KR101100716B1 - Antibiotics-producing a novel strain of Streptomyces sp. BCNU 1001 and composition using the same - Google Patents

Abstract

The present invention is a novel Streptomyces genus isolated from soil sp.) BCNU 1001 strain and antimicrobial composition using the same.
Streptomyces sp. BCNU 1001 strain of the present invention exhibits a broad antimicrobial spectrum against various bacteria and fungi, and thus can be widely applied as an antimicrobial composition, especially against fungi that cause opportunistic sensitization in the human body. As it shows antimicrobial activity, it can be used as a skin treatment.

Description

A novel strain of Streptomyces genus BNC 1001 producing an antimicrobial active substance and an antimicrobial composition using the same {Antibiotics-producing a novel strain of Streptomyces sp. BCNU 1001 and composition using the same}

The present invention is a novel Streptomyces genus isolated from soil sp.) BCNU 1001 strain and antimicrobial composition using the same.

In relation to antibiotics, including the treatment of infectious diseases that cause various infections in the human body, research on microbial free natural bioactive substances is being actively conducted. In particular, fungi cause various fungi when the body's immune function is weakened, or antibiotics, hormones, and anticancer drugs are used, resulting in a variety of fungal diseases. Candidiasis caused by Candida sp. ), Aspergillus sp. Fungi, Aspegillosis, yeast fungus caused by Crytococcus neoformans , Mucor sp. And Rhizopus sp. ) bonding by fungi, such as fungi increases (zygomycosis), epi more fur tone flow koseom (Epidermophyton floccosum ) and Trichophyton sp.) Dematophytosis caused by fungi is a known fungal disease.

The discovered polyene compounds produced by the microorganisms of the various Streptomyces sp. Can form complexes with ergosterol to weaken the fungal cell membrane, Bacillus cereus Cispentacin isolated from is also known to have antimicrobial activity. In addition, amphotericin B, flucytosine and griseofulvin are widely used as natural antifungal agents. However, some antifungal agents, including amphotericin B, cause human toxicity, and the griseofulvin has a narrow antifungal spectrum, which necessitates the study of new natural antifungal substances that can replace the side effects of existing antifungal agents due to the emergence of resistant strains. It is true.

Accordingly, the present inventors have conducted research and efforts to provide microorganisms having excellent antifungal activity against various fungi, and have completed the present invention by finding that BCNU 1001 strain of Streptomyces genus isolated and identified in soil has excellent antifungal activity.

Therefore, the present invention is Streptomyces genus having antibacterial activity sp.) BCNU 1001 strain and an antimicrobial composition comprising the same.

The present invention is characterized by a novel Streptomyces sp. BCNU 1001 strain (Accession No .: KCTC 18186P) with antimicrobial activity.

In another aspect, the present invention is characterized by an antimicrobial composition comprising a strain, strain suspension, strain culture or culture supernatant of the strain.

In addition, the present invention,

Streptomyces sp. Streptomyces sp. BCNU 1001 strain in a complex medium of pH 6.0 ~ 7.0, 25 to 30 ℃, 5 to 15 days to obtain a culture;

Centrifuging the culture solution to obtain a culture supernatant; And

Extracting the culture supernatant with an organic solvent

Streptomyces sp. ( Streptomyces sp.) Having an antimicrobial activity comprising a method of producing a culture supernatant extract of BCNU 1001 strain is another feature.

Streptomyces sp. BCNU 1001 strain of the present invention exhibits a broad antimicrobial spectrum against various bacteria and fungi, and thus can be widely applied as an antimicrobial composition, especially against fungi that cause opportunistic sensitization in the human body. As it shows antimicrobial activity, it can be used as a skin treatment.

1 is a schematic diagram based on 16S rRNA analysis of Streptomyces sp. BCNU 1001 strain of the present invention.
Figure 2 is a photograph confirming the size of the inhibitory ring by plate medium diffusion method in order to measure the antimicrobial activity of the Streptomyces sp. BCNU 1001 strain of the present invention.
A: B. subtilis KACC 10111 B: M. luteus KACC 10488
C: Sta . aureus IMSNU 11088 D: E. coli KACC 10080
E: P. aeruginosa IMSNU 10191 F: P. fluorescens KACC 10071
G: C. albicans KACC 30062 H: Filobasidium neoformans KCTC 7902
I: Sa. cerevisiae KCTC 7968.
Figure 3 is a photograph showing the antifungal effect of human pathogenic fungi of Streptomyces sp. BCNU 1001 strain of the present invention.
a: A. niger b: T. mentagrophytes
c: T. rubrum

Hereinafter, the present invention will be described in detail.

