CN109706187B - Method for synthesizing nano-silver by using streptomyces parvulus K-15 and application - Google Patents
Method for synthesizing nano-silver by using streptomyces parvulus K-15 and application Download PDFInfo
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Abstract
The invention relates to a method for synthesizing nano-silver by utilizing streptomyces microformis K-15 and application thereof, belonging to the technical field of biosynthesis of nano materials. The nano silver synthesized by the method is spherical or approximately spherical, the average particle size is 28.56nm, and the polydispersity index (PDI) is 0.274. Bioactive molecules in the extracted supernatant of the streptomyces microformis K-15 thalli are coated on the surface of formed nano silver particles besides the reduction process of mediated silver ions, so that the nano silver particles have good stability and biological affinity, and are combined with the unique physicochemical property of the nano particles, so that the nano silver particles can more easily permeate into microbial cells, and the microbial cells are killed by interfering the normal metabolism of the microbes. In addition, the combined use of the nano silver and the antibacterial drug can generate synergistic bacteriostatic effects with different degrees on microorganisms, so that the nano silver antibacterial agent has wider application prospect in the field of biomedicine.
Description
Technical Field
The invention belongs to the technical field of biosynthesis of nano materials, and particularly relates to a method for synthesizing nano silver by utilizing streptomyces microformis K-15 and application thereof.
Background
The nano material refers to various nano structure materials with the property of nano size range, the unique characteristics are endowed by the small size of the nano material, and the nano material is widely applied to the fields of optics, electronics, catalysis, biomedicine, mechanics, magnetism, energy science, agriculture, environment and the like.
Silver is a rare precious metal element that has been used since ancient times for the production of tableware, currency, jewelry, photography, dental materials, explosives and anti-infective treatments. With the rapid development of nanotechnology, the nano-silver simple substance (nano-silver) exhibits unique physicochemical and biological properties, and becomes one of the most promising nano-materials.
In recent years, as the demand of nano silver in industry is continuously increased, the defects of the conventional nano silver synthesis technology are gradually exposed. The physical synthesis method of nano silver comprises an electric arc discharge method, a physical vapor condensation method, an energy ball milling method, a direct current magnetron sputtering method and the like, and has high requirements on instruments and equipment, high production cost and high energy consumption. Chemical methods can be divided into chemical reduction methods, electrochemical methods, radiation-assisted chemical methods, pyrolysis methods and the like, and although the chemical methods are widely applied to commercial production, the chemical methods not only increase the cost and pollute the environment, but also possibly enhance the toxicity of the nano-silver due to the addition of toxic chemical reducing agents, stabilizing agents and dispersing agents in the process of synthesizing the nano-silver, thereby limiting the application range of the nano-silver in the biomedical field.
With the popularization of green chemistry concepts and the improvement of environmental awareness of countries in the world, the development of a nano-silver synthesis method towards green, non-toxic, environmental-friendly and energy-saving is also needed urgently, so that a nano-silver synthesis method based on biological materials is produced at the same time. As the microorganism has the advantages of wide distribution, easy culture, rapid propagation and the like, the microorganism has great potential as a natural biological material for the intracellular or extracellular controllable synthesis research of the nano-silver.
Fungi are common starting strains for synthesizing nano-silver microorganisms, but the fungi can secrete potential mycotoxins, so that the safety of the synthesized nano-silver is reduced; meanwhile, the nano-silver synthesized by the fungi has large particle size and polydispersity, and the effect of the nano-silver is influenced to a certain extent. Actinomycetes (particularly streptomyces) are a class of microorganisms known for their excellent ability to synthesize secondary metabolites, have a long history of safe production and industrial application, and have great advantages as microbial materials for nano-silver synthesis. In addition, as the streptomyces is a main fermentation strain for industrial production of antibiotics, the antibacterial active substances secreted by the streptomyces can be coated on the surface of the synthesized nano-silver particles, so that the antibacterial activity of the nano-silver is enhanced. Therefore, the streptomyces is expected to become a dominant strain for large-scale industrial synthesis of the nano-silver.
Disclosure of Invention
Aiming at the problems in the prior art, the invention provides a method for synthesizing nano-silver by utilizing streptomyces parvulus K-15 and application thereof. The synthesis method is simple, convenient, efficient, environment-friendly and energy-saving, and does not need to add any chemical reducing agent, stabilizer or dispersing agent. The nano silver synthesized by the method has high yield, small and uniform particle size, and has strong antibacterial activity on gram positive/negative bacteria and fungi.
