of the DPPH solution and 1 ml of the culture solution were mixed and reacted in the dark for 30 minutes, and the absorbance was measured at 517 nm. L-ascorbic acid 0.5% (w / v) (Sigma) was used as a positive control and MRS broth, a lactic acid culture medium, was used as a negative control. The measured value was calculated as DPPH free radical scavenging activity (%) of the following formula (1).

The DPPH free radical scavenging activity (antioxidative effect), one of skin health related indicators, was measured by the culture of lactic acid bacteria derived from kimchi. As a result, Lactobacillus sakei WiKim0066 showed 79% DPPH free radical scavenging activity (Fig. 6).

Example  7: Confirmation of anti-aging activity

One) UVB  Inductive MMP -1 activation inhibition measurement

HaCaT cells were cultured in a 24-well plate at a density of 1 × 10 ⁵ cells / well, and cultured in an incubator (37 ° C., 5% CO ₂ ) for 24 hours. The medium was discarded and washed with PBS. were put in culture for 18 hours CN-6 were irradiated UVB 20 mJ / cm ² in the cabinet away with PBS and then by the semi-skin cells treated lactic acid bacteria lysate samples washed twice with 1X PBS treated by new 1㎖ each well. As a positive control, 0.5% ascorbic acid was used. 100 μl of the culture supernatant was then measured using a 96-well plate of Amersham Matrix Metalloproteinase-1 (MMP-1), Human, Biotrak ELISA System (RPN2610).

2) Gelatin Zymography  ( MMP -9) activation inhibition measurement

HaCaT cells were seeded at a density of 3 × 10 ⁵ cells / well in a 12-well plate, and cultured in an incubator (37 ° C., 5% CO ₂ ) for 24 hours. The medium was discarded and washed with PBS. and a put process CN-6 were irradiated UVB 20 mJ / cm ² in the cabinet discard the PBS by the quasi following skin cells treated lactic acid bacteria culture fluid and the lysate 1X samples washed twice with fresh PBS to each well 1ml were incubated for 72 hours. . As a positive control, 1 μM of retinoic acid was used. The culture supernatant was mixed 1: 1 with 2X Zymogram sample buffer (Bio-rad, 1610764) to prepare samples. Each sample was electrophoresed at 120 ° C for 90 minutes at 4 ° C using 10% zymogram gel (gelatin, Bio-rad, 1611167). Then, the gel was reacted with 100 ml of zymogram renaturing buffer (Bio-rad, 1610765) twice for 30 minutes to remove the SDS. Then, the gel was washed with distilled water for a while, and then 100 ml of zymogram development buffer (Bio-rad, 1610766) And reacted at 37 캜 for 24 hours. After completion of the reaction, the gel was stained with 100 ml of coomassie blue R-250 staining solution (Bio-rad, 1610436) for 30 minutes and then washed with 100 ml of coomassie blue R-250 destain solution (Bio-rad, 1610438) (72 kDa) and MMP-9 (92 kDa), respectively.

Skin exposed to ultraviolet light is known to increase the expression of Matrix metalloproteinases (MMPs), the degradation enzyme of extracellular matrix proteins. Among them, MMP-1 degrades collagen type 1 and type 3, and gelatinases MMP-2 and MMP-9 decompose collagen fragments degraded by MMP-1 more finely and use it as an important biomarker of skin aging . Therefore, the smaller the amount of expression, the higher the anti-aging effect (Fig. 8). In the anti-aging test, the amount of UVB-induced MMP-1 expression was measured by irradiating the skin cell (HaCaT cell) with UVB ultraviolet ray at a dose of 30 mJ / cm ^{2. As} a result, Lactobacillus sakei WiKim0066 confirmed that the activity of MMP-1 was reduced by 87% (Fig. 7).

Example  8: Hoechst  33258 Skin cells using dyeing method Protection ability  Research

HaCaT cells were seeded at a density of 1 × 10 ⁵ cells / well on a 12-well plate, and cultured in an incubator (37 ° C., 5% CO ₂ ) for 24 hours. After washing with PBS, 100 μl of PBS was added and irradiated with UVB 20 mJ / cm ² in a CN-6 cabinet. PBS was discarded and washed twice with fresh PBS. Then, And cultured for 18 hours. After incubation, the cells were fixed by reacting in a 3.7% (v / v) formaldehyde solution at 37 ° C for 10 minutes. Cells were washed twice with cold PBS and 0.5 ml of Hoechst 33258 solution (Sigma, 94403) at a concentration of 5 μg / ml was added to each well. Cells were stained for 1 hour in an incubator (37 ° C, 5% CO ₂ ) Respectively. One or two drops of ProLong® Gold antifade reagent (Invitrogen, P36934) were then placed on the slide glass. Samples were observed and photographed using ZEISS LSM 510 laser fluorescence microscope (Carl Zeiss, Inc., Thornwood, NY).

