CN110272922B - Method for synthesizing nano-silver by utilizing pseudomonas praecox cell-free supernatant, product and application thereof - Google Patents
Method for synthesizing nano-silver by utilizing pseudomonas praecox cell-free supernatant, product and application thereof Download PDFInfo
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Abstract
The invention discloses a method for synthesizing nano-silver by utilizing pseudomonas praecox cell-free supernatant, which comprises the following steps: 1) culturing pseudomonas herbaceum in NB culture medium, collecting bacterial suspension; 2) centrifuging the bacterial suspension, and collecting supernatant to obtain cell-free supernatant; 3) adding cell-free supernatant to AgNO3And (4) solution, namely centrifuging, washing and vacuum freeze-drying the obtained reaction solution to obtain the nano silver particles. The nano silver particles prepared by the method have stable structure, good dispersibility and good biocompatibility, have strong in-vitro and in-vivo antibacterial activity on Dickeya dadantii CZ1501, can be prepared into an eco-friendly nano metal bacteriostatic agent with good biocompatibility for preventing and treating the sweet potato stem rot, reduce the burden of the traditional chemical bactericide on the environment and have good popularization prospect in the field of agricultural production.
Description
Technical Field
The invention belongs to the technical field of green biosynthesis of nano materials, and particularly relates to a method for synthesizing nano silver by utilizing pseudomonas praecox cell-free supernatant and application of the method in agriculture.
Background
The nano material has a structural unit within a range of 1-100 nm, and has wide development potential in the fields of science, technology and the like due to unique physical and chemical properties such as small size, large specific surface area and the like. The nano Silver (AgNPs) has strong antibacterial property and potential antibacterial activity on gram-negative and gram-positive bacteria such as escherichia coli and staphylococcus aureus.
At present, the traditional method for synthesizing the nano silver mainly comprises a physical method, a chemical method and a microbiological method, the physical method and the chemical method have simple feasibility, but the energy requirement in the synthesis process is large, the cost is high, and the environmental pollution is easily caused. In contrast, microorganisms have the advantages of strong vitality, wide adaptability, easy availability, high propagation speed, convenient genetic modification and the like, and are often used as raw materials for biosynthesis of nano metals.
The Chinese patent application with publication number CN107904262A discloses a method for preparing nano-silver based on bacterial extracts, which adopts Deinococcus radiodurans (Deinococcus radiodurans) to prepare a nano-silver synthetic agent, and then adds AgNO into the nano-silver synthetic agent3And reacting in the solution to obtain the nano silver particles.
The chinese patent application publication No. CN105039419A discloses a method for synthesizing nano-silver by Trichoderma asperellum, which comprises adding silver ions and nitrate into the supernatant of Trichoderma asperellum, and culturing in dark to obtain nano-silver, wherein the obtained nano-silver has uniform particle size distribution and better properties.
The bacterial supernatant contains Extracellular Polymers (EPS) produced by itself, and the EPS is some high molecular polymers secreted in vitro by microorganisms, mainly bacteria, under certain environmental conditions, such as protein, nucleic acid, lipid, polysaccharide and other components, and plays an important role in the formation of nano silver particles. However, the types of microorganisms disclosed in the prior art that can be used for synthesizing nano silver particles are few, and therefore, the synthesis of nano silver based on bacterial cell-free fermentation supernatant (CFCS) has great development prospects.
Furthermore, Dickeya dadantii (d.dadantii) is a pathogen causing stem rot of sweet potatoes, and this bacterial disease causes enormous economic loss to the fields and storage of sweet potatoes, and has raised great attention worldwide. In order to control the disease, many compounds such as chlorine dioxide glutaraldehyde, bizall ammonium chloride, cefaclor and the like have been used in field tests, but the effect is not obvious.
Disclosure of Invention
The invention aims to provide a method for synthesizing nano-silver by using pseudomonas praecox supernatant as a reducing agent, the preparation process is environment-friendly, the production cost is low, the equipment is simple, and the obtained nano-silver particles are small in size and good in dispersibility.
The invention also aims to provide the application of the nano silver particles in preventing and treating the stem rot of the sweet potato in the field, and the nano silver particles have stronger in-vitro and in-vivo antibacterial activity on Dickeya dadanti CZ1501 which is a pathogenic factor of the stem and root rot of the sweet potato, can replace the traditional chemical prevention and treatment method, and are environment-friendly.
