NOVEL PEDIOCOCCUS PENTOSACEUS AB 160011 STRAIN AND COMPOSITION CONTAINING SAME

Abstract

The present invention relates to a novel lactic acid bacteria strain and a composition comprising the same. In addition, the present invention relates to an antibacterial composition, a cosmetic composition, and a functional food, which contain the lactic acid bacterial stain and a culture medium thereof.

Claims

1. A novel Pediococcus pentosacus AB160011 strain (Deposit no.: KCCM11954P).

2. A health functional food for improving inflammatory bowel disease comprising the strain of claim 1.

3. An antimicrobial composition comprising the cell-free culture supernatant of the strain of claim 1.

4. An antifungal composition comprising the cell-free culture supernatant of the strain of claim 1.

5. A cosmetic composition comprising the cell-free culture supernatant of the strain of claim 1.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0015] FIG. 1 is a microscopic picture of Pediococcus pentosaceus AB160011 strain of the present invention.

[0016] FIG. 2 is a phylogenetic tree showing the molecular biological associations of the strain of the present invention.

[0017] FIG. 3 is a graph showing the DNA sequence of the strain of the present invention.

[0018] FIG. 4 is a graph showing a growth curve of the strain of the present invention.

[0019] FIG. 5 is a graph showing pH changes according to the growth time of the strain of the present invention.

[0020] FIG. 6 shows the results obtained by inducing the expression of Claudin-4 by the strain of the present invention in CaCo2 cells.

[0021] FIG. 7 shows the results obtained by inducing the expression of Claudin-1 by the culture supernatant of the strain of the present invention in HaCaT cells.

[0022] FIG. 8 shows selective inhibitory activities against intestinal pathogens and beneficial bacteria of the strain of the present invention.

[0023] FIG. 9 is a result of measuring the amount of change in Nitric Oxide, an inflammatory response indicator, by the culture supernatant of the strain of the present invention.

[0024] FIG. 10 is a result of measuring the amount of change in Nitric oxide, an inflammatory response indicator, by the strain of the present invention.

[0025] FIG. 11 shows the anti-inflammatory effect by the culture supernatant of the strain of the present invention.

[0026] FIG. 12 shows the antioxidant effects by the culture supernatant of the strain of the present invention.

[0027] FIG. 13 shows a skin irritation replacement test by the culture supernatant of the strain of the present invention.

[0028] FIG. 14 shows the skin moisture improvement effects by the culture supernatant of the strain of the present invention.

BEST MODE

[0029] The present invention will now be described in more detail with reference to the following examples. The following examples are merely illustrative of the present invention, and it is to be understood that the scope of the invention is not limited to the following examples.

[0030] Isolation and Identification, and Examples of Manufacture

[0031] (1) Isolation of Lactic Acid Bacteria

[0032] Various kinds of lactic acid bacteria were isolated from fermented products including kimchi, soy sauce, and soybean paste prepared in Korea in order to screen lactic acid bacteria strains having antibacterial and antifungal activities, and cultured in a MRS (Difco 288110) solid medium containing 2% agar using a 10-fold dilution method to obtain the colonies. The resulting colonies were classified according to size, color, and characteristics, and then streaked onto another MRS medium to separate a total of 1,785 lactic acid bacteria of the target strains. Each colony of the isolated lactic acid bacteria was streaked again onto MRS agar plates and incubated at 37° C. for 24 hours. The isolated lactic acid bacteria were inoculated into the sterilized MRS broth and incubated at 37□ for 24 hours, followed by the addition of 20%(v/v) glycerol and stored in a ultra-low temperature freezer at −80° C.

[0033] (2) Strain Identification

[0034] Morphology Analysis

[0035] In order to identify the strains, the morphology of the colonies was observed under a microscope after being cultured in MRS plate media. The colonies' morphology is shown in Table 1, and FIG. 1 is a microscopic picture of the strain of the present invention.