In order to achieve the purpose, the invention provides the following technical scheme:
a method for synthesizing nano-silver by utilizing streptomyces microformis K-15 and an application thereof comprise the following steps:
(1) activation of Streptomyces microfeatus K-15 strain: streaking streptomyces microformis K-15 slant strain stored at 4 ℃ on a solid culture medium for culture to obtain spores, and scraping a proper amount of spores to obtain a monospore suspension;
(2) obtaining streptomyces microfeatus K-15 thalli: inoculating the monospore suspension into a liquid culture medium, obtaining thallus of streptomyces microformis K-15 through fermentation culture, and thoroughly washing the thallus by using sterile deionized water to remove residual culture medium components;
(3) preparation of the cell leaching supernatant: dispersing the wet thalli in sterile deionized water again, oscillating and leaching, centrifuging and filtering a leaching solution to completely remove the thalli, and collecting thalli leaching supernatant containing bioactive substances;
(4) and (3) synthesis of nano silver: taking the extracted supernatant of the streptomyces microfeatus K-15 thallus obtained in the step (3) as a reaction substrate, adding a silver nitrate solution into the reaction substrate to perform a synthesis reaction of nano-silver, and centrifuging, washing and drying the reaction solution to obtain the nano-silver.
Preferably, in step (1), the medium slant is a solid culture medium of Gao's number one.
More preferably, the formula of the Gao's first solid medium is as follows: the following components are contained in each 1L of distilled water: 20g of soluble starch, 0.5g of NaCl and KNO31 g，K2HPO4.3H 2O 0.5g，MgSO4.7H2O 0.5g，FeSO4.7H20.01g of O and 15-20 g of agar powder, and high-temperature and high-pressure sterilization is needed before use.
More preferably, the pH of the solid culture medium of kaowski No. one is 7.6.
Preferably, in step (1), the culture conditions are: the culture was inverted at 30 ℃ for 7d at initial pH 7.6.
Preferably, in step (1), the concentration of the monospore suspension is 8X 106spores/mL。
Preferably, in the step (2), the liquid medium is a Gao's No. one liquid medium.
More preferably, the formula of the Gao's No. one liquid culture medium is as follows: the following components are contained in each 1L of distilled water: 20g of soluble starch, 0.5g of NaCl and KNO31 g，K2HPO4.3H2O 0.5g，MgSO4.7H2O 0.5g，FeSO4.7H 20.01g of O, and high-temperature and high-pressure sterilization is required before use.
More preferably, the pH of the gao's No. one liquid medium is 7.6.
Preferably, in step (2), the fermentation culture conditions are: the culture was carried out for 7d with shaking at an initial pH of 7.6, 30 ℃ and a rotation speed of 160 rpm.
Preferably, in the step (3), the mass-to-volume ratio of the streptomyces microfeatus K-15 wet cells re-dispersed in sterile deionized water to water is 10g:100 mL.
Preferably, in the step (3), the conditions of the re-dispersion of the wet streptomyces microfeatus K-15 thalli in sterile deionized water and the shaking extraction are as follows: shaking and leaching at 30 deg.C and 160rpm for 72 h.
Preferably, in the step (3), after the water extraction of the streptomyces microfeatus K-15 thalli is finished, the extract is centrifuged for 5min at 5000rpm, and filtered for 3 times by Whatman No.1 filter paper to collect thalli extraction supernatant containing bioactive substances.
More preferably, the pH of the supernatant from the cell leaching is adjusted to 8.0.
Preferably, in the step (4), the volume ratio of the streptomyces microfeatus K-15 thallus leaching supernatant to the silver nitrate solution is 9: 1, the final concentration of the silver ions in the reaction system is 1 mmol/L.
Preferably, in the step (4), the synthesis reaction conditions of the nano silver are as follows: the reaction was carried out for 72h at an initial pH of 8.0, 30 ℃ and 160rpm protected from light with shaking.
Preferably, in the step (4), the reaction solution is centrifuged at 12000rpm for 20min to collect the precipitate, the precipitate is repeatedly washed with absolute ethyl alcohol for 3 times, and the precipitate is dried in a vacuum drying oven at 20 ℃ overnight to obtain the nano-silver powder.
As a most preferred possible embodiment, the synthesis method of nano silver specifically comprises the following steps:
(1) activation of Streptomyces microfeatus K-15 strain: streaking streptomyces microformis K-15 slant strain stored at 4 ℃ on a Gao's first solid culture medium with pH of 7.6, and performing inverted culture at 30 ℃ for 7 d; scraping appropriate amount of spore after culturing to obtain 8 × 10 spore6spores/mL of a single spore suspension;
(2) obtaining streptomyces microfeatus K-15 thalli: inoculating the monospore suspension into a Gao's No. I liquid culture medium with the pH of 7.6, and performing shaking culture at 30 ℃ and 160rpm for 7 d; thoroughly washing thallus of streptomyces microformis K-15 obtained by fermentation culture with sterile deionized water to remove residual culture medium components;
(3) preparation of the cell leaching supernatant: re-dispersing wet thalli in sterile deionized water according to the volume ratio of the wet weight of the thalli to water of 10g:100mL, and carrying out shaking extraction for 72h at the temperature of 30 ℃ and the rotating speed of 160 rpm; centrifuging the leaching solution at 5000rpm for 5min, filtering with Whatman No.1 filter paper for 3 times to collect the thallus leaching supernatant containing bioactive substances;
(4) and (3) synthesis of nano silver: taking the supernatant of the thallus leaching with the pH of 8.0 as a reaction matrix, and mixing the reaction matrix according to the volume ratio of 9: 1, slowly dripping silver nitrate solution into the mixture to ensure that the final concentration of silver ions in a reaction system is 1mmol/L, and carrying out vibration reaction for 72 hours at 30 ℃ and 160rpm in a dark place; centrifuging the reaction solution at 12000rpm for 20min to collect precipitate, washing the precipitate with anhydrous ethanol repeatedly for 3 times, and drying in a vacuum drying oven at 20 deg.C overnight to obtain nano-silver powder.
