KR102542231B1 - Silver nanoparticles derived from Flavobacteriaceae bacterium F202Z8, and uses thereof - Google Patents

Abstract

The present invention relates to silver nanoparticles derived from the Flavobacteriaceae bacterium strain F202Z8 and uses thereof, wherein the Flavobacteriaceae bacterium F202Z8 strain is silver nanoparticles under conditions containing silver nitrate (AgNO ₃ ) , and confirming that the silver nanoparticles derived from the bacterium F202Z8 strain exhibit antibacterial activity against gram-positive bacteria and gram-negative bacteria, the Flavobacteriaceae bacterium F202Z8 strain Provided are silver nanoparticles derived from silver, a method for preparing the same, and an antibacterial composition including the same.

Description

Silver nanoparticles derived from Flavobacteriaceae bacterium F202Z8 strain and uses thereof {Silver nanoparticles derived from Flavobacteriaceae bacterium F202Z8, and uses thereof}

The present invention relates to silver nanoparticles derived from the Flavobacteriaceae bacterium strain F202Z8 and uses thereof.

Nanoparticles generally refer to a size of 100 nm or less, and as particles larger than micro are nanosized, their size, shape, distribution, etc. change into unique properties that are completely different from those of the original material. Metal and metalloid nanoparticles are applied in various fields such as antibacterial activity, drug delivery, cancer treatment, medical diagnosis, and bioimaging. For this reason, research on metal and metalloid nanoparticles has attracted attention in recent years. Although traditional methods of physicochemical methods have been used for many years to synthesize nanoparticles, they are expensive and may be exposed to toxic substances caused by various reducing agents and stabilizers in the synthesis process. Research on the synthesis of biological nanoparticles using bacteria, fungi, and plants has been attempted as an environmentally friendly method that can easily increase productivity at low cost. In particular, research on biological synthesis using bacteria showing high adaptability in extreme environments is in the limelight. are receiving

Although the synthesis mechanism using biological nanoparticles has not been precisely identified, biological synthesis using bacteria can be divided into two types: intracellular and extracellular. Intracellular synthesis is produced by interactions between intracellular proteins and cofactors by attracting metal ions into cells through electrical interactions between carboxyl groups located on cell walls. Extracellular synthesis is synthesized by reduction of metal ions through proteins, enzymes, and organic substances secreted in cell wall components or culture medium. Extracellular synthesis is preferred because it is less costly than intracellular synthesis, is easy to extract without destroying cell walls, and has high efficiency, and also stabilizes nanoparticles due to various biomolecules secreted extracellularly during the synthesis process.

Silver nanoparticles are applied in various industrial fields from medicine to electronics. In particular, applications in the field of medicine (joint disease, wound treatment, etc.) account for more than 23% of all nanoparticles, and are widely used as effective antibacterial agents such as prevention of skin diseases, textile materials, and antibacterial, antifungal, and antiviral. Until now, continuous research has been conducted to utilize metal nanoparticles such as gold, silver, copper, titanium, and zinc as antibacterial agents. It is most suitable as an antibacterial material. Research on the synthesis of biological silver nanoparticles with advantages of low cost, non-toxicity, efficient productivity, and environmental friendliness has been continuously reported. At the same time, the diversity of new source materials for the synthesis of various nanoparticles also requires continuous research.

Korean Patent Registration No. 10-1905026 (2018. 10. 08. Notice)

In order to solve the above problems, an object of the present invention is to confirm that the bacterium F202Z8 strain in Flavobacteria produces silver nanoparticles under conditions containing silver nitrate (AgNO ₃ ), and the bacteria in Flavobacteria By confirming that silver nanoparticles derived from strain Leeum F202Z8 exhibit antibacterial activity against gram-positive and gram-negative bacteria, silver nanoparticles derived from Flavobacteriaceae bacterium strain F202Z8, a method for preparing the same, and including the same It is to provide an antibacterial composition.

The present invention provides silver nanoparticles derived from the Flavobacteriaceae bacterium strain F202Z8 deposited under accession number KCCM12947P.

In addition, the present invention provides a method for preparing silver nanoparticles comprising culturing the Flavobacteriaceae bacterium F202Z8 strain deposited under accession number KCCM12947P in a solution containing silver nitrate (AgNO ₃ ).

In addition, the present invention provides an antibacterial composition comprising a Flavobacteriaceae bacterium strain F202Z8 as an active ingredient.

In addition, the present invention provides an antibacterial composition comprising, as an active ingredient, silver nanoparticles derived from the Flavobacteriaceae bacterium strain F202Z8 deposited under Accession No. KCCM12947P.