TABLE-US-00001 TABLE 1 Pediococcus pentosaceus AB160011 phenotype circular size 0.8~1.0 .sup.mm color white opacity opaque cell surface protrusion morphology surface smoothness

[0036] Identification by 16S rRNA

[0037] The novel strain of the present invention was found to have a 16S rRNA sequence as shown in SEQ ID NO: 1. Based on this, the isolated strain was found to have high similarity to Pediococcus pentosaceus AJ3053 and named Pediococcus pentosaceus AB160011, and deposited with deposition accession no. KCCM11954P at Korean Culture Center of Microorganisms (KCCM) on Dec. 7, 2016.

[0038] Identification by DNA Sequence Analysis

[0039] It was confirmed that the novel strain of the present invention has 98.97% homology with Pediococcus pentosaceus (ATCC25745 ATP), according to DNA sequence analysis (Gepard, Nucleotide sequences Dot plot) (see FIG. 3).

[0040] Cell Culture Property

[0041] The novel lactic acid bacteria strains of the present invention were inoculated into a sterile MRS broth medium and incubated at 37° C. to observe changes in turbidity (Ultrospec2100pro, OD625 nm) and acidity (Orion star A211, pH). FIGS. 4 and 5 show the growth curve and acidity change of the strain of the present invention.

[0042] Cultivation of Lactic Acid Bacteria Strains and Freeze Drying of the Cell-Free Culture Supernatant

[0043] The composition of the medium for culturing lactic acid bacteria is shown in Table 2, and the manufacturing process is largely classified into as follows: seed culture, main culture, separation of strains and cell-free culture supernatant, and freeze-drying. The seed culture refers to the process of culturing the strains to a volume of 10% of the final culture volume, depending on the size of the target final culture volume. After the seed culture is completed, the obtained seed culture solution including the strains is inoculated into another sterilized culture medium having larger volume and cultured, which is main culture. After the completion of the said main culture, the lactic acid bacteria and the culture supernatant are separated by centrifugation and/or using ceramic filter to obtain cell-free strain culture supernatant. The separated lactic acid bacteria and the cell-free culture supernatant are freeze-dried through a pre-freezing process and processed into a final powder form stored at −20□ for the following use.

TABLE-US-00002 TABLE 2 Concentration (g/L) Glucose dextrose 10 MgSO.sub.4 0.1 Yeast extract 5 Whey 2.5 Sodium acetate 5 Trisodium citrate 2 K.sub.2HPO.sub.4 2 MnSO.sub.4 0.05

BEST MODE FOR INVENTION

Example

Example 1: Culture of Pathogens

[0044] In order to select lactic acid bacteria inhibiting the growth of pathogenic bacteria, the following bacteria were obtained from KCCM and cultured in the solid media presented in Table 3 below. Bacteria: Staphylococcus aureus (KCCM11335), Escherichia coli (KCCM11234), Bacillus cereus (KCCM40935), Staphylococcus epidermidis (KCCM40416), Pseudomonas (KCCM11321), Propionibacterium aeruginosa (KCCM41747), Salmonella enterica subsp. enterica (KCCM11806), Listeria monocytogenes (KCCM40307). Fungi (including fungi): Cryptococcus neoformans (KCCM50785), Candida albicans (KCCM11282), Aspergillus fumigatus (ATCC Mya-4609), Aspergillus niger (KCCM60332), Fusarium oxysporum (KCCM44187), Penicillium chrysogenum (KCCM60353) and Malassezia furfur (KCCM12679). Next, each colony of the pathogen was inoculated into a nutrient broth medium and incubated for 24 hours at an appropriate temperature (37° C. for E coli, Staphylococcus aureus and Bacillus; 30° C. for Cryptococcus and Candida), followed by the addition of 20% glycerol, and then stored at −80□ cryogenic freezer. In the case of Aspergillus, Fusarium and Penicillium, the spores were harvested with PBS (phosphate buffer saline) from YPD medium (Sigma-Aldrich) after 7 days incubation at 30° C., followed by the addition of 20% glycerol, and then stored at −80□, cryogenic freezer.