Provided novel Streptomyces sp. BCNU 1001 strain derived from the soil of the present invention, the strain was deposited on January 27, 2010 to the Korea Institute of Bioscience and Biotechnology (Accession Number: KCTC) 18186P).

The strain, strain suspension, strain culture medium or culture supernatant of the strain according to the present invention exhibits a broad antimicrobial spectrum against various bacteria and fungi, and claims the antimicrobial composition comprising the strain, strain suspension, strain culture medium or culture supernatant of the strain. Include in the range. The antimicrobial composition is Bacillus subtilis ( Bacillus subtilis ), Micrococcus luteus ), Staphylococcus aureus ), Escherichia coli), Pseudomonas kid fishing not labor (Pseudomonas aeruginosa), Pseudomonas sense Floresta (Pseudomonas fluorescens), Aspergillus and this (Aspergillus niger), Candida albicans (Candida albicans ), Epidermophyton floccosum , Filobasidium neoformus neoformans ), Saccharomyces cerevisiae ), Trichophyton mentagrophytes and Trichophyton rubrum ) shows antimicrobial activity against the selected strain.

Streptomyces genus of the present invention sp.) The antimicrobial active material produced by the BCNU 1001 strain can be extracted from strain suspensions, strain cultures or culture supernatants of strains, but preferably from culture supernatants of strains.

The present invention is the genus Streptomyces (Streptomyces sp.) How to extract antimicrobial active material from the culture supernatant of strain BCNU 1001 is included in the scope.

First, Streptomyces sp. BCNU 1001 strain is cultured in a complex medium of pH 6.0-7.0 at 25-30 ° C. for 5-15 days to obtain a culture solution. Complex media for culturing Streptomyces sp. BCNU 1001 strain include International Streptomyces Project (ISP) media, nutrient agar (NA) media, brain heart infusion agar (BHI) media, sabouroud dextrose agar (SDA), PDA (potato dextrose agar) medium, NB (nutrient broth) medium and the like can be used, preferably ISP medium, BHI medium, SDA medium, NB medium is preferably used.

After centrifugation of the culture solution to obtain a culture supernatant, the culture supernatant is extracted with an organic solvent, and filtered and concentrated to obtain a culture supernatant extract from which an antimicrobial substance is optimally extracted. In this case, as the organic solvent, it is preferable to use an organic solvent selected from nucleic acids, dichloromethane and ethyl acetate according to polarity, and more preferably ethyl acetate.

Hereinafter, the present invention will be described by the following examples. The following examples are only examples for describing the present invention, and thus the scope of the technical spirit of the present invention is not changed or reduced.

Example 1 Identification of Strains Through Isolation, Physiology, Biochemical Characteristics and 16S rRNA Sequencing of Strains

The strain of the present invention was isolated from soil samples in the open mountains of Pyeongchang-gun, Gangwon-do.

After collecting the collected soil samples, they were refrigerated at 4 ℃. 1 g of the soil sample was placed in 10 ml of nutrient broth (NB) medium and pre-cultured at 28 ° C. for 3 weeks, followed by starch casein agar, oatmeal agar, PDA (potato dextrose agar), and NA. 100 μl each was plated in (nutrient agar) medium and incubated at 28 ° C. for 3 weeks to isolate pure actinomycetes.

For the identification of the isolated strains, the physiological and biochemical properties were examined by referring to Bergey's manual of systematic bacteriology, and the results are shown in Table 1 below.

Test Items Characteristics (+: positive reaction,-: negative reaction) Cultivation in Anaerobic Conditions － Gram Dyeing positivity shape Long filament, aerial growth motility － Production of Diffuse Pigments
(Production of diffusible pigment) + Melanin formation + Incubation temperature range 25-35 Incubation temperature 28 Culture pH pH 6.0-8.2 Culture pH pH 6.4 NaCl resistance 0.025M-0.7M Culture Optimal NaCl Concentration 0.05M Amylase + Protease － Cellulase － Lipase － Chitinase － Gelatinase + Catalase + Oxidase － Urease + H2S production + Nitrate reductase － Culture in the presence of phenol (0.01%) + Culture in the presence of sodium azide (0.01%) － Application of arabinose + Fructose ± Galactose ± Glucose + Glycerol + Lactose ± Maltose + Mannitol ± Mannose ± Rhamnose ± Starch + Sucrose ± Xylose + Sensitivity to 10 ug of Streptomycin sensitive Sensitivity to Ampicillin 10 ug tolerance Sensitivity to 10 ug of Tetramycin tolerance Sensitivity to Gentamicin 10 ug sensitive Sensitivity to Chlorampenicol 5 ug tolerance