In addition, the application of the nano silver synthesized by the method, the nano silver itself or the nano silver and antibacterial drugs which are combined to be used as or for preparing antibacterial agents and antifungal agents is also within the protection scope of the invention.
Preferably, the antibacterial drugs refer to a plurality of antibacterial drugs and antifungal drugs such as azithromycin, gentamicin, vancomycin, ampicillin, ciprofloxacin, amphotericin B and nystatin.
Preferably, the antibacterial preparation refers to antibacterial preparations against various pathogenic bacteria such as pseudomonas aeruginosa, bacillus subtilis and the like.
Preferably, the antifungal preparation is an antifungal preparation for various pathogenic fungi such as candida parapsilosis, aspergillus fumigatus and the like.
The invention has the beneficial effects that: the invention provides a method for synthesizing nano-silver by utilizing streptomyces microformis K-15 and application thereof. The method mainly uses the extracted supernatant of the streptomyces microformis K-15 thallus as a reaction substrate to synthesize the nano-silver, and the whole process is simple and convenient to operate, green, nontoxic, environment-friendly and energy-saving. The nano silver synthesized by the method is spherical or approximately spherical, the average particle size is 28.56nm, and the polydispersity index (PDI) is 0.274. Bioactive molecules in the extracted supernatant of the streptomyces microformis K-15 thalli are coated on the surface of formed nano silver particles besides the reduction process of mediated silver ions, so that the nano silver particles have good stability and biological affinity, and are combined with the unique physicochemical property of the nano particles, so that the nano silver particles can more easily permeate into microbial cells, and the microbial cells are killed by interfering the normal metabolism of the microbes. In addition, the combined use of the nano silver and the antibacterial drug can generate synergistic bacteriostatic effects with different degrees on microorganisms, so that the nano silver antibacterial agent has wider application prospect in the field of biomedicine.
Drawings
In order to make the object, technical scheme and beneficial effect of the invention more clear, the invention provides the following drawings for explanation:
FIG. 1 is a graph showing the color change of the reaction solution before and after the synthesis of nano-silver in example 1;
FIG. 2 is a diagram showing an ultraviolet absorption spectrum of the synthesized nano-silver in example 1;
FIG. 3 is a transmission electron microscope image of the synthesized nano-silver in example 1;
FIG. 4 is a TEM-EDX spectrum of the synthesized nano-silver in example 1;
FIG. 5 is an XRD pattern of the synthesized nano-silver in example 1;
FIG. 6 is a FTIR spectrum of the synthesized nanosilver in example 1;
fig. 7 is a bacteriostatic effect diagram of the synthesized nano silver in example 1.
Detailed Description
Preferred embodiments of the present invention will be described in detail below with reference to the accompanying drawings.
Materials and reagents: the microbial strain for synthesizing the nano-silver is streptomyces microformis K-15 which is classified and named as streptomyces parvulus, the preservation number is CGMCC No.17019, the preservation unit is the common microorganism center of China Committee for culture Collection of microorganisms, the preservation unit address is No. 3 of Xilu No.1 of Beijing Korean district, and the preservation date is 12 months and 21 days in 2018. The tested bacteria used for the nanosilver antibacterial property test were Pseudomonas aeruginosa (Pseudomonas aeruginosa, ATCC-15442) and Bacillus subtilis (Bacillus subtilis, ATCC-6633), the tested fungi were Candida parapsilosis (ATCC-10231) and Aspergillus fumigatus (ATCC-96918), both from the culture collection of microbial cultures in guangdong province, silver nitrate was from Sigma-Aldrich in usa, all the media components for fungal and bacterial culture were from Oxoid corporation and hangzhou shoi chemical agents in the united kingdom, and drug sensitive paper sheets and standard drug sensitive paper sheets containing specific blank concentrations of antibacterial drugs were from microbial agents limited in state.