TABLE-US-00003 TABLE 3 Pathogenic Microorganisms Nutrient media E. coli LB (Luria Bertani) Staphylococcus aureus TSB (Tryptic Soy Broth) Bacillus cereus NB (Nutrient Broth) Cryptococcus neoformans YM (Yeast Mold) Candida albicans YM (Yeast Mold) Aspergillus fumigatus YPD (Yeast Extract, Peptone, Dextrose) Aspergillus niger PDA (Potato Dextrose Agar) Fusarium oxysporum PDA (Potato Dextrose Agar) Penicillium chrysogenum PDA (Potato Dextrose Agar)

Example 2: Detection of Antimicrobial and Antifungal Activities

[0045] For the isolated strains, their antimicrobial and antifungal activities were detected. First, each isolated strain stored at −80° C. was inoculated onto MRS solid medium and incubated at 37□ for 24 hours, and then the respective colonies were taken and suspended in 500 μl of

[0046] PBS to prepare a sample solution for subsequent antibacterial and antifungal activity experiments. Next, media for antimicrobial and antifungal activity tests of the isolation strains were prepared. To this end, the bacteria and fungi cultured and stored in Example 1 were inoculated into a nutrient media suitable for each growth and incubated for 24 hours. Next, the soft agar medium containing MRS (1%, w/w) was sterilized at 121□ for 15 minutes, cooled to 55□, and each culture medium including bacteria or fungi obtained above was inoculated with 1% (v/v) on the said MRS medium. The soft agar MRS medium inoculated with the bacteria or fungi was then poured into a disposable petri dish and placed at room temperature to prepare an overlay medium for antibacterial or antifungal activity experiments. A sterilized 8 mm paper disk (Adventec, 51020693) was placed on the obtained overlay medium, 50 μl of the isolated lactic acid bacteria or cell-free culture supernatant of the present invention was added, and incubated at the appropriate growth temperature of each bacteria or fungi for 24 hours. After the culture was completed, the diameter of the clear zone for the pathogens was measured to determine the antibacterial activity and antifungal activity of the isolated lactic acid bacteria. The antibacterial activity and antifungal activity above was indicated according to the diameter of the clear zone as follows: 8 mm or more: +, 13 mm or more: ++, 15 mm or more: +++, 17 mm or more: ++++.

Example 3: Acid Resistance and Bile Resistance of the Lactic Acid Bacteria of the Present Invention

[0047] The isolated lactic acid bacteria strains were cultured in Lactobacilli MRS broth for more than 16 hours and then the 100 μl of the bacterial culture was inoculated into 100 μl of MRS broth with different pH conditions (pH7.0, pH2.5) in a 96 well plate, followed by incubation at 37□ for 24 hours. The survival rate of the lactic acid bacteria was measured by comparing the absorbances of the pH7.0 culture medium and the pH2.5 culture medium at 620 nm using Microplate reader. The cell viability for the acid resistance test was measured in the following manner.

Viability (%)=OD.sub.24 h, pH 2.5/OD.sub.24 h, pH 7.0×100

[0048] To confirm bile resistance, the isolated lactic acid bacteria strain was cultured in Lactobacilli MRS broth for more than 16 hours and then 100 μl of the bacterial culture was inoculated into 100 μl of MRS broth with two conditions (MRS broth, MRS broth+0.3% oxgall) in a 96 well plate, followed by incubation at 37□ for 24 hours. The survival rate was measured by comparing the absorbance at 620 nm using Microplate reader. The cell viability for the bile resistance test was measured in the following manner.

Viability Measurement (%)=OD.sub.24 h, MRS+0.3% Oxgall/OD.sub.24 h, MRS×100

[0049] The acid resistance and bile resistance of the novel lactic acid bacteria of the present invention were 86.87% and 99.37%, respectively, and were found to have excellent properties exceeding the general standard of 70%.

