KR101878288B1 - Paenibacillus tianmuensis jr103 strain having antimicrobial activity and uses thereof - Google Patents

Abstract

The present invention relates to a Paenibacillus tianmuensis JR103 strain having antimicrobial activity or to an antimicrobial composition comprising a culture of the strain as an active ingredient, wherein the strain produces a surfactant and poly-L-lysine to have excellent antimicrobial activity, thereby being effectively used for an antimicrobial use.

Description

FIELD OF THE INVENTION [0001] The present invention relates to a FANIBACILLUS THIAMMUNISIS JR103 strain having antimicrobial activity and a use thereof,

The present invention relates to an antimicrobial composition comprising as an active ingredient a strain of Fanny Bacillus thyramnensis JR103 (Accession No. KCTC18519P) or a culture of the strain.

Fungi cause various mycoplasms when the immune function of the human body is weakened or when resistance to antibiotics occurs due to overuse of antibiotics, hormones, and anticancer drugs. Examples thereof include candidiasis caused by Candida sp., Aspegillosis caused by Aspergillus sp., Yeast infection caused by Cryptococcus neoformans , Zygomycosis caused by Mucor sp. And Rhizopus sp., Dermatophytosis caused by Epidermophyton floccosum and Trichophyton sp., And the like .

Studies on natural antimicrobial substances derived from microorganisms related to antibiotics for the treatment of infectious diseases have been actively conducted. Regarding the antibacterial substance using microorganisms, Korean Patent No. 10-1594446 discloses Bacillus subtilis RX7 strain having antibacterial activity against harmful microorganisms (Patent Document 1). In addition, Patent No. 10-1190901 discloses a Bacillus subtilis CSY191 strain having excellent antimicrobial activity against a food-harmful microorganism, but discloses a Bacillus subtilis strain having antimicrobial activity against food-borne bacteria such as Escherichia coli and Salmonella Patent Document 2).

The present inventors isolated a novel strain of the genus Fanny-Bacillus in the soil, and confirmed that the strain has excellent antimicrobial activity.

1. Korean Patent No. 10-1594446 2. Korean Patent No. 10-1190901

 1. Isolation and screening of antimicrobial lactic acid bacteria from fermentating millet gruel. International Journal of Microbiology (IRJM) Vol. 3 (2) pp. 072-079, February 2012  2. Characterization of a novel biosurfactant produced by Staphylococcus sp. strain 1E with potential application on hydrocarbon bioremediation. J Basic Microbiol. 2012 Aug; 52 (4): 408-18. Epub 2011 Nov 4.  3. Growth Inhibition of Some Plant-Pathogenic Fungi by Streptomyces vellosus HR29. Proceedings of the World Congress on New Technologies (NewTech 2015) Barcelona, Spain - July 15-17, 2015 Paper No. 128

One aspect of the present invention is to provide a strain of Fanny Bacillus thyramansis JR103 (Accession No. KCTC18519P) having antibacterial activity.

Another aspect of the present invention is to provide an antimicrobial composition comprising a culture of the strain or strain.

Another aspect of the present invention is to provide a cosmetic composition comprising a culture of the strain as an active ingredient.

One aspect of the present invention provides Fanny Bacillus thyramansis JR103 (Accession No. KCTC18519P) strain having antibacterial activity.

According to one embodiment of the present invention, the Fanny Bacillus thyramansis JR103 strain of the present invention is a microorganism isolated from soil and confirmed to be a novel strain contained in the genus Fanny Bacillus through 16S rDNA sequencing. Therefore, the above strain was deposited with the BRC at the Korea Biotechnology Research Institute and received the deposit number of KCTC18519P.

According to one embodiment, the Fanny Bacillus thyramansis JR103 strain of the present invention is a strain of Bacillus genus Bacillus , genus Staphylococcus , genus Aspergillus , genus Pseudomonas , A genus Escherichia , and a genus Candida . ≪ Desc / Clms Page number 2 > More specifically, there can be mentioned Bacillus subtilis , Staphylococcus aureus , Aspergillus niger , Pseudomonas aeruginosa , Escherichia coli, it is preferable that the microorganism has antibacterial activity against a microorganism selected from the group consisting of E. coli and Candida albicans .