The purpose of the invention is realized by the following technical scheme:
a method for synthesizing nano-silver by utilizing pseudomonas praecox supernatant comprises the following steps:
(1) culturing Pseudomonas aeruginosa (Pseudomonas poae) in NB medium, and collecting bacterial suspension;
(2) centrifuging the bacterial suspension, and collecting the supernatant to obtain a cell-free supernatant (CFCS);
(3) adding cell-free supernatant to AgNO3And (4) solution, namely centrifuging, washing and vacuum freeze-drying the obtained reaction solution to obtain the nano silver particles.
The invention uses pseudomonas herbaceus cell-free supernatant as reducing agent and AgNO3The solution is mixed to prepare the nano-silver which has small average particle size and stable structure and can effectively inhibit the activity of the sweet potato stem rot, thereby further improving the antibacterial effect of the nano-silver and effectively preventing and treating the sweet potato stem rot in the field.
In the step (1), the NB medium is composed of 1% tryptone, 1% sucrose, 0.3% peptone and 0.1% yeast powder, i.e. 10g tryptone, 10g sucrose, 3g peptone and 1g yeast powder are weighed to prepare 1L NB medium.
In the steps (2) and (3), in the process of centrifuging the bacterial suspension to obtain the supernatant and centrifuging the reaction liquid to obtain the precipitate, the rotating speed is 8000-10000 rpm, the centrifuging time is 10-20 min, and energy waste can be caused by long centrifuging time and high rotating speed; the short centrifugation time and the slow rotation speed can cause the supernatant to contain impurities, and the purity of the nano particles is influenced.
In the step (3), the AgNO3The concentration of the solution is 0.5-2 mM, the concentration is not enough to synthesize nano particles, and the concentration is too high, so that the stability and the synthesis efficiency of a product structure are influenced, and unnecessary waste of raw materials is caused.
In the step (3), the bacterial supernatant is mixed with AgNO3The volume ratio of the solution is 2-3: 10, too high a metal oxide solution will result in waste of material resources, while too low a metal oxide solution will result in a reduction in raw material input, ultimately reducing synthesis efficiency.
And (3) placing the mixed reaction solution in a shaking table at the temperature of 30-32 ℃ for vibration in a dark place, and stopping the reaction when the color of the mixed solution is changed from light yellow to dark brown.
The invention also discloses the nano silver prepared by the preparation method, and the particle size of the nano silver is 20-100 nm.
The nano silver can be attached to the surface of bacterial cells, the cell morphology is damaged, and the cell permeability is increased through the cell wall, which is caused by the fact that the nano silver can oxidize the surface protein of a plasma membrane. Experiments show that the novel nano-silver particles prepared by the method have stable structure, good dispersibility, good biocompatibility and stronger in-vitro and in-vivo antibacterial activity on Dickeya dadantii CZ 1501.
The invention also discloses application of the nano-silver in preventing and treating sweet potato stem rot. The nano silver particles obtained by the invention have strong in-vitro and in-vivo antibacterial activity on Dickeya dadanti CZ1501 which is a pathogenic factor of the stem and root rot of the sweet potato, so that the nano silver particles can be prepared into an eco-friendly nano metal bacteriostatic agent with good biocompatibility, can play a good role in preventing and treating, also reduces the burden of the traditional chemical bactericide on the environment, and has good popularization prospect in the field of agricultural production.
Compared with the prior art, the invention has the following beneficial effects:
(1) the synthetic method is simple, low in price, safe and harmless, belongs to a green synthetic process, and is suitable for large-scale production;
(2) the invention utilizes the supernatant of the pseudomonas praecox and AgNO for the first time3The solution is mixed to prepare the nano silver which has small average particle size and stable structure and can effectively inhibit the activity of the sweet potato stem rot bacteria;
(3) the nano silver particles prepared by the invention can be prepared into nano metal bacteriostat for preventing and treating the stem rot of the sweet potato, and the prevention and treatment method is environment-friendly, does not pollute the environment and does not bring potential harm to human bodies.