Example 4: Expression of TJ (Tight Junction) Proteins in Caco-2 Cells or HaCaT Cells by the Lactic Acid Bacteria of the Present Invention

[0050] The induction of expression of the intestinal TJ constituent proteins was confirmed by using Caco-2 cells (obtained from Korean Cell Line bank), intestinal epithelial cells. As shown in FIG. 6, it was confirmed that the cell-free supernatant of the lactic acid bacteria of the present invention effectively induced the expression of ZO-1, Occludin, Claudin-1 and Claudin-4 proteins. Next, HaCaT cells (obtained from Korean Cell Line Bank) were used to confirm the induction of expression of TJ constituent proteins. As a result, as shown in FIG. 7, it was confirmed that the said cell-free supernatant of the present invention effectively induced the expression of ZO-1 ATP, Occludin, Claudin-1, and Claudin-4 proteins in the HaCaT cells. Specifically, the freeze-dried powder of the said cell-free culture supernatant was first quantified and dissolved in PBS. To prevent microbial contamination, 0.2 μm syringe filtration was performed and treated at a concentration suitable for 6 well plate. The cell lysis was then performed using a RIPA/PI buffer, after 24 hours of incubation in a 370, CO.sub.2 incubator. The protein was quantified via Bradford assay, and then SDS-PAGE was carried out. SDS-PAGE was loaded with 20 μg per lane, and performed at 150 V for 1 h 30 min running, at 100 V for 1 h transfer (NC membrane) followed by blocking for 1 h (4% SKIM milk). Then, Claudin-1 (1:1000) as a primary antibody was treated for 16 hours and beta-actin (1:1000) was treated for 1 hour. As a secondary antibody, Claudin-1(anti-rabbit, 1:2000) was treated for 1 hour and beta-actin (anti-mouse, 1:2000) was treated for one hour. The band was detected using ECL kit.

Example 5: Antimicrobial Activity Test of the Lactic Acid Bacterial Culture Supernatant of the Present Invention (MIC, Minimum Inhibitory Concentration)

[0051] The minimum inhibitory concentration (MIC) measurement was performed by a standard test method (Wiegand et al. Nat Protoc. 2008). First, E. coli, S. aureus or B. cereus was inoculated onto a sterile test solid nutrient medium (MHB, Muller Histone Broth, Difco) and cultured. The C. neoformans or C. albicans was inoculated on YM nutrient medium (Difco) and A. fumigatus was inoculated on YPD nutrient medium (Difco), and incubated at 37□ for 24 hours. The bacteria were then inoculated into sterile liquid medium (MHB) and the fungus was inoculated into RPMI1640 medium, respectively. Next, the cell-free bacterial culture supernatant of the present invention was added to MHB or RPMI1640 medium obtained above and incubated at 37° C. for 24 hours to measure the turbidity of the pathogenic microorganisms. The cell-free culture supernatant solution was prepared by dissolving the freeze-dried lactic acid bacteria culture supernatant in the triple distilled water. The serial 2×dilution of the supernatant solution said was used to confirm the lowest concentration of the culture supernatant inhibiting the visible growth of pathogens. To confirm the antibacterial and antifungal activities, the test above was repeated three times, and the OD (620 nm) value of the culture supernatant was detected using microplate spectrophotometer (Multiskan FC A28696). As shown in Table 4, the lactic acid bacteria culture supernatant of the present invention exhibited extensive inhibitory activity against Gram-positive and Gram-negative bacteria and fungi.

Example 6: Antifungal Activity Test of the Lactic Acid Bacterial Culture Supernatant of the Present Invention (MIC)

[0052] Antifungal activity and MIC test were performed according to the standard test method (CLSI document M38-A2, 2008). The culture supernatant prepared in the example above was serially 2×diluted for this experiment. Each well of each 96 well was inoculated with mold or fungi spores at a concentration of 2×10.sup.3 spores/ml. The plate was incubated at 35° C. for 48 hours. The MIC was determined by microscopic observation to the lowest concentration of the cell-free culture supernatant inhibiting the visible growth of the fungi or mold. The experiment was repeated three times to confirm the antifungal activity. Table 4 shows the results.

