CN114210995A - Method for preparing nano-silver particles by using burdock root extracting solution, product and application - Google Patents
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Abstract
The invention discloses a method for preparing nano-silver particles by using a burdock root extracting solution, which comprises the following steps: (1) cleaning and drying burdock root, grinding to obtain burdock root powder, uniformly stirring the burdock root powder and deionized water, and centrifuging to obtain a burdock root extracting solution; (2) mixing the burdock root extract in the step (1) with AgNO3And (3) mixing the solutions, reacting at 20-30 ℃ in the dark to obtain a mixed solution, and centrifuging, washing and vacuum freeze-drying the mixed solution to obtain the nano-silver particles. The method has simple process, low energy consumption, easily available raw materials, and no need of additional reductionThe agent and the catalyst do not need operations such as illumination, ultrasound, heating and the like, and are suitable for large-scale production, the prepared nano silver particles have stable structures and small average particle diameters of 5-50 nm, the biofilm formation and mobility inhibition rate on rice bacterial leaf blight protobacteria is high, and the excellent prevention and treatment effect on rice bacterial leaf blight is achieved.
Description
Technical Field
The invention relates to the field of preparation of nano materials, in particular to a method for preparing nano silver particles by using a burdock root extracting solution, a product and application.
Background
The rice bacterial leaf blight is a common rice disease caused by pathogenic bacteria of the rice bacterial leaf blight, is easy to spread and causes huge economic loss to agricultural production in China, and the traditional chemical pesticide is easy to pollute the environment, causes drug resistance of pathogenic bacteria and has potential harm to human bodies, so that an economic, efficient, safe and environment-friendly product is urgently needed to be developed to prevent and treat the rice bacterial leaf blight.
Nano silver (AgNPs) is now widely used in the food packaging, environmental biology and medical industries. In agriculture, the nano silver has strong insecticidal, antifungal, antiviral and bactericidal effects. AgNPs have large specific surface area and crystal surface structure, and are potential antibacterial agents. In addition, AgNPs also have a synergistic antimicrobial effect when mixed with other metal nanoparticles or oxides. Due to broad-spectrum antibacterial activity and good water solubility, AgNPs can inhibit various plant diseases by spraying on leaf surfaces, and can also complete the rapid detection of the plant diseases by using the AgNPs.
The plant nanotechnology is an important component of the biological nanometer synthesis technology and has the characteristics of biocompatibility, environmental protection, safety, easy availability and the like. The nano synthesis reaction rate is high, the synthesis condition is simple, and the method is more suitable for large-scale production. The plant extract contains alkaloid, polysaccharide, protein, flavonoid, terpenoid, steroid, saponin and tannin, and can be used as reducing agent and stabilizer for synthesis of nano-silver.
Chinese patent publication No. CN109702218A discloses a method for preparing nano silver particles by using an extract of emblic leafflower fruit; using emblic leafflower fruit extract and AgNO3The solution reacts under the condition of heating and stirring to prepare nano silver particles, and the prepared nano silver particles have stable structures and stronger bacteriostatic action on a rice bacterial brown streak pathogen RS-2.
Chinese patent publication No. CN107671305A discloses a method for rapidly preparing a nano silver bacteriostatic agent from a ligustrum quihoui fruit extract, which comprises collecting mature ligustrum quihoui fruit, and preparing the ligustrum quihoui fruit extract by heating and refluxing; mixing the extract of Ligustrum quihoui fruit with AgNO3And mixing the solutions, performing ultrasonic treatment, and irradiating by sunlight to obtain the nano-silver bacteriostatic agent.
In addition, the plant types which are disclosed in the prior art and can be used for synthesizing the nano silver particles are less, and aiming at the problem, the simple and convenient combination method of the nano silver particles with excellent antibacterial effect is urgently needed to be developed.
Disclosure of Invention
The invention provides a method for preparing nano-silver particles by using a burdock root extracting solution, which has mild reaction conditions, does not need heating, ultrasound, illumination and other operations, has simple preparation process and less energy consumption, and the prepared nano-silver particles have stable structure, small average particle size of 5-50 nm, high biofilm formation and mobility inhibition rate on rice bacterial blight pathogenic bacteria and excellent prevention and control effect on rice bacterial blight.