Drawings
FIG. 1 is a photograph showing a color change of the synthesized nano-silver particles in example 1;
FIG. 2 is a diagram of UV-Vis of the nano silver particles in example 1;
FIG. 3 is a FTIR chart of the nanosilver particles in example 1;
FIG. 4 is a TEM image of the nano-silver particles in example 1;
FIG. 5 is an XRD pattern of the nano-silver particles in example 1;
FIG. 6 is an SEM-EDS diagram of nano-silver particles in example 1;
FIG. 7 is a graph showing the effect of different concentrations of nanosilver solutions prepared from nanosilver on the growth of sweet potato phoma rot fungi in example 1;
FIG. 8 is a graph showing the effect of different concentrations of nanosilver solutions prepared from nanosilver on the formation of biofilm of sweet potato phoma rot pathogen in example 1;
FIG. 9 is a graph showing the effect of different concentrations of nanosilver solutions prepared from nanosilver on the zone of inhibition of sweet potato stem rot;
FIG. 10 is a graph showing the effect of the nano-silver particles prepared at a concentration of 2mg/ml in example 1 on the cell wall of Phoma nikoense of sweet potato;
FIG. 11 is a graph showing the in vivo bacteriostatic effect of the nano-silver solutions of different concentrations prepared from nano-silver in example 1 on sweet potato stem rot.
Detailed Description
The invention will be further elucidated with reference to the following specific examples.
The strain used in the following examples 1 to 3 was Pseudomonas herbacea (CGMCC) available from China general microbiological culture Collection center (CGMCC) No. 3 of Navy, Beijing, with the collection number: CGMCC No. 18734.
Example 1
(1) And (3) bacterial culture: picking single pseudomonas praecox colony on an NA agar plate into a 5ml test tube culture medium, carrying out shaking culture at 30 ℃ and 200rpm overnight, transferring the single pseudomonas praecox colony into an NB liquid culture medium according to the proportion of 1%, and carrying out overnight culture at the culture temperature of 30 ℃ and the shaking speed of 200 rpm;
(2) collecting the supernatant: centrifuging the bacterial suspension obtained in the step (1) for 10min according to the rotation speed of 8000rpm, and collecting the supernatant to obtain a cell-free supernatant (CFCS);
(3) preparing nano silver: taking the bacterial supernatant obtained in the step (2) and 1mM AgNO3The solution was prepared as follows 1: 3, placing the mixture into a shaking table with 200rpm for vibration for 48 hours in a dark place, centrifuging the obtained reaction solution for 15min at 8000rpm, washing the reaction solution for 3 times by using distilled water, and performing vacuum freeze drying to obtain the nano-silver particles.
Example 2
(1) And (3) bacterial culture: picking single pseudomonas praecox colony on an NA agar plate into a 5ml test tube culture medium, carrying out shaking culture at 30 ℃ and 200rpm overnight, transferring the single pseudomonas praecox colony into an NB liquid culture medium according to the proportion of 1%, and carrying out overnight culture at the culture temperature of 30 ℃ and the shaking speed of 200 rpm;
(2) centrifuging the bacterial suspension obtained in the step (1) for 10min according to the rotation speed of 10000rpm, and collecting supernatant to obtain cell-free supernatant (CFCS);
(3) taking the bacterial supernatant obtained in the step (2) and 0.5mM AgNO3The solution was prepared as follows 1: 5, mixing well, placing in a shaker at 200rpm, and shaking in dark for 48 h. And centrifuging the obtained reaction solution at 8000rpm for 10min, washing with distilled water for 3 times, and freeze-drying in vacuum to obtain the nano-silver particles.
Example 3
(1) And (3) bacterial culture: picking single pseudomonas praecox colony on an NA agar plate into a 5ml test tube culture medium, carrying out shaking culture at 30 ℃ and 200rpm overnight, transferring the single pseudomonas praecox colony into an NB liquid culture medium according to the proportion of 1%, and carrying out overnight culture at the culture temperature of 30 ℃ and the shaking speed of 200 rpm;
(2) centrifuging the bacterial suspension obtained in the step (1) for 15min at 9000rpm, and collecting supernatant to obtain cell-free supernatant (CFCS);
(3) taking the bacterial supernatant obtained in the step (2) and 1.5mM AgNO3The solution was prepared as follows 1: 4, and placing the mixture into a shaker at 200rpm to carry out dark shaking for 48 hours. And centrifuging the obtained reaction solution at 10000rpm for 15min, washing the reaction solution for 3 times by using distilled water, and performing vacuum freeze drying to obtain the nano silver particles.
The performance and bacteriostatic effect of the nano-silver particles obtained by utilizing pseudomonas praecox mediation in example 1 were tested, and the obtained results are as follows:
characterization of (I) Nano silver Properties
FIG. 1 is a photograph showing the color change of nano-silver during the preparation process, and shows that the liquid is milky before the supernatant of Pseudomonas aeruginosa is added, the supernatant is added and oscillated in dark, the color of the liquid gradually becomes dark with the increase of time, and the liquid is reddish brown after 48 h.