TABLE-US-00004 TABLE 4 MIC (mg/ml) Escherichia coli 0.7 Staphylococcus aureus 0.9 Staphylococcus epidermidis 1.6 Propioni bacterium acnes 0.8 Bacillus cereus 0.7 Pseudomonas aeruginosa 0.8 Salmonella 0.8 Listeria 0.8 Cryptococus neoformans 25~50 Candida albicans 25~50 Aspergillus niger 25~50 Fusarium oxysporum 25~50 Penicillium chrysogenum 25~50 Malassezia furfur 25~50

Example 7: Thermal Stability

[0053] Experiments were conducted to determine whether the antibacterial and antifungal activity against the pathogens by the cell-free culture supernatant of the lactic acid bacteria has thermal stability. The concentrated culture supernatant dissolved solution was prepared in the same manner as in the above example and was divided to two groups of non-heat treatment and heat treatment to determine the MIC showing antibacterial and antifungal activities. For the heat-treated group, the culture supernatant solution was heat-treated in 800 water bath (Biofree, BF60AC) for 30 minutes or in 121□ autoclave for 15 minutes. The results of the experiments are shown in Table 5.

TABLE-US-00005 TABLE 5 Minimum Inhibitory Concentration (mg/ml) non-heat 80□, 121□, pathogens treatment 30 min. 15 min. G(−) E. coli 0.7 0.7 0.7 G(+) S. aureus 0.9 0.9 0.9 G(+) B. cereus 0.7 0.7 0.7 fungi C. neoformans  6.25  6.25  6.25 fungi C. albicans 25.00 25.00 25.00

Example 8: pH Stability

[0054] Experiments were conducted to determine whether the antibacterial and antifungal activity against pathogens of the lactic acid bacteria culture supernatant has pH stability. The concentrated culture supernatant solution prepared in the same manner as in the above example was treated with non-pH adjusted group or pH adjusted group to measure the MIC showing antibacterial and antifungal activities.

[0055] The pH was adjusted using lactic acids and NaHCO.sub.3. The results of the experiments are shown in Table 6 below.

TABLE-US-00006 TABLE 6 Minimum Inhibitory Concentration (mg/ml) non- adjusted adjusted adjusted (pH (pH pathogens (pH 4.3) 4.3 .fwdarw. 7 .fwdarw. 4.5) 4.3 .fwdarw. 7 .fwdarw. pH 5) G(−) E. coli 0.7 0.7 0.7 G(+) S. aureus 0.9 0.9 0.9 G(+) B. cereus 0.7 0.7 0.7 fungi C. neoformans  6.25  6.25  6.25 fungi C. albicans 25.00 25.00 25.00

Example 9: Selective Inhibition Activity of the Cell-Free Culture Supernatant of the Lactic Acid Bacteria

[0056] The Pediococcus pentosaceus AB160011 of the present invention, Lactobacillus brevis and E. coli were cultured until OD600 reached 0.8, respectively. Then, 0.5, 1 and 1.5 ml of Pediococcus pentosaceus AB160011, 1 ml of Lactobacillus brevis and 1 ml of E. coli were used to remove the supernatant using centrifuge, and the precipitated cells were pooled and washed with distilled water (DW). After performing centrifugation again, the supernatant was removed and Pediococcus pentosaceus AB160011 was pooled in 5 μl of DW, and each Lactobacillus brevis and E. coli were pooled in 1 ml of DW. Then, each Lactobacillus brevis and Escherichia coli, which were pooled in 1 ml of DW, were spread evenly in the MRS solid media. Then, Pediococcus pentosaceus AB1600115, which was pooled in 5 μl of DW, was spotted on the MRS medium above where Lactobacillus brevis or Escherichia coli, was spread, at a constant distance. The results were confirmed after 6˜20 hours of incubation and are shown in FIG. 8.