The technical scheme is as follows:
a method for preparing nano-silver particles by using a burdock root extracting solution comprises the following steps:
(1) cleaning and drying burdock root, grinding to obtain burdock root powder, uniformly stirring the burdock root powder and deionized water, and centrifuging to obtain a burdock root extracting solution;
(2) mixing the burdock root extract in the step (1) with AgNO3And (3) mixing the solutions, reacting at 20-30 ℃ in the dark to obtain a mixed solution, and centrifuging, washing and vacuum freeze-drying the mixed solution to obtain the nano-silver particles.
Burdock belongs to the order of platycodon, also named: the malignant transformation and the powerful seed are mainly distributed in China, Western Europe, the Kaishmil region, Europe and the like. Burdock is rich in vitamins, minerals, and amino acids. In addition, burdock has functions of treating anemia, leukemia, headache, jaundice, gastropathy and intermittent malaria fever.
The burdock root contains rich inulin and volatile oil, burdock acid, various polyphenol substances and aldehydes, and is rich in cellulose, amino acid and other substances, the substances play an important role in reducing silver nitrate, and can also be used as a capping agent and a stabilizing agent for synthesizing nano silver particles, and the nano silver particles prepared by adopting the burdock root extracting solution are beneficial to better extracting nano silver. In addition, the method is carried out under the condition of keeping out of the sun, the possibility that silver nitrate is directly decomposed under the condition of illumination is avoided, and the silver nitrate is continuously stirred in the reaction process, so that the contact area is increased, and the active substances in the burdock root extract can more quickly reduce silver ions in the solution into nano silver particles.
According to the method, burdock from Xuzhou Jiangsu is selected, the root extracting solution of the burdock is utilized to prepare the nano-silver particles, the prepared nano-silver particles are stable in structure and have strong bacteriostatic activity on the bacterial blight pathogenic bacteria of rice, and the inhibitory effect of the nano-silver on the bacterial blight pathogenic bacteria of rice is remarkably improved.
Preferably, in the step (1), the mass ratio of the burdock root powder to the deionized water is 1: 100-200.
Further preferably, the burdock root powder and the deionized water are uniformly mixed by using a mobile stirrer, the power of the stirrer is 100 +/-10W, the stirring time is 1-2 h, the extraction efficiency of effective components of the burdock root is low due to too low power, and unnecessary energy consumption is caused due to too high power.
Preferably, in step (2), the AgNO is3The concentration of the solution is 0.5-2 mM, the burdock root extract andAgNO3the volume ratio of the solution is 1-2: 7, the generation of nano-silver particles can be influenced by the excessively high content of the effective components of the burdock roots, and the synthesis efficiency of the nano-silver particles is low due to the excessively low content of the effective components of the burdock roots.
Preferably, in the step (2), the burdock root extract and AgNO are mixed3And (3) immediately placing the mixed solution in a dark environment at 20-30 ℃ for reaction after mixing, and stopping the reaction when the color of the mixed solution is changed from reddish brown to dark brown.
Preferably, in the step (2), continuous stirring is required in the light-shielding reaction process, the stirring speed is 300-1000 rpm/min, and the stirring time is 2-3 h. Too short stirring time can cause insufficient contact, insufficient reaction and poor synthesis effect, and finally cause overlarge particle size and uneven distribution of the nano silver, thereby affecting the bacteriostatic effect of the product; too long a stirring time is liable to deteriorate and causes unnecessary energy consumption.
Preferably, in the step (2), the centrifugation conditions are as follows: the rotation speed is 10000-14000 rpm, and the time is 10-15 min. The centrifugation rate is too low, the time is too short, the supernatant contains impurities, the stability of the finally obtained nano silver particles is low, and the bacteriostatic effect is further influenced.
The invention also provides the nano-silver particles prepared by the method for preparing the nano-silver particles by using the burdock root extracting solution, wherein the particle size of the nano-silver particles is 5-50 nm. The nano silver particles prepared by the method have small average particle size and stable structure.
The invention also provides application of the nano silver particles as an agricultural bactericide in preventing and treating rice bacterial leaf blight.
Preferably, the application method comprises the following steps: and dissolving the nano silver particles in water to obtain AgNPs solution, and uniformly spraying the AgNPs solution on the rice seedlings.
Further preferably, the concentration of the AgNPs solution is 5-20 mu g/ml. The AgNPs solution within the concentration range has good inhibition effect on bacterial blight pathogenic bacteria of rice when being used as an agricultural bactericide.