FIG. 2 is a UV-Vis diagram obtained by detecting nano-silver particles by using an ultraviolet spectrophotometer, a single, strong and wide surface plasma resonance peak is shown around 420nm, the formation and the stability of nano-silver are proved, and in many researches, the initial characterization peak value of spherical nano-silver particles is between 410 and 450nm, so that the synthesis of the spherical nano-silver is indirectly proved.
Fig. 3 is an FTIR plot of the synthesis of nanosilver using pseudomonas aeruginosa supernatant, confirming that the presence of protein in the supernatant may be responsible for the rapid reduction and increased stability of silver nanoparticles by the presence of amide groups.
Fig. 4 is a TEM image of the synthesized nano silver, and it can be seen that the synthesized nano silver is relatively uniform spherical particles.
Fig. 5 is an XRD pattern of nano silver particles, XRD being an analytical technique for measuring crystallinity, qualitatively detecting various compounds, and qualitatively distinguishing chemical species, size, shape, and structure of crystalline materials, the position of the peak indicating the presence of nano silver particles having an average particle diameter of 49.5 nm.
FIG. 6 is SEM-EDS image of the synthesis of nano silver by using Pseudomonas praecox supernatant, surface imaging is performed by using SEM for analyzing the particle size, particle size distribution, shape and surface morphology of nano material, and qualitative and quantitative analysis of element state is performed by using EDS. The result shows that the spherical shape of the nano particles is consistent with the transmission electron microscope image result, the nano particles are uniformly distributed, the agglomeration phenomenon is avoided, and the average particle size of the nano particles is 21.8-99.4 nm. In addition, as the matrix of the scanning electron microscope is made of tinfoil, Ag element appears in an EDS spectrogram to ensure the existence of nano silver particles.
(II) in-vitro bacteriostatic effect of nano-silver with different concentrations on sweet potato stem rot bacteria
The nano-silver powder prepared in the example 1 is respectively prepared into solutions with the concentrations of 0.012mg/ml, 0.025mg/ml and 0.05mg/ml, and the antibacterial mechanism of the synthesized nano-silver is explained and the antibacterial performance of the nano-silver with different concentrations is evaluated by measuring the growth density, the biofilm formation capability, the diameter of an antibacterial zone and the change of cell morphology of bacteria.
FIG. 7 is a graph showing the effect of different concentrations of nano-silver solution on the growth of sweet potato stem rot, and it can be seen from FIG. 7A that the bacterial cell-free fermentation supernatant (CFCS) alone has no bacteriostatic effect, while the nano-silver concentrations of 0.012mg/ml, 0.025mg/ml and 0.05mg/ml can cause OD600 values to be respectively reduced by 65.5%, 70.8% and 90.0% compared with the control group, and 0.05mg/ml AgNO alone is added3The antibacterial ability of the nano-silver can be reduced by 63.5%, which shows that the antibacterial ability of the nano-silver is basically in direct proportion to the concentration of the nano-silver, and the synthetic nano-silver is proved to have stronger antibacterial effect compared with the single bacterial supernatant by the same method shown in FIG. 7B.
In addition, researches show that the biomembrane can protect pathogens from the influence of external adverse environment, and the swimming activity of phytopathogens is related to the growth and the toxicity of the phytopathogens and the formation of the biomembrane, so that the invasion and the colonization of host plants by bacterial pathogens are facilitated, and the forming capability and the swimming activity of the biomembrane can show the antibacterial activity of the nano-silver.
As shown in FIG. 8, the concentrations were 0.012mg/ml, 0.025mg/ml and 0.05mg/ml for the nano meterThe silver can reduce the light absorption value of 46.28%, 58.52% and 76.90% compared with the control group, and 0.05mg/ml AgNO is added separately3Also has certain bacteriostatic effect because of its strong oxidizing property.
FIG. 9 shows the results for a control group of 2.3cm diameter, 0.05mg/ml AgNO3The nano silver reduces the diameter of the bacteria to 1.1cm, and the nano silver reduces the diameter of the bacteria to 1.08cm, 1.02cm and 0.9cm respectively by 0.012mg/ml, 0.025mg/ml and 0.05 mg/ml. Fig. 8 and fig. 9 show that the higher the nano silver concentration, the better the effect of inhibiting the growth of the biofilm of pathogenic bacteria.