Example 10: Inflammation Induction of the Cell-Free Culture Supernatant of the Lactic Acid Bacteria

[0057] The amount of Nitric Oxide, an inflammatory response indicator, was measured. First, RAW264.7 cells were seeded in 12 well plates with 1×10.sup.5 cells per well and were incubated overnight. The 0.2 g of the lyophilized sample of the cell-free culture supernatant of Pediococcus pentosaceus AB160011 was dissolved in 1 ml of DMEM. The RAW264.7 cells were treated with 0.25, 0.3, 0.35, 0.4, 0.45 and 0.5% (wt/v) of the cell-free culture supernatant dissolved in DMEM above, and incubated for 24 hours. The amount of NO was determined by measuring the absorbance at 540 nm using Nitric Oxide Colorimetric Assay kit (BioVision). As a result, the said culture supernatant of the present invention did not induce an inflammatory response. The results are shown in FIG. 9.

[0058] RAW R264.7 cells were seeded into 12 well plates at a concentration of 1×10.sup.5 cells per well. One day after the seeding, each well was treated with Pediococcus pentosaceus AB160011 at each concentration of 5×10.sup.7, 1×10.sup.8 and 2×10.sup.8 CFU/ml for 2 hours and then the amount of NO was measured at 540 nm using Nitric Oxide Colorimetric Assay kit (Biovision, Korea). As a result, the lactic acid bacteria of the present invention did not induce an inflammatory response. The results are shown in FIG. 10.

Example 11: Anti-Inflammatory Effect of Lactic Acid Bacteria of the Present Invention

[0059] One day after the seed inoculation of 1×10.sup.5 cells per well in a 12 well plate of RAW R264.7 cells, 1×10.sup.8 or 2×10.sup.8 cfu/ml of Pediococcus pentosaceus AB160011 were added to each well, and each well was treated with 1 μg/ml of LPS for 24 hours to induce inflammation. The amount of NO was determined at 540 nm using Nitric Oxide Colorimetric Assay kit (Biovision, Korea). As a result, the lactic acid bacteria of the present invention have been shown to inhibit inflammation reactions. The results are shown in FIG. 11.

Example 12: Measurement of Antioxidant Effect

[0060] The DPPH (1,1-diphenyl-2-picrylhydrazyl), which is a water-soluble material having a chemically stabilized free radical, has a maximum absorbance between 515 nm and 520 nm, and the radical (DPPH) is extinguished and discolored when the material encounters a material having an antioxidant activity. It is known that a chemically stable 1-diphenyl-2-picylhydryl radical (DPPH) can be used to analyze an antioxidant effects of the extracts containing several antioxidant components, a beverage, an oil, a pure phenol compound, and the like. The cell-free bacterial culture supernatant of the present invention was added to 500 ml of 0.2 mM DPPH, wherein the said culture supernatant was applied at each concentration of 0.1, 0.25, 0.3, 0.35, 0.4, 0.45 and 0.5 (wt/v)% (or 0.1, 0.25, 0.5, 0.75, 1, 1.5 and 2 (wt/v)%) while adjusting the final volume of the mixture to 1 ml. The absorbance was measured at 540 nm after the reaction in the dark room for 30 minutes. As a result, as shown in FIG. 12, the present lactic acid bacterial culture supernatant was shown to increase the antioxidant effects in a concentration-dependent manner.

Example 13: Skin Irritation Replacement Test

[0061] A skin irritation test using a SKINETHIC human skin model (EPISKIN) was carried out at Korea Testing & Research Institute (KTR) according to the guidelines for testing toxicology for cosmetics and animal replacement tests (V)/skin irritation test method (Ministry of Food and Drug Safety, MFDS; Guideline-0752-01). The 16 μl of the test article of the cell-free bacterial culture supernatant was applied to the said human skin model with 1 minute intervals per tissue and exposed for 42 minutes (±1) in the clean bench. The test material was then removed and washed a total of 25 times with PBS (1 ml at one-time). The reversible damage to the said human skin model was evaluated by incubation the treated human skin at 37□ for 42 hours±1 hour in a 5% CO.sub.2 incubator. Each group of the three groups of the negative control, the positive control, and the test material group was set to have three repetitive samples. The cell viability of the test article was determined to be 86.3±2.9% over 50% of the cell viability baseline and determined to be non-irritating to the skin. The results are shown in FIG. 13.