As shown in Table 1, the strain can be cultured at a temperature range of 25 ~ 35 ℃, pH 6.0 ~ 8.2, it was found that it can be cultured at a salt concentration of 0.025 M ~ 0.7 M. It also produces amylase, gelatinase, catalase, and urease, and it has been shown to use a variety of carbon sources in its magnetization ability, especially glucose, maltose, Growth was good in starch. And 10 ug of ampicillin, tetracycline and 5 ug of chlorampenicol.

In addition, 16S rRNA sequencing of the strain was performed, and PCR analysis of 16S rRNA was performed using 16F (5'-AGTTTGATCCTGGCTCAG-3 ') and 1492R (5'-ACGGCTACCTTGTTACGA CTT-3') primers.

The nucleotide sequence was analyzed by Blast search and found to have 98% homology with S. nojiriensis and S. cinnamonensis . Systemically, S. virginiae and S. lavendulae And of S. spororaveus It was identified as a strain belonging to the subcluster ( s ubcluster), which was named Streptomyces sp. BCNU 1001 strain, and was deposited on January 27, 2010 to the Korea Institute of Bioscience and Biotechnology. Number: KCTC 18186P).

The phylogenetic tree of the Streptomyces sp. BCNU 1001 strain was made through a neighbor joining method and bootstrap analysis, and the schematic diagram thereof is shown in FIG. 1.

In addition, the culture characteristics of the Streptomyces sp. BCNU 1001 strain were characterized by ISP (International Streptomyces Project) medium, NA medium, brain heart infusion agar (BHI) medium, sabouroud dextrose agar (SDA), and PDA medium. At 28 ℃, was confirmed while incubating for 14 days, the results are shown in Table 2 below.

badge growth Aerial mycelium
(Aerial
mycelium) Substrate mycelia
(Substrate
mycelium) Spores Pigment of substrate mycelia
(Pigmentation of
substrate
mycelium) Soluble pigment
(Soluble
pigment) ISP-1 middle Medium, slightly white
cream color existence Bad, purple Pale gray none ISP-2 Good Cornucopia, reddish brown existence Medium, purple Yellow brown none ISP-3 Good Rich, pale red
Brown existence Medium, purple Yellow brown none ISP-4 Good Rich, Pink Cream existence Medium, purple Yellow brown none ISP-5 middle Bad existence Bad, purple Red orange none ISP-6 Good Bad existence Bad, purple Red orange bluish
purple ISP-7 Good Medium, pale brown
grey existence Good purple Red orange none NA Good Poor, pale yellow
Brown existence none Brown none BHI Good Bad existence Bad, purple Red orange none SDA Good Rich, pale red
Brown existence Good purple Brown none PDA middle Abundance, Gray Black existence Medium, purple Yellow brown none

ISP 2, ISP 3, ISP 4 and SDA medium to form a substrate mycelia and spores were found to be good growth, and ISP 6 medium was confirmed to represent a bluish violet (Bluish Violet) pigment.

Example 2 Investigation of Antimicrobial Activity

In order to determine the antimicrobial activity of Streptomyces sp. BCNU 1001 strain was tested as follows.

First, in order to measure the antimicrobial activity, gram positive bacteria Bacillus subtilis (Bacillus subtilisKACC 10111 (ATCC 465), Micrococos Luteus (Micrococcus luteus) KACC 10488 (ATCC 4698) and Staphylococcus aureus (Staphylococcus aureus) IMSNU 11088 (ATCC 6538) and gram negative bacteria E. coli (Escherichia coliIMSNU 10080 (ATCC 10798), Pseudomonas Aeroginosa (Pseudomonas aeruginosa) IMSNU 10191 (ATCC 10145) and Pseudomonas florence (Pseudomonas fluorescensKACC 10071 (ATCC 13525), Aspergillus niger as a human pathogenic fungusAspergillus nigerKACC 40280 (ATCC 4695), Candida Albicans (Candida albicansKACC 30062 (ATCC 10231), Epidermopython Flocosum (Epidermophyton floccosumKCTC 6921, Philobariumium neo-performance (Filobasidium neoformansKCTC 7902, Saccharomyces cerevisiaeSaccharomyces cerevisiaeKCTC 7968, trichophyton mentagropiteTrichophyton mentagrophytesKCTC 6077 (IFO 6202) and Trichophyton rubrum (Trichophyton rubrumKCTC 6375 was distributed by the Korea Agricultural Microbial Resources Center, the Korea Institute of Biological Resources, and the Microbiological Research Institute of Seoul National University.