FA2004 electronic analytical balance (Shanghai Puchun Meter Co.), LDZ vertical pressure steam sterilizer (Shenan medical instrument factory in Shanghai city), DHG-9101-1SA digital display electric heating constant temperature drying cabinet (Tantan Shenglan instruments manufacturing Co., Ltd.), YJ-S/VD type super clean bench (Wuxi Yijing clean cleaning Equipment Co., Ltd.), PYX-250S-A biochemical incubator (Shaokuan City Taihong medical instrument Co., Ltd.), ZD-88 air bath full temperature oscillator (Jintanke Konji instruments factory), 3-18K type high speed refrigerated centrifuge (Germany SigmA Co., Ltd.), TU-1901 double light beam ultraviolet-visible spectrophotometer (Beijing general instruments Co., Ltd.), DGF-6020 vacuum drying cabinet (Jiangyi City responsibility instruments Co., Ltd.), KQ2200DE type numerical control ultrasonic cleaner (Kunshan Shumei ultrasonic instruments Co., Ltd.), G2F 20 field emission transmission electron microscope (FEI, USA), Zetasizer Nano nanometer particle size analyzer (Malvern, UK), D8 Advanced X-ray diffraction analyzer (Bruker, Germany), Frontier Fourier transform infrared spectrometer (PerkinElmer, USA), ICE 3500 atomic absorption spectrometer (Thermo Scientific, USA).
Example 1
Process for synthesizing nano silver by streptomyces microformis K-15
(1) Activation of Streptomyces microfeatus K-15 strain: selecting a streptomyces microformis K-15 slant strain stored at 4 ℃ by using an inoculating loop, streaking and inoculating the strain on a Gao's No. I solid culture medium with pH of 7.6, and carrying out inverted culture at 30 ℃ for 7d to activate the strain; after the culture, 3mL of sterile water was added to the solid culture medium of Gao's I, and an appropriate amount of spores was scraped off with an inoculating loop to obtain a culture medium having a concentration of 8X 106spores/mL of a single spore suspension;
(2) obtaining streptomyces microfeatus K-15 thalli: inoculating 200 μ L of the monospore suspension obtained in step (1) into 100mL of Gao's No. I liquid culture medium with pH 7.6, and performing shake culture at 30 deg.C and 160rpm for 7 d; after the fermentation culture is finished, filtering and collecting the thallus of the streptomyces microformis K-15 by Whatman No.1 filter paper, and washing for 3 times by using sterile deionized water to thoroughly remove the residual culture medium components on the thallus;
(3) preparation of the cell leaching supernatant: re-dispersing the streptomyces microfeatus K-15 thallus obtained in the step (2) into sterile deionized water according to the volume ratio of wet weight of the thallus to water of 10g:100mL, and carrying out oscillating extraction for 72h at the temperature of 30 ℃ and the rotating speed of 160 rpm; after the oscillating extraction is finished, centrifuging the extract at 5000rpm for 5min, and filtering with Whatman No.1 filter paper for 3 times to collect the thallus extraction supernatant containing bioactive substances;
(4) and (3) synthesis of nano silver: adjusting the pH value of the thallus leaching supernatant prepared in the step (3) to 8.0, and mixing the thallus leaching supernatant with the water according to the volume ratio of 9: 1, slowly dripping 10mmol/L silver nitrate solution into the reaction system to ensure that the final concentration of silver ions in the reaction system is 1mmol/L, and carrying out vibration reaction for 72 hours at 30 ℃ and 160rpm in the dark. And (3) observing the color change of the reaction liquid after the reaction is finished, simultaneously carrying out full-wavelength scanning on the nano-silver synthesis reaction liquid obtained in the step (4) by using an ultraviolet-visible spectrophotometer, wherein the detection range is 300-700 nm, the scanning interval is 0.5nm, comparing the full-wavelength scanning results of the silver nitrate solution and the extracted supernatant of the streptomyces microfeatus K-15 thallus which is not added with silver nitrate by using the silver nitrate solution as a reference. As shown in FIG. 1, after the silver nitrate and the cell leaching supernatant were subjected to a light-shielding oscillation reaction for 72 hours, it was observed that the reaction solution gradually changed from light purple to red brown, but the silver nitrate solution and the cell leaching supernatant to which no silver nitrate was added did not change. Meanwhile, as shown in fig. 2, an ultraviolet-visible (UV-vis) spectrum shows that a characteristic Surface Plasmon Resonance (SPR) peak of the nano-silver appears at a wavelength of 430nm in the reaction solution, and neither the silver nitrate solution nor the bacterial leaching supernatant to which the silver nitrate is not added has a characteristic SPR peak of the nano-silver. The phenomena show that a large amount of nano-silver particles are formed in the reaction system, and the streptomyces microformis K-15 thallus leaching supernatant has stronger nano-silver synthesis capacity.