Example 14: Skin Moisturizing Improvement Effects of a Cosmetic Essence Containing the Cell-Free Lactic Acid Bacteria Culture Supernatant of the Present Invention

[0062] In order to evaluate the effects of improving skin moisturizing of the cosmetic essence product containing the cell-free bacterial supernatant of the present invention, a clinical test was performed at KTR (Korea). The skin moisturizing assay and abnormal response assay were performed for 22 females aged between 29 years and 59 years using Corneometer both before and after the use of the product. The results of skin moisturization have shown a statistically significant increase of the moisture content in the forehead, both cheeks and jaw areas two and four weeks after use compared to before the product was used (see FIG. 14). The evaluation of adverse effects by dermatologists did not report any special skin adverse events to the test subjects during the period of use of the test article, nor did any abnormalities be observed in the scientific examination by dermatologists.

INDUSTRIAL APPLICABILITY

[0063] The present invention confirms that a novel lactic acid bacterial strain isolated from soybean paste, a Korean traditional fermented food, and the cell-free bacterial culture supernatant thereof have excellent antibacterial and antifungal activities, and induce the expression of proteins involved in TJ (tight junctions) of skin and intestinal epithelial cells. In addition, the bacterial strain and the supernatant of present invention have anti-inflammatory and antioxidant effects, and have effects of enhancing skin moisturization according to clinical trials. Therefore, the lactic acid bacterial strain of the present invention could be widely used in a wide range of industrial sectors such as health functional foods, cosmetics and preservatives.

Deposit No

[0064] Deposit institution name: Korean Culture Center of Microorganisms (overseas).

[0065] Deposit No.: KCCM11954P

[0066] Deposit Date: 20161207

[0067] <110>AmtixBioCo., Ltd.

[0068] <120>A novel Pediocococcus pentosaceus AB160011 and composition comprising thereof