According to one embodiment of the present invention, the Fanny Bacillus thyramuneis JR103 strain of the present invention may be a surfactant producing strain, preferably a strain producing a rhamnolipid surfactant.

The term "surfactant" as used herein refers to an amphipathic substance including both a hydrophilic group and a lipophilic group in a molecule, and is a substance excellent in washing power, dispersing power, emulsifying power, solubilizing power, wetting power, sterilizing power, foaming power and penetration power.

In addition, according to one embodiment, the Fanny Bacillus thyramansis JR103 strain of the present invention may be a strain producing poly-L-lysine. Poly-L-lysine is a substance widely used as a preservative because it exhibits remarkable antimicrobial activity with a structure in which about 20 to 30 L-lysine is linearly linked.

Another aspect of the present invention relates to an antimicrobial composition comprising at least one selected from the group consisting of Fanny Bacillus thyramnensis JR103 (Accession No. KCTC18519P), a culture of the strain, a concentrate of the strain or the culture, and a dried product thereof to provide.

The antimicrobial composition may have the same meaning as an antibiotic, which is generically referred to as an antimicrobial agent, and may have the same meaning as an antimicrobial agent, a bactericide, an antiseptic, a preservative or a bactericidal agent, ≪ / RTI > or a substance capable of inhibiting or inhibiting < RTI ID =

As used herein, the term "culture product" refers to a culture medium obtained by culturing a culture medium for a predetermined period of time, a culture medium containing the same, metabolites thereof, extra nutrients or the like, do.

According to one embodiment, the culture of the Fanny Bacillus thyramansis JR103 strain of the present invention can be selected from a medium easily used by a person skilled in the art in the culture medium for microbial culture, and preferably used for the culture of Fanny Bacillus Medium or more preferably the NA or LB medium described in the following Examples 1 and 2 can be selected and used.

According to one embodiment, the culture of the Fanny Bacillus thyramansis JR103 strain of the present invention can be prepared by inoculating the strain of the present invention into the above microorganism culture medium and culturing it in a microorganism culture method known in the art (for example, stationary culture, ). ≪ / RTI > The Fanny Bacillus thyramansis JR103 strain or the concentrate of the culture broth and the dried product thereof can be easily prepared by a method of concentrating or drying the microorganism or culture medium known in the art.

In the antimicrobial composition of the present invention, the components other than Fannibacillus thyramnisis JR103 strain, the culture of the strain, the concentrate of the strain or the culture solution, and the dried components thereof are components that promote microbial activity and antibacterial activity, ≪ / RTI > may be included.

Another aspect of the present invention provides a cosmetic composition comprising at least one selected from the group consisting of a culture of a strain of Fanny Bacillus thyramnisis JR103 (Accession No. KCTC18519P), a concentrate of a culture solution, and a dried product.

The cosmetic composition of the present invention may contain, as an active ingredient, components commonly used in cosmetic compositions in addition to the culture of the Fanny Bacillus thyramansis JR103 strain, the concentrate or the dried product of the culture, and may contain, for example, a stabilizer, , Customary adjuvants such as vitamins, pigments and flavoring agents, and carriers.

Meanwhile, the cosmetic composition of the present invention may be prepared in any form conventionally produced in the art, and examples thereof include a solution, a suspension, an emulsion, a paste, a gel, a cream, a lotion, a powder, a soap, Containing cleansing oil, powdered foundation, emulsion foundation, wax foundation, and spray, but is not limited thereto. More particularly, the present invention relates to a cosmetic composition for the preparation of a cosmetic composition, which comprises at least one component selected from the group consisting of a soft lotion, a gel, a water-soluble liquid, a milk lotion, a cream, an essence, a skin toner, an oil-in-water emulsion, , Pack, body oil, body lotion, underwater type make-up base, in-water type make-up base and foundation.

The Fanny Bacillus thyramnensis strain JR103 (accession number KCTC18519P) of the present invention produces a surfactant and poly-L-lysine and is thus useful for antibacterial use because of its excellent antimicrobial activity.