Example 2
Characterization analysis of the nanosilver synthesized in example 1
(1) Centrifuging the nano silver synthesis reaction solution at 12000rpm for 20min to collect precipitate, repeatedly washing the precipitate with absolute ethyl alcohol for 3 times, and drying in a vacuum drying oven at 20 ℃ overnight to obtain gray brown nano silver powder.
(2) Transmission Electron Microscopy (TEM) characterization analysis: and (2) re-dispersing the nano silver powder obtained in the step (1) in absolute ethyl alcohol, performing ultrasonic treatment for 20min, sucking 5 mu L of ethanol dispersed liquid of nano silver, dripping the ethanol dispersed liquid on a copper net coated with a carbon film, sucking residual liquid on the copper net by using filter paper after the solution is completely absorbed, and detecting the copper net by using a TEM (transmission electron microscope) after the copper net is naturally air-dried for 1-2 h. The conditions are that the accelerating voltage is 200KV, the maximum magnification is 105 ten thousand times, the dot resolution is 0.24nm, and the line resolution is 0.102 nm. As shown in fig. 3, the TEM photograph shows that the nano silver synthesized by example 1 is spherical or nearly spherical, and has a small and uniform particle size.
(3) Particle size distribution of nano silver particles: and (3) selecting a certain number of nano particles in different visual field ranges to perform particle size measurement and particle size distribution statistics according to the nano silver TEM picture obtained in the step (2). Meanwhile, the polydispersity index (PDI) of the nano silver particles was measured by a nano particle sizer based on a Dynamic Light Scattering (DLS) method under the conditions of 25 ℃ and a scattering angle of 90 ℃. The detection result shows that the particle size distribution range of the nano silver particles is 2.0-52.9 nm, the average particle size is 28.56nm, the PDI is 0.274, and the nano silver particles are all in moderate polydispersity (0.08-0.7) in aqueous solution.
(4) Energy dispersive X-ray (EDX) characterization analysis: and (3) placing the carbon-coated copper mesh loaded with the nano silver particles obtained in the step (2) on a TEM-EDX for detection and analysis. The condition is 5B-92U; the energy resolution was 130eV (Mn K alpha line). As shown in fig. 4, the energy spectrum of nano silver shows that a characteristic absorption peak signal belonging to silver element appears at about 3 keV. In addition, signals of elements such as phosphorus (P), sulfur (S) and carbon (C) appear on the spectrogram, and the surface of the nano silver particle is possibly coated with components from bioactive molecules such as protein.
(5) Characterization and analysis by X-ray powder diffraction (XRD): drying the nano-silver powder obtained in the step (1), grinding the dried nano-silver powder into fine powder with uniform granularity, and performing XRD detection analysis under the condition that CuK alpha is used as a radiation source
The voltage of the Cu target X light tube is less than or equal to 40kV, and the current is less than or equal to 40 mA; the 2 theta angle scanning range is 20-80 degrees; the precision of the goniometer is 0.0001 degree, and the accuracy is less than or equal to 0.02 degree. As shown in fig. 5, the XRD pattern of nano-silver shows characteristic diffraction absorption peaks of elemental silver at 38.252 °, 44.135 °, 64.511 ° and 77.582 °, respectively corresponding to (111), (200), (220) and (311) crystal planes, and no obvious impurity diffraction peak appears, which is highly matched with elemental silver standard X-ray diffraction card (JCPDS No.04-0783), confirming that the nano-silver synthesized in example 1 is a high-purity face-centered cubic silver crystal.
(6) Fourier Transform Infrared (FTIR) spectral characterization analysis: and (2) mixing the nano-silver powder obtained in the step (1) with potassium bromide according to a ratio of 1:100, uniformly grinding, tabletting, and then placing in an infrared spectrometer for detection and analysis. The conditions were mid-infrared beam splitter: 4000-500 cm-1(ii) a The resolution of the potassium bromide light splitting beam is 0.4cm-1(ii) a The detector is DTGS; the signal-to-noise ratio was 11000: 1. As shown in FIG. 6, the FTIR spectrum of the nano-silver is 4000-500 cm in the wavelength range-1There are some distinct absorption peaks that are caused by stretching vibrations of different types of chemical bonds. 3439.52cm-1The vibration peak is from-O-H which can mediate the reduction of silver ions into nano silver; 1615.67cm-1And 1745.18cm-1The vibrational peaks at (a) are from-N-H and-C ═ O of the amide bonds in protein amide I and amide II, respectively; in addition, at 1387.69cm-1The vibrational peaks at (A) are from the-C-N of aromatic and aliphatic amines in the protein. This shows that the bioactive components (such as protein) secreted by the streptomyces microformis K-15 thallus may play a key role in the reduction process of silver ions, and may also coat the surface of the formed nano silver particles, thereby preventing aggregation and endowing unique biological characteristics to the nano silver particles.