Antimicrobial activity against the strain was determined using agar diffusion method. The strain was incubated with NB medium or LB medium until Mcfaland 0.5 (1.5 × 10 ⁸ ) under optimal culture conditions. In addition, 100 μl of the strain culture medium adjusted to Mcfaland 0.5 in ISP 1, ISP 2, BHI, SDA and NA medium was evenly spread and 10 μl of ISP 2 medium culture medium of Streptomyces sp. BCNU 1001 was instilled. . After incubation for 18 to 24 hours, the size of the inhibitory (clear zone) was measured, and the results are shown in Table 3 and FIG. 2.

badge Ring size (mm) ISP1 ISP2 BHI SDA NA B. subtilis 11.80 ± 1.93 15.20 ± 1.06 10.87 ± 1.21 15.33 ± 1.51 13.60 ± 0.53 M. luteus 17.80 ± 1.93 27.33 ± 3.06 11.87 ± 0.23 28.00 ± 6.08 15.20 ± 1.06 Sta . aureus 9.27 ± 0.64 8.22 ± 0.03 - - 9.33 ± 1.45 E. coli - - - - - P. aerusinosa - - - - - P. fluorescens - - - - - C. albicans 11.87 ± 0.23 12.53 ± 2.20 9.87 ± 1.03 13.47 ± 1.29 12.53 ± 2.20 F. neoformans 10.10 ± 0.21 11.80 ± 0.14 8.50 ± 0.35 9.00 ± 0.42 8.80 ± 0.14 Sa . cerevisiae 12.13 ± 2.50 13.87 ± 1.80 10.33 ± 0.58 15.33 ± 1.50 13.53 ± 0.50

As shown in Table 3, Streptomyces sp. BCNU 1001 strain of the present invention had a broad antimicrobial spectrum against Gram-positive bacteria and human pathogenic fungi, especially 27.33 mm against M. luteus in ISP 2 medium. It showed high activity of and inhibited 12.53 mm and 13.87 mm of pathogenic fungi C. albicans and S. cerevisiae , respectively. Gram-negative bacteria did not show antimicrobial activity.

In addition, the main body of pathogenic fungi Aspergillus and this (Aspergillus niger ), Trichophyton mentagrophytes and Trichophyton rubrum ), Whipps (Whipps, J. M (1987), Effect of media on growth and interactions between a range of soil-borne glasshouse pathogens and antagonistic fungi. Phytol. 107, 127-142.) The antimicrobial spectrum was investigated by the replacement culture method described in (Fig. 1). The inhibition capacity was expressed by growth inhibition (GI) (Equation 1). The results are shown in Table 4 and FIG. 3.

GI: growth inhibition (%)

R1: Distance between bacteria after incubation, R2: Inoculation distance before incubation

A. niger T. rubrum T. mentagrophytes GI (%) 46.97 ± 0.13 46.10 ± 1.09 45.59 ± 1.01

As shown in Table 4, the Streptomyces sp. BCNU 1001 strain of the present invention has a high inhibitory effect of 45-46% against Aspergillus niger, Trichophyton mentagrophyte, and Trichophyton rubrum. As shown, the Streptomyces sp. BCNU 1001 strain of the present invention was confirmed to have excellent antibacterial activity against human pathogenic fungi.

Example 3 Extraction of Antimicrobial Active Materials and Measurement of Minimum Inhibitory Concentration (MIC) thereof

The culture medium was obtained by culturing BCNU 1001 strain of Streptomyces sp. For 7 days at 28 ° C. and pH 6.4 in ISP 2 medium.