Figure 1 is a schematic representation of the < RTI ID = 0.0 > Paenibacillus & 0.0 > tianmuensis ) < / RTI > JR103 strain.
Fig. 2 is a graph showing the results of measuring the minimum inhibitory concentration of P. tianmuensis JR103 against B. subtilis and S. aureus .
Fig. 3 shows the result of confirming the production of surfactant of P. tianmuensis JR103 strain.
Fig. 4 is a graph showing the results of analysis of the kinds of surfactants produced by P. tianmuensis JR103 strain.
FIG. 5 shows the results of P. tianmuensis And the concentration of the surfactant produced by the strain JR103 is measured.
Fig. 6 shows the results of confirming the production of poly-L-lysine by P. tianmuensis JR103 strain.
Fig. 7 shows the effect of P. tianmuensis L-lysine produced by the strain JR103.
Fig. 8 is a graph showing the effect of P. tianmuensis Figure 10 shows the result of measuring the antimicrobial activity of JR103 strain.

Hereinafter, one or more embodiments will be described in more detail by way of examples. However, these embodiments are intended to illustrate one or more embodiments, and the scope of the present invention is not limited to these embodiments.

Example  1: Isolation and Identification of Strain

1-1: Isolation and Culture of Strain

In order to isolate the microorganisms exhibiting antimicrobial activity, samples were collected from soil in Kangwon National University, Chuncheon City, Kangwon Province. 10 g of the collected soil sample was immersed in a conical tube of 50 ml, and then PBS (NaCl, 8.5%) was added to a total volume of 30 ml, followed by shaking for 30 minutes. After that, the samples were serially diluted (1/10 ³ , 1/10 ⁴ ) by using PBS, and Nutrient agar (peptone 5, beef extract 1.5, yeast extract 1.5, NaCl 5 and agar 15 g / Lt; / RTI > plate) was plated. Plates on which the samples were stained were cultured at 30 ° C for 24 hours to isolate grown bacterial colonies, and the separated colonies were subcultured in a new medium.

1-2: Identification of the strain

As a result of analysis of the 16S rDNA sequence (SEQ ID NO: 1) of the strain isolated in Example 1-1 above to Macrogen, the strain was confirmed to have 99% homology with Fanny Bacillus thyramansis. The isolated strain was finally transformed into Fanny Bacillus (Hereinafter referred to as JR103). The strain Fanny Bacillus thyramunense JR103 was deposited on December 2, 2016 at the Korea Research Institute of Bioscience and Biotechnology, and received the microorganism deposit number KCTC18519P.

Example 2: Confirmation of antimicrobial activity of strain JR103

2-1: Identification of microorganism growth inhibition effect of JR103 strain

In order to confirm the antibacterial activity of the JR103 strain isolated in Example 1, agar well diffusion was used.

Candida albicans (Candida albicans), Bacillus subtilis (Bacillus subtilis), Staphylococcus aureus (Staphylococcus aureus), rugi labor in this or Aspergillus (Aspergillus niger), Pseudomonas (Pseudomonas aeruginosa and Escherichia coli were cultured in LB medium for 48 hours. Thereafter, 100 占 퐇 of the 6 kinds of microorganism cultures adjusted to OD ₆₀₀ to 1 was streaked onto NA plates, and the culture of JR103 strain cultured in LB medium (Luria Bertani; tryptone 10, yeast extract 5 and NaCl 10 g / L) 100 [mu] l of the culture solution were each spotted. Plates were incubated for 18-24 hours and the diameter of the resulting zona pellucida was measured.

As a result, as shown in Table 1 and FIG. 1, it was confirmed that JR103 inhibited the growth of six microorganisms, and in particular, B. subtilis , S. aureus And C. albicans were excellent in antimicrobial activity.

As compared with the Lactobacillus plantarum FL9 strain disclosed in the non-patent document 1, since the FL9 strain expresses the serums of 18 ㎜, 14 ㎜ and 18 ㎜ against S. aureus , E. coli and P. aeruginosa , the microorganism growth inhibition of JR103 strain The activity is remarkably excellent.