Example 3
Antibacterial Properties of the nanosilver synthesized in example 1
(1) Activation of the test strain: pseudomonas aeruginosa (ATCC-15442) and Bacillus subtilis (ATCC-6633), Candida parapsilosis (ATCC-10231) and Aspergillus fumigatus (ATCC-96918) were selected as representatives of gram-negative bacteria, gram-positive bacteria, yeast and mold, respectively, and 4 test bacteria stored on a 4 ℃ slant were inoculated into nutrient broth (bacteria), MGYP liquid medium (yeast) and Potato Dextrose Broth (PDB) medium (mold), respectively, and cultured for 24h (bacteria) or 48h (yeast and mold) with shaking to activate the strains.
(2) Preparation of a detection plate: suspending the activated tested bacteria in a proper amount of 0.9% sterile physiological saline to prepare a bacterial suspension or a spore suspension, diluting the bacterial suspension to 0.5 McLeod turbidity by using a blood counting plate, dipping the bacterial suspension or the spore suspension of the tested bacteria by using a sterile cotton swab, uniformly coating the bacterial suspension or the spore suspension on the surface of a Muller Hinton Agar (MHA) culture medium, and drying at room temperature for 3-5 min for later use.
(3) Preparation of a sample to be tested: after the nano-silver powder prepared in example 1 was completely nitrated with concentrated nitric acid, the nitrated solution was diluted to a certain multiple, and the mass fraction of silver in the nano-silver powder prepared in example 1 was 58.65% by atomic absorption spectrometry. An appropriate amount of the silver nanopowder prepared in example 1 was weighed and dissolved in sterile deionized water, and subjected to ice bath ultrasound for 1 hour to prepare nano-silver solutions with final concentrations of 50. mu.g/mL, 100. mu.g/mL and 200. mu.g/mL, respectively. Meanwhile, the supernatant of the leaching of the Streptomyces microfeatus K-15 strain prepared in example 1 without adding silver nitrate and a silver nitrate solution of 200. mu.g/mL were set as controls.
(4) The Kirby-Bauer paper diffusion method is used for detecting the antibacterial performance of the sample: respectively sucking 30 mu L of each sample obtained in the step (3), dripping the sample on a circular sterile filter paper sheet with the diameter of 6mm to prepare a drug sensitive paper sheet, after the drug sensitive paper sheet is dried, carefully clamping the dried drug sensitive paper sheet by using sterile forceps, paving the drug sensitive paper sheet on the surface of an MHA culture medium, rightly placing the drug sensitive paper sheet for 1h at the temperature of 4 ℃, then inversely culturing the drug sensitive paper sheet for 24h (bacteria and yeasts) or 48h (molds) at the temperature of 37 ℃ (bacteria) or 35 ℃ (yeasts and molds), and performing three groups of experiments in parallel. After all tested bacteria grow out, the diameter of the inhibition zone around each drug sensitive paper sheet is measured and recorded, the diameter is expressed in the form of 'mean +/-Standard Deviation (SD)' of three parallel experiment results, and 'NI' indicates that no inhibition zone appears. The results are shown in FIG. 7 and Table 1.
TABLE 1 results of antibacterial tests on 4 test bacteria using different samples to be tested
As can be seen from FIG. 7 and Table 1, the supernatant of the leaching of Streptomyces microfeatus K-15 without silver nitrate has no significant zone of inhibition on 4 kinds of test bacteria, while the nano-silver synthesized in example 1 has significant antibacterial effect on different test bacteria and fungi, and is increased in dose-dependent manner within the range of 50-200. mu.g/mL. In addition, the nano silver with the same concentration (200 mug/mL) has better bacteriostatic activity compared with silver nitrate, and the reason probably is that the bioactive substances in the extracted supernatant of the streptomyces microfeatus K-15 thalli are coated on the surface of the formed nano silver particles to endow the nano silver particles with stronger bioaffinity; meanwhile, the unique physicochemical properties of the nanoparticles are combined, so that the nano-silver can be more efficiently and directly contacted with the surfaces of microbial cells to further permeate into the microbial cells, and the normal metabolism of the microbes is interfered by releasing free silver ions to cause the microbes to die, thereby showing better antibacterial activity.