Compared with the prior art, the invention has the beneficial effects that:
(1) the preparation method has the advantages of simple preparation process, less energy consumption, easily obtained raw materials, no need of additionally adding a reducing agent and a catalyst, no need of operations such as illumination, ultrasound, heating and the like, and suitability for large-scale production;
(2) the nano-silver particles obtained by the method have stable structure and small average particle size of 5-50 nm, have high biofilm formation and mobility inhibition rate on bacterial blight of rice, and have excellent prevention and treatment effect on bacterial blight of rice;
(3) the nano silver particles prepared by the invention are used as an agricultural antibacterial agent to be applied to preventing and controlling the bacterial leaf blight of rice, and have good prevention and control effects; the plant-derived agricultural bactericide is environment-friendly, high in environmental compatibility and high in safety, and has wide application and popularization prospects in the field of agricultural production.
Drawings
Fig. 1 is an ultraviolet spectrum, an infrared spectrum and an X-ray diffraction pattern of the nano silver particles prepared in example 1, wherein a is the ultraviolet spectrum, B is the infrared spectrum and C is the X-ray diffraction pattern.
Fig. 2 is a result of analyzing the morphology of the nano-silver particles prepared in example 1, wherein a is a scanning electron microscope image, B is a transmission electron microscope image, and C is a particle size statistical chart.
FIG. 3 shows the bacteriostatic performance of AgNPs solutions with different concentrations on rice bacterial blight pathogenic bacteria, wherein A is a bacteriostatic effect graph, B is a bacteriostatic circle diameter statistical graph, C is a light absorption value change graph, and a, B and C show significant differences.
FIG. 4 is a graph showing the effect of AgNPs solutions with different concentrations on the biofilm formation and the motility of bacterial blight pathogenic bacteria of rice, wherein A is a graph showing the effect on the biofilm formation, B is a graph showing the effect on the motility, and a, B, c and d show significant differences.
In FIG. 5, A is the cell wall structure of normal rice bacterial blight germ; b is the cell wall structure of the rice bacterial leaf blight original bacteria treated by 20g/ml AgNPs solution; c is SEM picture of rice bacterial leaf blight original bacteria treated by 20 mu g/ml AgNPs solution; d is an EDS analysis chart of the bacterial blight pathogenic bacteria of rice treated by 20 mu g/ml AgNPs solution.
Detailed Description
The invention is further elucidated with reference to the figures and the examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention.
Example 1
(1) Selecting fresh burdock root, cleaning, drying and grinding to obtain burdock root powder, wherein the burdock root powder can be stored in a refrigerator at the temperature of-20 ℃ in vacuum; uniformly stirring the burdock root powder and deionized water according to the mass ratio of 1: 150 by using a stirrer, wherein the stirring rate power is 100 +/-10W, the stirring time is 1h, then placing the burdock root powder and the deionized water in a centrifuge for centrifugation at 10000rpm for 10min, and taking the supernatant to obtain a red-brown burdock root extracting solution;
(2) mixing the radix Arctii extractive solution with 1.25mM AgNO3And (3) mixing the solutions according to the volume ratio of 1: 4, immediately placing the mixed solutions in a dark condition at 25 ℃ for reaction, continuously stirring the mixed solutions for 2 hours at 600rpm/min in the reaction process until the color of the mixed solution is changed from reddish brown to dark brown, stopping the reaction, centrifuging the mixed solution at 14000rpm/min for 10 minutes, taking the precipitate, washing the precipitate twice with deionized water, and preparing powdery particles by adopting a vacuum freeze-drying method to obtain the nano-silver particles.
Example 2
(1) Selecting fresh burdock root, cleaning, drying and grinding to obtain burdock root powder, wherein the burdock root powder can be stored in a refrigerator at the temperature of-20 ℃ in vacuum; uniformly mixing the burdock root powder and deionized water according to the mass ratio of 1: 120 by using a stirrer, wherein the stirring rate power is 100 +/-10W, the stirring time is 1.5h, placing the mixture in a centrifuge for centrifugation at 10000rpm for 10min, and taking the supernatant to obtain a red-brown burdock root extracting solution;
(2) mixing the radix Arctii extractive solution with 1.5mM AgNO3And (3) mixing the solutions according to the volume ratio of 1: 3, immediately placing the mixed solutions in a dark condition at 25 ℃ for reaction, continuously stirring the mixed solutions for 2 hours at 800rpm/min in the reaction process until the color of the mixed solution is changed from reddish brown to dark brown, stopping the reaction, centrifuging the mixed solution at 14000rpm/min for 10 minutes, taking the precipitate, washing the precipitate twice with deionized water, and preparing powdery particles by adopting a vacuum freeze-drying method to obtain the nano-silver particles.