[0069] <130>AMTIX17P01KR

[0070] <160>1

[0071] <170>KoPatent In 3.0

[0072] <210>1

[0073] <211>2849

[0074] <212>DNA

[0075] <213>Pediococcus pentosaceus

[0076] <400>1

TABLE-US-00007 tcatggctca ggatgaacgc tggcggcgtg cctaatacat gcaagtcgaa cgaacttccg 60 ttaattgatt atgacgtact tgtactgatt gagattttaa cacgaagtga gtggcgaacg 120 ggtgagtaac acgtgggtaa cctgcccaga agtaggggat aacacctgga aacagatgct 180 aataccgtat aacagagaaa accgcatggt tttcttttaa aagatggctc tgctatcact 240 tctggatgga cccgcggcgt attagctagt tggtgaggta aaggctcacc aaggcagtga 300 tacgtagccg acctgagagg gtaatcggcc acattgggac tgagacacgg cccagactcc 360 tacgggaggc agcagtaggg aatcttccac aatggacgca agtctgatgg agcaacgccg 420 cgtgagtgaa gaagggtttc ggctcgtaaa gctctgttgt taaagaagaa cgtgggtaag 480 agtaactgtt tacccagtga cggtatttaa ccagaaagcc acggctaact acgtgccagc 540 agccgcggta atacgtaggt ggcaagcgtt atccggattt attgggcgta aagcgagcgc 600 aggcggtctt ttaagtctaa tgtgaaagcc ttcggctcaa ccgaagaagt gcattggaaa 660 ctgggagact tgagtgcaga agaggacagt ggaactccat gtgtagcggt gaaatgcgta 720 gatatatgga agaacaccag tggcgaaggc ggctgtctgg tctgcaactg acgctgaggc 780 tcgaaagcat gggtagcgaa caggattaga taccctggta gtccatgccg taaacgatga 840 ttactaagtg ttggagggtt tccgcccttc agtgctgcag ctaacgcatt aagtaatccg 900 cctggggagt acgaccgcaa ggttgaaact caaaagaatt gacgggggcc cgcacaagcg 960 gtggagcatg tggtttaatt cgaagctacg cgaagaacct taccaggtct tgacatcttc 1020 tgacagtcta agagattaga ggttcccttc ggggacagaa tgacaggtgg tgcatggttg 1080 tcgtcagctc gtgtcgtgag atgttgggtt aagtcccgca acgagcgcaa cccttattac 1140 tagttgccag cattaagttg ggcactctag tgagactgcc ggtgacaaac cggaggaagg 1200 tggggacgac gtcaaatcat catgcccctt atgacctggg ctacacacgt gctacaatgg 1260 atggtacaac gagtcgcgag accgcgaggt taagctaatc tcttaaaacc attctcagtt 1320 cggactgtag gctgcaactc gcctacacga agtcggaatc gctagtaatc gcggatcagc 1380 atgccgcggt gaatacgttc ccgggccttg tacacaccgc ccgtcacacc atgagagttt 1440 gtaacaccca aagccggtgg ggtaaccttt taggagctag ccgtctaagt gacagagttg 1500 gagacaaata aaaaaggata atatctccta agatattctc tttaaaaaca agggctttgg 1560 atatcaattt ccgggacgga ccccggaggg gttttttggc tagatcgtgg aggtaaaagg 1620 ctcccccaag gcattggatc agtagcaggc cttgagaggg gtatttgggc cccattggga 1680 atgaaacacg gccccagatt cctacgggag gcagcagtta tgaatttttc cccaatggac 1740 gcaagtctga tcgacccacc cccgcgtgag tgaagaaggg ttttcggctc gtaaagctct 1800 tgttgttaaa gaagaacgtg ggtaagagta actgtttacc cagtgacggt atttaaccag 1860 aaagccacgg ctaattacgt gccagcagcc gcggtaatac gtaggtggca agcgttatcc 1920 ggatttattg ggcgtaaagc gagcgcaggc ggtcttttaa gtctaatgtg aaagccttcg 1980 gctcaaccga agaagtgcat tggaaactgg gagacttgag tgcagaagag gacagtggaa 2040 ctccatgtgt agcggtgaaa tgcgtagata tatggaagaa caccagtggc gaaggcggct 2100 gtctggtctg caactgacgc tgaggctcga aagcatgggt agcgaacagg attagatacc 2160 ctggtagtcc atgccgtaaa cgatgattac taagtgttgg agggtttccg cccttcagtg 2220 ctgcagctaa cgcattaagt aatccgcctg gggagtacga ccgcaaggtt gaaactcaaa 2280 agaattgacg ggggcccgca caagcggtgg agcatgtggt ttaattcgaa gctacgcgaa 2340 gaaccttacc aggtcttgac atcttctgac agtctaagag attagaggtt cccttcgggg 2400 acagaatgac aggtggtgca tggttgtcgt cagctcgtgt cgtgagatgt tgggttaagt 2460 cccgcaacga gcgcaaccct tattactagt tgccagcatt aagttgggca ctctagtgag 2520 actgccggtg acaaaccgga ggaaggtggg gacgacgtca aatcatcatg ccccttatga 2580 cctgggctac acacgtgcta caatggatgg tacaacgagt cgcgagaccg cgaggttaag 2640 ctaatctctt aaaaccattc tcagttcgga ctgtaggctg caactcgcct acacgaagtc 2700 ggaatcgcta gtaatcgcgg atcagcatgc cgcggtgaat acgttcccgg gccttgtaca 2760 caccgcccgt cacaccatga gagtttgtaa cacccaaagc cggtggggta accttttagg 2820 agctagccgt ctaagtgaca gagttggag 2849