(5) Detecting the Minimum Inhibitory Concentration (MIC) of the nano-silver by a trace broth dilution method: MIC values of the nanosilver synthesized in example 1 were measured for bacteria (Pseudomonas aeruginosa, Bacillus subtilis), yeast (Candida parapsilosis) and mold (Aspergillus fumigatus), respectively, with reference to the broth dilution method (M07-A8, M27-A2, M38-A) recommended by the national institutes of Clinical and Laboratory Standards Institute (CLSI). An appropriate amount of the nano-silver powder prepared in example 1 was redissolved in Muller Hinton Broth (MHB) and RPMI1640 medium to prepare a stock solution with a final concentration of 800. mu.g/mL (calculated as silver). Taking a sterile 96-hole enzyme label plate as a trace drug sensitive plate, respectively adding 200 mu L MHB culture medium (bacteria) or RPMI1640 culture medium (yeast and mould) into No. 1-12 holes, respectively setting the No.1 hole and the No. 12 hole as a blank control hole and a growth control hole without adding nano-silver, respectively adding 200 mu L of the prepared 800 mu g/mL nano-silver solution into No. 2 hole, and continuously diluting the No. 3-11 holes by times to ensure that the final concentration of the nano-silver in each No. 2-11 hole is respectively: 400.00, 200.00, 100.00, 50.00, 25.00, 12.50, 6.25, 3.12, 1.56, and 0.78 μ g/mL. And (3) adding 2 mu L of the diluted bacterial suspension or spore suspension prepared in the step (2) into the No. 2-12 holes except the No.1 hole. The prepared drug sensitive reaction plate is respectively stood under 37 ℃ (bacteria) or 35 ℃ (yeast and mold) to be cultured for 24h (bacteria and yeast) or 48h (mold), and then MIC values of the nano silver to different microorganisms can be read. Triplicate wells were set for each concentration and the experiment was repeated three times. When the MIC value result is judged, the clear, turbid and macroscopic thallus growth of the No.1 blank control hole is ensured, and the microorganism growth of the No. 12 growth control hole is good; and comparing each experimental hole No. 2-11 with the hole No. 12, wherein the lowest drug concentration hole is the MIC value of the nano-silver to the microorganism when the growth of the microorganism is completely inhibited. The MIC value can be finally determined when it can be accurately repeated or only differs by one concentration, and the value with higher concentration is taken as the final determination result of the MIC value, and the result is shown in table 2.
TABLE 2 MIC values of nanosilver and silver nitrate for 4 test bacteria
As shown in Table 2, the MIC values of the nano-silver synthesized in example 1 for 4 test bacteria are lower than that of silver nitrate, and particularly the MIC values of the nano-silver synthesized in example 1 for Pseudomonas aeruginosa and Aspergillus fumigatus are the lowest (6.25 mug/mL), which further indicates that the nano-silver has better bacteriostatic activity for the test bacteria and fungi than the silver nitrate.
Example 4
Example 1 study of antibacterial Properties of the synthesized Nanosilver in combination with an antibacterial agent
With reference to the method described in the steps (1) to (4) of example 3, 4 bacterial suspensions or spore suspensions of the test bacteria were uniformly coated on the surface of the MHA medium, and 30. mu.L of 200. mu.g/mL nano-silver solution was respectively dropped on the MHA medium containing 5 antibacterial drugs, azithromycin (15. mu.g/tablet), gentamicin (10. mu.g/tablet), vancomycin (30. mu.g/tablet), ampicillin (10. mu.g/tablet),Ciprofloxacin (5. mu.g/tablet), 2 antifungal drugs amphotericin B (30. mu.g/tablet) and nystatin (100. mu.g/tablet), and a drug-sensitive paper containing only the supernatant of the extracted bacterial cells of Streptomyces parvus K-15 prepared in example 1 without silver nitrate, a nano-silver solution of 200. mu.g/mL, and an antibacterial drug were used as a control group. After the drug sensitive paper is dried, the dried drug sensitive paper is carefully clamped by a sterile forceps and spread on the surface of an MHA culture medium, the MHA culture medium is rightly placed for 1h at 4 ℃, then inverted and cultured for 24h (bacteria and yeast) or 48h (mold) at 37 ℃ (bacteria) or 35 ℃ (yeast and mold), and three groups of experiments are performed in parallel. After all the tested bacteria grow out, the diameter of the inhibition zone around each drug sensitive tablet is measured and recorded, and is expressed in the form of the mean value plus or minus Standard Deviation (SD) of the results of three parallel experiments. By the formula (b)2-a2)/a2The fold area increase times of the inhibition zones generated when the nano-silver and the antibacterial agent are used in combination (a: the diameter of the average inhibition zone generated when the antibacterial agent is used alone, and b: the diameter of the average inhibition zone generated when the nano-silver and the antibacterial agent are used in combination) compared with that generated when the antibacterial agent is used alone are calculated, so that the antibacterial activity of the nano-silver and the antibacterial agent on different microorganisms is evaluated when the nano-silver and the antibacterial agent are used in combination, and the results are shown in tables 3 and 4.