Example 3
(1) Selecting fresh burdock root, cleaning, drying and grinding to obtain burdock root powder, wherein the burdock root powder can be stored in a refrigerator at the temperature of-20 ℃ in vacuum; uniformly mixing the burdock root powder and deionized water according to the mass ratio of 1: 100 by using a stirrer, wherein the stirring rate power is 100 +/-10W, the stirring time is 2 hours, then placing the mixture in a centrifuge for centrifugation at 10000rpm for 10min, and taking the supernatant to obtain a red-brown burdock root extracting solution;
(2) mixing the radix Arctii extractive solution with 2mM AgNO3And (3) mixing the solutions according to the volume ratio of 1: 6, immediately placing the mixed solutions in a dark condition at 25 ℃ for reaction, continuously stirring the mixed solutions for 3 hours at 700rpm/min in the reaction process until the color of the mixed solution is changed from reddish brown to dark brown, stopping the reaction, centrifuging the mixed solution at 14000rpm/min for 10 minutes, taking the precipitate, washing the precipitate twice with deionized water, and preparing powdery particles by adopting a vacuum freeze-drying method to obtain the nano-silver particles.
Sample analysis
(1) The radix Arctii extract is red brown and AgNO3The solution is colorless and transparent, and the burdock root extract and AgNO are3When the solution is mixed and reacts away from light to prepare the nano-silver, the color of the mixed solution is changed from reddish brown to dark brown.
(2) The physical and chemical properties and structural characteristics of the nano-silver particles prepared in example 1 were evaluated by using ultraviolet-visible absorption spectroscopy (UV-VIS), fourier transform infrared spectroscopy (FTIR), X-ray diffraction (XRD), Transmission Electron Microscope (TEM), and Scanning Electron Microscope (SEM), and the specific results were as follows:
a in FIG. 1 is an ultraviolet-visible (UV-Vis) absorption spectrum diagram of the nano-silver particles, which shows an absorption peak at 438nm in the range of 300-700nm, and preliminarily proves the generation of the nano-silver particles;
b in FIG. 1 is Fourier Transform Infrared (FTIR) spectrum of the nano-silver particles, 3396cm-1The absorption peak of (a) is caused by N-H and O-H stretching vibration; 2928cm-1Is caused by C-H tensile vibration; 1601cm-1Is caused by a C ═ O amide group; 1376cm-1Absorption peak of (A) is represented by COO-Induce, 1026cm-1Is caused by C-N tensile vibration. Therefore, the nano silver particles are successfully synthesized;
c in fig. 1 is an X-ray diffraction pattern (XRD) of the nano-silver particles, and characteristic peaks of (111), (200), (220), and (311) appear at positions 2 θ of 38.117 °, 44.294 °, 64.503 °, 77.411 °, respectively, which are consistent with the characteristic peaks of the reported nano-silver powder diffraction pattern, confirming that stable nano-silver particles are synthesized;
a in fig. 2 is a Scanning Electron Microscope (SEM) image of the nano silver particles, the nano silver particles are spherical, and the nano silver particles have a high specific surface area and surface energy and are prone to agglomeration;
b in fig. 2 is a Transmission Electron Microscope (TEM) of the nano silver particles, which shows that the synthesized nano silver particles are spherical particles with uniform size;
c in FIG. 2 is a particle size statistical chart of the nano-silver particles, and it can be seen that the diameters of the nano-silver particles are smaller and are between 8nm and 36 nm.
(3) Dissolving nano-silver particles prepared in different masses in example 1 in deionized water to prepare AgNPs solutions of 5 microgram/ml, 10 microgram/ml and 20 microgram/ml, measuring the growth, biofilm formation and mobility of the bacterial blight bacterial of rice, staining viable and dead bacteria, performing a loss cytometry detection test, observing a TEM image of the cell membrane of the bacterial blight bacterial of rice under normal and AgNPs solution treatment, and detecting the bacteriostatic activity of AgNPs, wherein the detection result is as follows.
Firstly, the antagonism of the nano silver particles to the rice bacterial leaf blight pathogenic bacteria is evaluated through a bacteriostasis zone and a bacteria liquid light absorption value measured on a 0.5% nutrient agar double-plate.