According to the present invention, the bacterium F202Z8 strain in Flavobacteria can mass-produce silver nanoparticles by producing silver nanoparticles in the presence of silver nitrate (AgNO ₃ ). Since the silver nanoparticles of exhibit antibacterial activity against gram-positive and gram-negative bacteria, it is possible to provide an antibacterial composition containing the bacterium F202Z8 strain or silver nanoparticles derived from the bacterium F202Z8 in case of Flavobacteria.

1 is a photograph of solid-phase culture (left) and liquid-phase culture (right) of the bacterium F202Z8 strain in Flavobacteria.
Figure 2 is a phylogenetic analysis result of the bacterium F202Z8 strain in Flavobacteria.
FIG. 3 is a result of analyzing the absorbance (A) and concentration (B) of silver nanoparticles (AgNPs) for evaluating the ability of the bacterium F202Z8 strain to produce silver nanoparticles in Flavobacteria.
4 shows TEM (Transmission Electron Microscopy) images (A, B) and EDS (Energy-Dispersive X-ray Spectroscopy) profile (C) results of silver nanoparticles derived from the bacterium F202Z8 strain in Flavobacteria.
5 is a disk diffusion method (A) and minimum inhibitory concentration (B) results for evaluating the antibacterial activity of silver nanoparticles derived from the bacterium F202Z8 strain in Flavobacteria.

The terms used in this specification have been selected from general terms that are currently widely used as much as possible while considering the functions in the present invention, but these may vary depending on the intention of a person skilled in the art, precedent, or the emergence of new technologies. In addition, in a specific case, there is also a term arbitrarily selected by the applicant, and in this case, the meaning will be described in detail in the description of the invention. Therefore, the term used in the present invention should be defined based on the meaning of the term and the overall content of the present invention, not simply the name of the term.

Unless defined otherwise, all terms used herein, including technical or scientific terms, have the same meaning as commonly understood by one of ordinary skill in the art to which the present invention belongs. Terms such as those defined in commonly used dictionaries should be interpreted as having a meaning consistent with the meaning in the context of the related art, and unless explicitly defined in this application, it should not be interpreted in an ideal or excessively formal meaning. don't

Numerical ranges are inclusive of the values defined therein. Every maximum numerical limitation given throughout this specification includes every lower numerical limitation, as if such lower numerical limitations were expressly written. Every minimum numerical limitation given throughout this specification includes every higher numerical limitation, as if such higher numerical limitations were expressly written. Every numerical limitation given throughout this specification will include every better numerical range within the broader numerical range, as if the narrower numerical limitations were expressly written.

Hereinafter, the present invention will be described in more detail.

The inventors of the present invention predicted that there would be a metal-related metabolic process as a new rapid species isolated from coastal rusty iron, and purified the Flavobacteriaceae bacterium F202Z8 strain, a rapid species, derived from the F202Z8 strain. The present invention was completed by isolating silver nanoparticles and evaluating their antibacterial activity against four strains of pathogenic bacteria , Bacillus subtilis, Escherichia coli, Salmonella typhimurium, and Staphylococcus aureus .

The present invention provides silver nanoparticles derived from the Flavobacteriaceae bacterium strain F202Z8 deposited under accession number KCCM12947P.

The F202Z8 strain is a new rapid species isolated from coastal rusty iron, has a metal-related metabolic process, and exhibits antibacterial activity against four strains of pathogenic bacteria , Bacillus subtilis, Escherichia coli, Salmonella typhimurium, and Staphylococcus aureus .

The silver nanoparticles derived from the F202Z8 strain have a diameter ranging from 10 to 103 nm, and exhibit antibacterial activity against Gram-negative bacteria such as Escherichia coli and Salmonella typhimurium and Gram-positive bacteria such as Bacillus subtilis and Staphylococcus aureus .

In addition, the present invention provides a method for preparing silver nanoparticles comprising culturing the Flavobacteriaceae bacterium F202Z8 strain deposited under accession number KCCM12947P in a solution containing silver nitrate (AgNO ₃ ).

In addition, the present invention provides an antibacterial composition comprising a Flavobacteriaceae bacterium strain F202Z8 as an active ingredient.

In addition, the present invention provides an antibacterial composition comprising, as an active ingredient, silver nanoparticles derived from the Flavobacteriaceae bacterium strain F202Z8 deposited under Accession No. KCCM12947P.

Hereinafter, examples will be described in detail to aid understanding of the present invention. However, the following examples are merely illustrative of the contents of the present invention, but the scope of the present invention is not limited to the following examples. The embodiments of the present invention are provided to more completely explain the present invention to those skilled in the art.