2-2: Determination of minimum inhibitory concentration (MIC) of JR103 strain

The JR103 strain was cultured at 30 DEG C for 3 days using LB medium, and 40 mL of the culture solution was recovered and centrifuged (7,500 g, 10 minutes, 4 DEG C) to obtain a culture supernatant. 40 ml of ethyl acetate (99.5% by weight) was added to the obtained culture supernatant, and the mixture was extracted at 25 캜 for 30 minutes. The extract was concentrated under reduced pressure at 40 DEG C by using a rotary evaporator to obtain a culture supernatant extract. The minimum inhibitory concentrations of B. subtilis and S. aureus were measured using the culture supernatant of the JR103 strain obtained.

100 .mu.l of B. subtilis or S. aureus culture adjusted to a Mcfaland turbidity of 0.5 by NB medium was dispensed into a 96 well plate and the JR103 culture supernatant extract was started at a concentration of 6.67 mg / After dilution step by step, 100 쨉 l of each solution was added. After incubation at 30 ° C for 24 hours, 30 μl of resazurin (Sigma-Aldrich co., USA) solution was added to each well, and further cultured at 30 ° C for 15 minutes. The inhibitory concentration was measured.

As a result, as shown in FIG. 2, the strain JR103 of the present invention was found to inhibit growth at a concentration of 0.104 mg / ml for B. subtilis and 0.4 mg / ml for S. aureus .

Non-Patent Document 2: Staphylococcus sp. Compared with the strain 1E, the MIC of the strain 1E is 6.75, indicating that the antibacterial activity of the strain JR103 is remarkably low because of the remarkably low MIC.

Example  3: Confirmation of production of biological surfactant of JR103 strain

3-1: Oil Spreading  Oil spreading test

The culture supernatant of JR103 strain was obtained in the same manner as in Example 2-2, and the extract was used for oil spreading test.

Specifically, 50 ml of distilled water was added to a petri dish, 10 쨉 l of crude oil was dropped, and 10 쨉 l of the JR103 culture supernatant was added to observe zeros generated.

As a result, it was confirmed that the zona pellucida was formed by the addition of the culture supernatant of the JR103 strain as shown in Fig. 3, and it was found that the JR103 strain produced the surfactant.

3-2: Biosurfactant analysis

The culture supernatant of JR103 strain was obtained in the same manner as in Example 2-2, and the extract was used to identify the surfactant component.

Specifically, the extract was dissolved in a chloroform-methanol mixed solvent (Chloroform: methanol = 9: 1) at a concentration of 10 mg / ml and analyzed by thin layer chromatography (TLC). The mixture was developed on a TLC (thin layer chrosilica gel, 60 F254) plate at intervals of 2 cm while being dotted, and sufficiently developed using a chloroform-methanol-acetic acid mixed solution (65: 15: 2) as a mobile phase. After the development, the TLC plate was immersed in 15% H ₂ SO ₄ (ethanol) solution and incubated at 100 ° C. for 2 to 5 minutes to stain the sample.

As a result, as shown in FIG. 4, it was confirmed that a red band was formed by staining the sample with an orcinol solution, and the JR103 strain of the present invention produced a rhamnolipid-based surfactant .

3-3: Rannolipid  Quantitative analysis

Using the LB medium, JR103 strain was cultured at 30 DEG C for 6 days, and 1 mL of the culture solution was recovered at intervals of 24 hours. After centrifugation (1000 g, 30 minutes, 4 DEG C), a culture supernatant was obtained. 1 ml of diethyl ether was added to 333 占 퐇 of the obtained culture supernatant, and the mixture was extracted for 10 minutes. The extract was depressurized and concentrated at 58 ° C using a rotary evaporator to obtain a JR103 culture supernatant extract. After repeating the above procedure twice, 0.5 ml of distilled water was added to the extract, and the mixture was diluted for 5 minutes. The diluted sample (0.1 ml) was mixed with 0.9 ml of the orcinol solution and reacted at 80 ° C for 30 minutes under dark condition. After the reaction was completed, the reaction solution was allowed to cool at room temperature and dark for 30 minutes, and the absorbance was measured at OD ₄₂₁ .

As a result, as shown in Fig. 5, the amount of rhamnorefid produced by the strain JR103 was increased to about 3 days after the incubation, and the maximum concentration was found to be about 228.3 mg / L.