FIG. 10 shows the effect of the synthesis of nano-silver from Pseudomonas solanacearum supernatant on the cell wall of sweet potato stem rot. FIG. 10A shows the normal and intact cell morphology of Dickeya dadantii CZ1501, compared to a negative control, and TEM images (FIG. 10B, C) from different fields show that the cell morphology of strain CZ1501 is significantly changed in the presence of 0.2mg/ml of nanosilver, the matrix distribution is inconsistent, and the nanosilver is responsible for the efflux of intercellular substance of strain CZ 1501.
(III) in-vivo bacteriostatic effect of nano-silver with different concentrations on sweet potato stem rot bacteria
The nano silver synthesized in example 1 was prepared into nano silver solutions of different concentrations. As shown in FIG. 11, after inoculating CZ1501 strain on the stem tuber of sweet potato and culturing in the greenhouse for 24h, the sweet potato tuber has lesion with diameter of 3.47cm, and after treating the sweet potato with bacterial supernatant alone and inoculating with bacterial liquid, the lesion diameter is almost the same as that without using supernatant. However, when nano silver with the concentration of 0.012mg/ml, 0.025mg/ml and 0.05mg/ml is used for treatment, the size of the lesion spots is obviously reduced, and the diameters are 1.80cm, 1.48cm and 1.10cm respectively. 0.05mg/ml AgNO alone3The size of the lesion spot is about 1.90cm during treatment, which is similar to the result of treatment with 0.012mg/ml nano silver.
Therefore, the inhibition effect of the bacterial supernatant on the sweet potato stem rot is not obvious, but the nano silver with different concentrations can reduce the diameter of the sweet potato tuber scab by more than 50.0 percent, which shows that the nano silver has great potential in controlling the sweet potato stem rot. This data is consistent with the results of AgNPs in vitro antibacterial activity, indicating that inhibition of sweet potato stem rot may be due at least in part to the in vitro direct antibacterial activity of nanosilver on d.dadantii strain CZ 1501.
Comparative example
The present invention additionally screens for a variety of environmental bacteria, including: bacillus amyloliquefaciens (Bacillus amyloliquefaciens), Bacillus methylotrophicus (Bacillus methylotrophicus), Bacillus subtilis (Bacillus subtilis) and Bacillus polymyxa (Paenibacillus polymyxa), wherein the specific strain types are shown in the following table 1.
The strains in the table 1 are synthesized into nano silver by the method described in the example 1, and the nano silver cannot be prepared, and the inhibition zone experiment shows that the strains have no inhibition effect on Dickeya dadantii CZ 1501.
TABLE 1 preparation of nanosilver by different bacteria on the inhibition of Dickeya dadantii CZ1501
Claims (6)
1. A method for synthesizing nano silver by utilizing pseudomonas praecox cell-free supernatant comprises the following steps:
(1) performing plate-coating activation culture on pseudomonas praecox, selecting pseudomonas praecox single colony on an NA agar plate to a test tube culture medium, performing overnight shaking culture, and transferring to an NB culture medium for amplification culture according to the transfer amount of 1-3% in volume ratio, wherein the culture conditions are as follows: rotating at 180-220 rpm and 30-32 ℃, and collecting the bacterial suspension;
(2) centrifuging the bacterial suspension, and collecting supernatant to obtain cell-free supernatant;
(3) adding cell-free supernatant to AgNO3Solution, centrifuging, washing and vacuum freeze-drying the obtained reaction solution to obtain nano silver particles; the AgNO3The concentration of the solution is 0.5-2 mM, and the cell-free supernatant fluidWith AgNO3The volume ratio of the solution is 2-3: 10;
the pseudomonas praecox (A) and (B)Pseudomonas poae) The microbial inoculum is preserved in China general microbiological culture preservation management center with the preservation number as follows: CGMCC No. 18734.
2. The method according to claim 1, wherein in step (1), the formula of the NB medium is: 1% tryptone, 1% sucrose, 0.3% peptone and 0.1% yeast powder.
3. The method according to claim 1, wherein in the step (2) and the step (3), the centrifugation speed is 8000-10000 rpm, and the centrifugation time is 10-20 min.
4. The method of claim 1, wherein in step (3), the cell-free supernatant is mixed with AgNO3And (3) after the solutions are mixed, placing the mixture in a shaking table at the temperature of 30-32 ℃ for vibration in a dark place, and ending the reaction when the color of the mixed solution is changed from light yellow to dark brown.
5. The nano-silver prepared by the method according to any one of claims 1 to 4, wherein the nano-silver has a particle size of 20 to 100 nm.
6. The application of the nano-silver according to claim 5 in preventing and treating sweet potato stem rot.
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