<Example 1> F202Z8 strain isolation and identification

On June 1, 2018, samples were taken from rusty iron on the coast of Gomseom Beach (36°35' 25"N, 126°17' 17"E), Sinon-ri, Nam-myeon, Taean-gun, Chungcheongnam-do. In a clean bench, the sample was suspended in sterile water, diluted 1/100, inoculated into ZoBell agar, and incubated at 20° C. for 5 days. A single strain with an orange pigment was inoculated into Marine agar again and isolated as a pure strain. To identify the F202Z8 strain, 1 ml of the strain cultured at 27 ° C for 3 days in marine liquid medium was taken and genomic DNA was extracted using the “Exgene DNA Extraction Kit” (GeneAll, South Korea) according to the manufacturer's protocol. PCR was performed using 27F and 1492R primers for 16S rRNA gene amplification. The PCR product was purified using “MEGAquick-spin Plus Total Fragment DNA Purification Kit” (iNtRON, South Korea) and then analyzed using a sequencer (Applied Biosystems 3730XL) from Macrogen (South Korea). The 16S rRNA gene base sequence (SEQ ID NO: 1) of the F202Z8 strain thus obtained was compared with information of previously reported strains through BLAST search of the EzBioCloud database. A phylogenetic tree of F202Z8 species and neighboring species was analyzed through the MEGA program.

As shown in Figure 1, the F202Z8 strain, a new rapid species isolated from coastal rusty iron, has an orange pigment (Fig. 1 left, right), and when grown in marine broth, the strains are entangled with each other (Fig. 1 right) appeared to have As a result of identification through the 16S rRNA gene, Flavobacteriaceae showed 94.5% similarity with the genus Maribacter belonging to the family. In addition, as a result of phylogenetic analysis of species F202Z8 and adjacent species, as shown in Figure 2, it was classified between Pricia antarctica ZS1-8 ^T and Costertonia aggregata KCCM 42265 ^T and belonged to the Flavobacteria family.

<Example 2> Synthesis of silver nanoparticles using F202Z8 strain

It was inoculated into a 50ml Erlenmeyer flask containing sterilized 20ml marine liquid medium and primary culture was performed at 150rpm for 3 days at 27°C. The primary cultured strain was re-inoculated with 1% in a 100ml Erlenmeyer flask containing fresh 60ml marine liquid medium, and then secondary culture was performed under the same conditions as above. 30ml of the secondary culture medium was divided into two 50ml falcon tubes and separated by centrifugation at 20°C at 7,000rpm for 10 minutes. After washing the strain pellet twice with sterilized PBS, about 0.4 g each was prepared with the same weight. For the synthesis of silver nanoparticles, 30 ml of 1 mM silver nitrate was added to one tube, and 30 ml of 1 mM silver nitrate (AgNO ₃ ) was added by heat treatment at 100 ° C. for 10 minutes to the other tube to be used as a control, and reacted at room temperature of 25 ° C. In addition, 30 ml of the supernatant separated from the secondary culture was sterilized with a 0.2 μm syringe, and a final concentration of 1 mM silver nitrate (AgNO ₃ ) was added thereto and used as a negative control. Brown change was observed after 36 hours of reaction, and centrifugation was performed at 4,000 rpm for 10 minutes to remove strains, and the supernatant was stored at 4°C. Ethanol was added to the isolated strain pellet in a ratio of 1:1 to confirm the presence/absence of intracellular synthesis and vortexing for 2 minutes. Additionally, 10 ml of hexane was added and vortexed for 2 minutes, and the supernatant was stored at 4°C.

<Example 3> Characteristic analysis of silver nanoparticles

3-1) Absorbance measurement

100 μl of each sample of the synthesized nanoparticles and two controls was added to a 96-well plate, and then measured at 5 nm intervals in a wavelength range of 300 to 700 nm using a UV-vis spectrophotometer (SpectrMas i3x, Molecular Devices, USA).

3-2) Analysis of shape, concentration and elemental composition

5 μl of the synthesized silver nanoparticles were fixed on a carbon-coated copper grid for 5 minutes, removed with an experimental towel, and then dried. The dried samples were analyzed for morphology and elemental components of nanoparticles using a spherical aberration corrected transmission electron microscope (JEM-ARM200F, JEOL Ltd, Japan). The size and concentration of silver nanoparticles were analyzed using a nanoparticle analyzer (LM10, Nanosight Ltd, UK).

As shown in FIG. 3 , after 36 hours of reaction with silver nitrate (AgNO ₃ ), it was observed that the silver nanoparticles were changed to a dark brown color (inner red line container in FIG. 3A). It is known that silver ions show absorbance at 400-450 nm due to surface plasmon resonance when converted to nanoparticles. From the above results, an absorption peak specific to silver nanoparticles (red line in FIG. 3A) was observed at 450 nm, suggesting that the newly isolated silver nanoparticles derived from the F202Z8 species were successfully synthesized. As a result of confirming the concentration and size of the synthesized silver nanoparticles using a nanoparticle analyzer, an average size of 48 nm (FIG. 3B) and 1.5×10 ⁹ /ml were synthesized.