Non-Patent Document 3: Streptomyces Compared with strain Lam vellosus HR29 HR29 Norris feed production strain is the strain JR103 to 102 ㎎ / L can be seen that generate at least about twice the amount of the surface active agent.

Example  4: strain of JR103 strain Poly -L- Lysine ( poly -L- lysine ) Confirm production

4-1: Poly -L- Lysine  Confirm production

The strain JR103 was cultured at 30 ° C. for 3 days using LB medium. Then, the culture medium was recovered and a medium containing 0.02% Remazol brilliant blue dye was prepared. The medium composition was 10 g of glycerol, 0.66 g of ammonium sulfate, 0.68 g of sodium dihydrogen phosphate, 0.25 g of magnesium phosphate, 0.1 g of yeast extract, 1 ml of Kirk's solution, 15 g of agar and pH 7. Next, 10 mm diameter wells were punched into the medium, and then 100 占 퐇 of the JR103 strain culture solution was dispensed into the wells. After culturing for 24 hours, formation of the rings was observed.

As a result, as shown in FIG. 6, it was confirmed that a strain was formed in the medium supplemented with the culture medium of JR103 strain, and thus it was confirmed that JR103 strain produced poly-L-lysine.

4-2: Poly -L- Lysine  dose

JR103 strains which had been preliminarily grown in liquid medium (30 ° C, 160 rpm) were inoculated with fresh liquid medium at a concentration of 2 × 10 ⁸ CFU / ml, cultured for 4 days, and cultures were collected at intervals of 24 hours. The culture supernatant was recovered by centrifugation (7,500 g, 10 min, 4 ° C), and the recovered culture supernatant (1 ml), phosphate buffer (2.88 ml) and trypan blue (0.12 ml) Lt; 0 > C for 60 minutes. After the reaction was completed, the reaction solution was centrifuged for 10 minutes, and the supernatant was taken and absorbance was measured at 580 nm.

As a result, as shown in FIG. 7, it was confirmed that the amount of poly-L-lysine produced by the JR103 strain was increased as the incubation time increased.

Example  5: Identification of time-dependent antimicrobial activity of JR103 strain (Time-killing assay)

The JR103 strain was cultured at 30 DEG C for 3 days using LB medium, and 40 mL of the culture solution was recovered and centrifuged (7,500 g, 10 minutes, 4 DEG C) to obtain a culture supernatant. The obtained culture supernatant was adjusted to pH 2 with 6N hydrochloric acid and centrifuged (10000 g, 20 minutes, 4 캜) to separate the pellet. 15 ml of methanol was added to the separated pellet and suspended. The methanol suspension was adjusted to pH 7 with 1N NaOH, and the extract was filtered using a hydrophobic filter (0.22 μm pore). The extract, which was filtered using a rotary evaporator, was decompressed and concentrated at 40 ° C to obtain a JR103 culture supernatant.

100 .mu.l (10 ⁵ CFU / ml) of the B. subtilis culture was dispensed into microtube, 100 .mu.l of the JR103 culture supernatant was added, and 100 .mu.l of the culture solution was recovered at intervals of 30 minutes for 2 hours. The recovered culture was inoculated on NB medium and cultured. The CFU of B. subtilis was measured.

As a result, as shown in FIG. 8, when the JR103 culture supernatant was added to the B. subtilis culture, the B. subtilis population was abruptly decreased, indicating that the JR103 strain had excellent antimicrobial activity.

The present invention has been described with reference to the preferred embodiments. It will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention as defined by the appended claims. Therefore, the disclosed embodiments should be considered in an illustrative rather than a restrictive sense. The scope of the present invention is defined by the appended claims rather than by the foregoing description, and all differences within the scope of equivalents thereof should be construed as being included in the present invention.