TABLE 3 results of antibacterial tests on different bacteria tested with antibacterial drugs alone and in combination with nanosilver
TABLE 4 results of antibacterial tests on different fungi tested with antibacterial drugs alone and in combination with nanosilver
As can be seen from tables 3 and 4, when the nano-silver synthesized in example 1 is used in combination with 5 antibacterial agents and 2 antifungal agents, which are clinically commonly used and have different antibacterial mechanisms, the folding area of the inhibition zone generated by the tested bacteria and fungi is increased by 0.25-8.01 times compared with that of the antibacterial agent used alone, which indicates that the nano-silver-antibacterial agent combined use shows an obvious synergistic antibacterial effect, so that the antibacterial effect on the tested bacteria and fungi is more significant than that of the antibacterial agent used alone, especially on drug-resistant bacteria (bacillus subtilis) and multi-drug-resistant bacteria (pseudomonas aeruginosa). Cytotoxic studies have demonstrated that the use of nanoparticles in combination with antibiotics is expected to reduce the toxicity of nanoparticles and antibiotics alone on mammalian cells. Therefore, the clinical use dosage of the nano silver and the antibiotics can be reduced by the combined mode of the nano silver and the antibiotics, and the toxicity and the drug resistance of the nano silver and the antibiotics to organisms are reduced. China is one of the most serious countries of antibiotics abuse in the world, infection caused by more and more drug-resistant bacteria and multiple drug-resistant bacteria poses serious threat to human health, and the nano silver synthesized in the example 1 has potential to be used as or prepared into novel, low-toxicity and efficient nano silver composite antibacterial preparations and antifungal preparations and is applied to the field of biomedicine.
Finally, it is noted that the above-mentioned preferred embodiments illustrate rather than limit the invention, and that, although the invention has been described in detail with reference to the above-mentioned preferred embodiments, it will be understood by those skilled in the art that various changes in form and detail may be made therein without departing from the scope of the invention as defined by the appended claims.
Claims (9)
1. A method for synthesizing nano-silver by utilizing streptomyces microformis K-15 is characterized in that the streptomyces microformis K-15 is classified and named as streptomyces microformis (S) (A)Streptomyces parvulus) The preservation number is CGMCC No.17019, the preservation unit is China general microbiological culture Collection center, the address of the preservation unit is No. 3 Xilu No.1 Beijing, Chaoyang, and the preservation date is 2018, 12 months and 21 days; the method comprises the following steps:
(1) streaking streptomyces microformis K-15 slant strain stored at 4 ℃ on a solid culture medium for culture to obtain spores, and scraping a proper amount of spores to obtain a monospore suspension;
(2) inoculating the monospore suspension into a liquid culture medium, obtaining thallus of streptomyces microformis K-15 through fermentation culture, and thoroughly washing the thallus by using sterile deionized water to remove residual culture medium components;
(3) dispersing the wet thalli in sterile deionized water again, oscillating and leaching, centrifuging and filtering a leaching solution to completely remove the thalli, and collecting thalli leaching supernatant containing bioactive substances;
(4) taking the extracted supernatant of the streptomyces microfeatus K-15 thallus obtained in the step (3) as a reaction substrate, adding a silver nitrate solution into the reaction substrate to perform a synthesis reaction of nano-silver, and centrifuging, washing and drying the reaction solution to obtain the nano-silver.
2. The method for synthesizing nano silver by using streptomyces microformis K-15 as claimed in claim 1, wherein in step (1), the concentration of the monospore suspension is 8 x 10 6 spores/mL。
3. The method for synthesizing nano silver by using streptomyces microformis K-15 as claimed in claim 1, wherein in step (2), the fermentation culture conditions are as follows: shaking and culturing at 30 deg.C and 160rpm for 7 d.
4. The method for synthesizing nano silver by using streptomyces microfeatus K-15 as claimed in claim 1, wherein the mass-to-volume ratio of wet bacteria of streptomyces microfeatus K-15 re-dispersed in sterile deionized water to water in step (3) is 10g:100 mL.
5. The method for synthesizing nano-silver by using streptomyces microformis K-15 as claimed in claim 1, wherein the conditions of the shaking leaching of the wet thallus of streptomyces microformis K-15 re-dispersed in sterile deionized water in step (3) are as follows: shaking and leaching at 30 deg.C and 160rpm for 72 h.
6. The method for synthesizing nano-silver by using streptomyces microfeatus K-15 as claimed in claim 1, wherein the volume ratio of the streptomyces microfeatus K-15 thalli leaching supernatant to the silver nitrate solution in step (4) is 9: 1, the final concentration of silver ions in the reaction system is 1 mmol/L.
7. The method for synthesizing nano silver by using streptomyces microformis K-15 as claimed in claim 1, wherein the reaction conditions for synthesizing nano silver in step (4) are as follows: the reaction was carried out at 30 ℃ and 160rpm in the dark for 72 h.
8. Use of nanosilver synthesized according to the method of claim 1 for the preparation of antibacterial and antifungal agents.
9. Use of nanosilver synthesized according to the method of claim 1 in the preparation of antibacterial and antifungal agents in combination with antibacterial agents.
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