For the morphological analysis of Kimchi lactic acid bacteria to protect against cell damage caused by UV treatment, the nucleated DNA was stained with Hoechst 33258 and the degree of damage of the cells was observed by optical focusing fluorescence microscope. The nuclei of the UV treated control were damaged and fragmented and showed strong fluorescence whereas Lactobacillus In the culture medium of sakei WiKim0066 and the disruption solution treatment, it was confirmed that the morphology was constant without damaging the nucleus like the control without UV treatment (Fig. 9).

Lactobacillus skeeti WiKim0066 cultured in MRS broth for 24 hours showed excellent UVB resistance through survival rate of UVB-stressed skin cells and showed the best antioxidative and anti-aging effects.

Agricultural Biotechnology Research Institute KACC92194 20171016

<110> Korea Food Research Institute <120> Lactobacillus sakei WiKim0066 <130> P17R10C1072 <160> 1 <170> KoPatentin 3.0 <210> 1 <211> 1430 <212> DNA <213> Unknown <220> <223> Lactobacillus sakei WiKim0066 <400> 1 tgcagtcgaa cgcactctcg tttagattga aggagcttgc tcctgattga taaacatttg 60 agtgagtggc ggacgggtga gtaacacgtg ggtaacctgc cctaaagtgg gggataacat 120 ttggaaacag atgctaatac cgcataaaac ctaacaccgc atggtgtagg gttgaaagat 180 ggtttcggct atcactttag gatggacccg cggtgcatta gttagttggt gaggtaaagg 240 ctcaccaaga ccgtgatgca tagccgacct gagagggtaa tcggccacac tgggactgag 300 acacggccca gactcctacg ggaggcagca gtagggaatc ttccacaatg gacgaaagtc 360 tgatggagca acgccgcgtg agtgaagaag gttttcggat cgtaaaactc tgttgttgga 420 gaagaatgta tctgatagta actgatcagg tagtgacggt atccaaccag aaagccacgg 480 ctaactacgt gccagcagcc gcggtaatac gtaggtggca agcgttgtcc ggatttattg 540 ggcgtaaagc gagcgcaggc ggtttcttaa gtctgatgtg aaagccttcg gctcaaccga 600 agaagtgcat cggaaactgg gaaacttgag tgcagaagag gacagtggaa ctccatgtgt 660 agcggtgaaa tgcgtagata tatggaagaa caccagtggc gaaggcggct gtctggtctg 720 taactgacgc tgaggctcga aagcatgggt agcaaacagg attagatacc ctggtagtcc 780 atgccgtaaa cgatgagtgc taggtgttgg agggtttccg cccttcagtg ccgcagctaa 840 cgcattaagc actccgcctg gggagtacga ccgcaaggtt gaaactcaaa ggaattgacg 900 ggggcccgca caagcggtgg agcatgtggt ttaattcgaa gcaacgcgaa gaaccttacc 960 aggtcttgac atcctttgac cactctagag atagagcttt cccttcgggg acaaagtgac 1020 aggtggtgca tggttgtcgt cagctcgtgt cgtgagatgt tgggttaagt cccgcaacga 1080 gcgcaaccct tattactagt tgccagcatt tagttgggca ctctagtgag actgccggtg 1140 acaaaccgga ggaaggtggg gacgacgtca aatcatcatg ccccttatga cctgggctac 1200 acacgtgcta caatggatgg tacaacgagt tgcgagaccg cgaggtttag ctaatctctt 1260 aaaaccattc tcagttcgga ttgtaggctg caactcgcct acatgaagcc ggaatcgcta 1320 gtaatcgcgg atcagcatgc cgcggtgaat acgttcccgg gccttgtaca caccgcccgt 1380 cacaccatga gagtttgtaa cacccaaagc cggtgaggta acccttcggg 1430

Claims (10)

Lactobacillus sakei WiKim0066 strain of Accession No. KACC 92194P containing 16s rRNA represented by SEQ ID NO: 1.
The method according to claim 1,
Antioxidant or UV-induced inhibition of skin aging.
delete The method according to claim 1,
A strain derived from kimchi.
A cosmetic composition for antioxidation comprising at least one selected from the group consisting of Lactobacillus saccharum WiKim0066 (Accession No. KACC 92194P) strain of claim 1, a disruption thereof, a culture thereof, a concentrate of the culture, and a dried product of the culture.
Skin aging caused by ultraviolet rays comprising at least one selected from the group consisting of Lactobacillus saccharum WiKim0066 (Accession No. KACC 92194P) strain of the present invention, a disruption product thereof, a culture thereof, a concentrate of the culture product and a dried product of the culture Inhibiting cosmetic composition.
delete The method according to claim 5 or 6,
Wherein the active ingredient is contained in an amount of 0.01 to 10% by weight based on the total weight of the cosmetic composition.
An external preparation for skin comprising as an active ingredient at least one selected from the group consisting of Lactobacillus saccharum WiKim0066 (Accession No. KACC 92194P) strain of claim 1, a disruption product thereof, a culture thereof, a concentrate of the culture product and a dried product of the culture.
10. The method of claim 9,
Wherein the active ingredient is contained in an amount of 0.01 to 10% by weight based on the total weight of the external preparation for skin.

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