Korea Biotechnology Research Institute KCTC18519P 20161202

<110> Kangwon University-Industry Cooperation Foundation <120> PAENIBACILLUS TIANMUENSIS JR103 STRAIN HAVING ANTIMICROBIAL          ACTIVITY AND USES THEREOF <130> PN160346 <160> 1 <170> KoPatentin 3.0 <210> 1 <211> 1608 <212> DNA <213> Paenibacillus sp. <400> 1 aacacacaca cacacacaca caacacacat aaaatataca tatacataac atatataaac 60 atatataaaa aaaaacacaa aaaaaccgtt agagtttgga tcatggctca ggacgaacgc 120 tggcggcgtg cctaatacat gcaagtcgag cgggctttgc cttcgggtaa agctagcggc 180 ggacgggtga gtaacacgta ggcaacctgc ctgtaaggct gggataacta ccggaaacgg 240 tagctaagac cggataagtg gtcttctcgc atgaggagat caagaaacac ggggcaacct 300 gtggcttaca gatggacctg cggcgcatta gctagttggt ggggtaacgg ctcaccaagg 360 cgacgatgcg tagccgacct gagagggtga tcggccacac tgggactgag acacggccca 420 gactcctacg ggaggcagca gtagggaatc ttccgcaatg gacgcaagtc tgacggagca 480 acgccgcgtg agtgatgaag gttttcggat cgtaaagctc tgttgccagg gaagaatgtc 540 gcggagagta actgctctgc gaatgacggt acctgagaag aaagccccgg ctaactacgt 600 gccagcagcc gcggtaatac gtagggggca agcgttgtcc ggaattattg ggcgtaaagc 660 gcgcgcaggc ggccgtttaa gtctggtgtt taagcccggg gctcaacccc ggttcgcact 720 ggaaactggg cggcttgagt gcaggagagg aaagcggaat tccacgtgta gcggtgaaat 780 gcgtagatat gtggaggaac accagtggcg aaggcggctt tctggcctgt aactgacgct 840 gaggcgcgaa agcgtgggga gcaaacagga ttagataccc tggtagtcca cgccgtaaac 900 gatgagtgct aggtgttagg ggtttcgata ctccttggtg ccgaagtaaa cacaataagc 960 actccgcctg gggagtacgc tcgcaagagt gaaactcaaa ggaattgacg gggacccgca 1020 caagcagtgg agtatgtggt ttaattcgaa gcaacgcgaa gaaccttacc aggtcttgac 1080 atcccgatga aagcactaga gatagtgccc ctcttcggag cattggagac aggtggtgca 1140 tggttgtcgt cagctcgtgt cgtgagatgt tgggttaagt cccgcaacga gcgcaaccct 1200 tgaacttagt tgccagcatt aagttgggca ctctaagttg actgccggtg acaaaccgga 1260 ggaaggtggg gatgacgtca aatcatcatg ccccttatga cctgggctac acacgtacta 1320 caatggccgg tacaacggga agcaaagtcg cgagatggag ccaatcctaa gaaagccggt 1380 ctcagttcgg attgcaggct gcaactcgcc tgcatgaagt cggaattgct agtaatcgcg 1440 gatcagcatg ccgcggtgaa tacgttcccg ggtcttgtac acaccgcccg tcacaccacg 1500 agagtttaca acacccgaag tcggtggggt aaccgtaagg agccagccgc cgaaggtggg 1560 gtagatgatt ggggtgaagt cgtaacaagg taaaccgtaa attccggc 1608

Claims (6)

Fanny Bacillus thyramnensis JR103 (Accession No. KCTC18519P) strain having antimicrobial activity.
The method according to claim 1, wherein the Fannibacillus thyramansis JR103 strain is selected from the group consisting of Candida albicans , Bacillus subtilis , Staphylococcus aureus , Aspergillus niger, niger , Pseudomonas aeruginosa , and Escherichia coli . The strain is characterized in that it has an antimicrobial activity against a microorganism selected from the group consisting of Pseudomonas aeruginosa , Escherichia coli , Pseudomonas aeruginosa and Escherichia coli .
The strain according to claim 1, wherein the Fannibacillus thyramansis JR103 strain produces a surfactant.
The strain according to claim 1, wherein the Fannibacillus thyramansis JR103 strain produces poly-L-lysine.
Wherein the composition comprises at least one selected from the group consisting of Fanny Bacillus thyramnensis JR103 (Accession No. KCTC18519P), a culture of the strain, a concentrate of the strain or the culture, and a dried product thereof.
Wherein the composition comprises at least one selected from the group consisting of a culture of a strain of Fanny Bacillus thyramnensis JR103 (accession number KCTC18519P), a concentrate of a culture solution, and a dried product.

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