As shown in FIG. 4, as a result of observing the shape of the synthesized silver nanoparticles using a spherical aberration corrected transmission electron microscope (Cs corrected Transmission Electron Microscopy), similar to previously reported silver nanoparticles, they had a range of about 10 to 103 nm. It had a spherical or near-spherical shape (Fig. 4A, B), and as a result of analyzing the elemental components of the synthesized silver nanoparticles by EDS (Energy-Dispersive X-ray Spectroscopy), an absorption peak was shown at 3 keV, and 91.11% A silver component of was observed (Fig. 4C).

<Example 4> Investigation of antibacterial activity

4-1) Disc Diffusion Aaasy

Four pathogens, Bacillus subtilis KCTC 3135, Escherichia coli KCTC 2441, Salmonella typhimurium KCTC 1925, and Staphylococcus aureus ATCC 6538, were inoculated into LB broth and incubated for 24 hours at optimum temperature. Each strain was plated on LB solid medium to a size of about 5 × 10 ⁶ , and then an 8 mm disk containing 10, 20, 30 μl of synthesized silver nanoparticles and 20 μl of negative control was placed thereon, and then each strain for 1 to 2 days. The inhibitory effect of them was observed.

4-2) Measurement of Minimum Inhibitory Concentration

Three pathogens, Bacillus subtilis KCTC 3135, Escherichia coli KCTC 2441, and Staphylococcus aureus ATCC 6538, were inoculated into LB broth and incubated for 24 hours at optimum temperature. After inoculating 0.1% of each strain in a dedicated 50ml falcon tube containing sterilized 10ml LB liquid medium and adding silver nanoparticles by concentration of 10 ~ 100μg (/ml) at the same time, the automatic microbial incubator (RTS-1, Biosan, UK) ) was measured at OD600nm while incubating at 30 ° C. for 36 hours and was performed in 3 repetitions.

As shown in FIG. 5, antimicrobial activity increased with the concentration of silver nanoparticles against both gram-negative bacteria ( Escherichia coli, Salmonella typhimurium ) and two positive bacteria ( Bacillus subtilis, Staphylococcus aureus ) (FIG. 5A). . For the four pathogenic strains, the antibacterial activity of silver nanoparticles was in the order of S. aureus, B. subtilis, E. coli, and S. typhimurium, especially against S. aureus , a representative antibiotic-resistant strain that can cause life-threatening diseases. showed the highest antibacterial activity against For three pathogenic strains, except for S. typhimurium , which has the lowest antibacterial activity, silver nanoparticles were treated by concentration and the minimum inhibitory concentration (MIC) was determined. As a result, the MIC values of the silver nanoparticles for E. coli, B. subtilis, and S. asureus were 80, 40, and 30 μg/ml, respectively, showing a pattern consistent with the above disc diffusion results (FIG. 5B).

Having described specific parts of the present invention in detail above, it is clear to those skilled in the art that these specific descriptions are only preferred embodiments, and the scope of the present invention is not limited thereby. do. That is, the substantial scope of the present invention is defined by the appended claims and their equivalents.

Korea Microbial Conservation Center (Overseas) KCCM12947P 20210202

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Claims (6)

Silver nanoparticles derived from Flavobacteriaceae bacterium strain F202Z8 deposited under accession number KCCM12947P. The silver nanoparticles of claim 1, wherein the silver nanoparticles have a diameter ranging from 10 to 103 nm. The silver nanoparticles of claim 1, wherein the silver nanoparticles exhibit antibacterial activity against Gram-negative bacteria such as Escherichia coli and Salmonella typhimurium and Gram-positive bacteria such as Bacillus subtilis and Staphylococcus aureus . A method for producing silver nanoparticles comprising culturing a Flavobacteriaceae bacterium strain F202Z8 deposited under accession number KCCM12947P in a liquid containing silver nitrate (AgNO ₃ ). delete An antimicrobial composition against gram-negative bacteria and gram-positive bacteria comprising, as an active ingredient, silver nanoparticles derived from the Flavobacteriaceae bacterium strain F202Z8 deposited under accession number KCCM12947P,
The silver nanoparticles exhibit antibacterial activity against Gram-negative bacteria such as Escherichia coli and Salmonella typhimurium and Gram-positive bacteria such as Bacillus subtilis and Staphylococcus aureus .

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