(Issue 1, ISP 2, BHI, SDA and NB were used as the basic medium to investigate the optimum production conditions of the antimicrobial material, and cultured for 7-14 days at various pH, temperature conditions and NaCl concentrations. The culture medium was collected at intervals of time and used as a sample for investigation of antimicrobial activity. 7 days of incubation at 6.4 was the most optimal production condition)

1 L of the culture solution was collected and centrifuged (7,500 × g, 10 minutes, 4 ° C.) to obtain a culture supernatant. 2 L of 99.5% by weight ethyl acetate was added thereto, and extracted at 25 ° C. for about 24 hours. The extract was filtered using Whatman no. 2 filter paper, concentrated under reduced pressure at 37 ° C. using a rotary evaporator to obtain 0.123 g of the culture supernatant extract.

Using the culture extract sample, the minimum inhibitory concentration was measured for three Gram-positive bacteria, three Gram-negative bacteria, and seven human pathogenic fungi listed in Example 1.

In order to determine the minimum inhibitory concentrations of Gram-positive bacteria and Gram-negative bacteria, 100 μl of the target strain cultures adjusted to Mcfaland 0.5 using ISP 2 medium in 96 well plates, and the culture supernatant extracts by ethyl acetate were doubled in steps. Dilutions were made for each concentration and added to 10 μl (National Committee for Clinical Laboratory Standards. (2004), Methods for antimicrobial susceptibility testing of anaerobic acteria: Approved Standard.6th ed., Vol. 24, NCCLS Document M11-A6.Pennsylvania , USA). Optimum temperature of each bacteria ( P. fluorescens : After 24 hours incubation at 26 ℃ / B. subtilis , M. lutes : 30 ℃ / S. aureus , E. coli : 35 ℃), the minimum inhibitory concentration was determined by measuring the absorbance at 570 nm, and Streptomycin and The results are shown in Table 5 below.

Strain
Streptomycin
(Μg / disc) Minimum Inhibitory Concentration (MIC) of Cultured Supernatant Extracts
(mg / disc) B. subtilis 1.25 0.25 M. luteus 0.625 0.125 Sta. aureus 0.625 0.125 E. coli 0.625 0.25 P. aeruginosa 5 0.25 P. fluorescens 1.25 0.125

As shown in Table 5, the growth of Gram-positive bacteria M. luteus , S. aureus and Gram-negative bacteria P. fluorescens were inhibited at low concentrations of 0.125 mg / ml, and were inhibited by B. subtilis, E. coli and P. aeruginosa . Growth was also inhibited at 0.25 mg / ml concentration.

In addition, the minimum inhibitory concentration of human pathogenic fungi was inoculated with the target fungi in PDA medium, and the culture supernatant extracts obtained by ethyl acetate were diluted two-fold step by step, and then dipped into sterilized paper discs by 10 μl. After culturing at 28 ° C. for 7 days, the minimum inhibitory concentration was measured by measuring absorbance at 570 nm, and compared with Itraconazol as a control, and the results are shown in Table 6 below.

Strain Itraconazol
(Μg / disc) Minimum Inhibitory Concentration (MIC) of Cultured Supernatant Extracts
(mg / disc) A. niger 0.0199 0.5 C. albicans 0.0796 0.125 Ep. floccosum 1.274 > 1 F. neoformans 0.0398 > 1 Sa. cerevisiae 0.159 0.25 T. mentagrophytes 2.548 0.5 T. rubrum 2.548 > 1

Was inhibited the growth of C. albicans in a low concentration of 0.125 mg / ml As shown in Table 6, Sa from 0.25 mg / ml. cerevisiae , inhibited growth of A. niger and T. mentagrophytes at concentrations of 0.5 mg / ml.

As a result, it was confirmed that the Streptomyces sp. BCNU 1001 strain of the present invention can exhibit a broad antimicrobial spectrum against various bacteria and fungi.

Korea Research Institute of Bioscience and Biotechnology KCTC18186 20100127

Claims (5)

delete delete delete delete Streptomyces sp. Streptomyces sp. BCNU 1001 strain in a complex medium of pH 6.0 ~ 7.0, 25 to 30 ℃, 5 to 15 days to obtain a culture;
Centrifuging the culture solution to obtain a culture supernatant;
Extracting the culture supernatant with an organic solvent; And
Filtering the extract using Whatman filter paper, concentrating under reduced pressure using a rotary evaporator,
The organic solvent is a method of producing a culture supernatant extract of Streptomyces sp. BCNU 1001 strain having antibacterial activity, characterized in that the solvent selected from nucleic acid, dichloromethane, ethyl acetate.

Images