A in figure 3 is a graph showing the effect of the diameter of inhibition zones of 5 mu g/ml, 10 mu g/ml and 20 mu g/ml AgNPs solution on bacterial blight pathogenic bacteria of rice, and B in figure 3 shows that the inhibition zones of the AgNPs with the concentrations of 5 mu g/ml, 10 mu g/ml and 20 mu g/ml respectively reach 9.1mm, 14.1mm and 16.5 mm. In the picture, a, b and c represent significant differences.
As is clear from C in FIG. 3, the growth inhibition rates of the bacterial blight pathogenic fungi of rice by the AgNPs solutions added at concentrations of 5. mu.g/ml, 10. mu.g/ml and 20. mu.g/ml were 45.08%, 69.28% and 72.10%, respectively, and a, b and C in the figure represent significant differences. The AgNPs solution has strong bacteriostatic activity, the higher the concentration is, the better the bacteriostatic effect is, and the bacteriostatic effect is obvious when the concentration is 10-20 mu g/ml.
Secondly, the bacteriostatic effect of the nano-silver particles can be indirectly evaluated by measuring the forming and swimming capacities of bacterial biofilms, as shown in A in figure 4, the growth of the biofilms is measured by a crystal violet staining method, the forming capacities of the bacterial biofilms are respectively reduced by 48.70%, 55.08% and 60.41% by AgNPs with the concentrations of 5 mug/ml, 10 mug/ml and 20 mug/ml compared with the biofilms generated by the bacterial blight pathogenic bacteria with the concentration of 0 mug/ml (without nano-silver treatment), and a, b, c and d in the picture represent significant differences.
As shown in B in FIG. 4, by measuring the bacterial motility on a 0.3% nutrient agar medium plate, the inhibition rates of the bacterial motility of AgNPs solutions with the concentrations of 5. mu.g/ml, 10. mu.g/ml and 20. mu.g/ml, compared with the bacterial motility with the concentration of 0. mu.g/ml (without nano-silver treatment), respectively reach 28.76%, 36.40% and 51.43%, and a, B, c and d in the picture represent significant differences. The inhibition of the bacterial motility can directly weaken the adhesion of the bacterial blight pathogenic bacteria to the rice surface, and inhibit the growth of the bacterial blight pathogenic bacteria and the attack of rice.
Thirdly, in order to visually judge the damage degree of the AgNPs solution to the bacterial biofilm, a TEM is used for observing the cell wall structure of the normal rice bacterial leaf blight pathogenic bacteria (A in figure 5) and the cell wall structure of the rice bacterial leaf blight pathogenic bacteria treated by the 20g/ml AgNPs solution (B in figure 5), and the cell wall damage is found, so that the content of the cell obviously flows out.
SEM (C in FIG. 5) and X-ray energy spectrum analysis (EDS) analysis (D in FIG. 5) of the bacterial blight of rice under the treatment of the AgNPs solution revealed that the surface of the bacterial blight of rice treated with the AgNPs solution had a peak of Ag element, indicating that the nano-silver particles killed the bacteria by adhering to the bacterial cells. The nano silver particles react with related protein on the surface of the bacteria and enzyme related to bacterial respiration, and break the internal and external osmotic pressure balance of the bacteria to break the cell membrane of the bacteria until the bacteria die, so that a better antibacterial effect is obtained.
Application example 1
Weighing 1g of the nano-silver particles obtained in the example 1, dissolving the nano-silver particles in 50L of water to prepare a 20 mu g/ml AgNPs solution, uniformly spraying the AgNPs solution on rice seedlings which are likely to produce rice bacterial blight in a greenhouse or a field, and observing that the rice seedlings sprayed with the AgNPs solution grow better than the rice seedlings which are not sprayed, so that the AgNPs solution can effectively prevent and treat diseases of the rice seedlings.
The embodiments described above are intended to illustrate the technical solutions of the present invention in detail, and it should be understood that the above-mentioned embodiments are only specific embodiments of the present invention, and are not intended to limit the present invention, and any modification, supplement or similar substitution made within the scope of the principles of the present invention should be included in the protection scope of the present invention.
Claims (10)
1. A method for preparing nano-silver particles by using a burdock root extracting solution is characterized by comprising the following steps:
(1) cleaning and drying burdock root, grinding to obtain burdock root powder, uniformly stirring the burdock root powder and deionized water, and centrifuging to obtain a burdock root extracting solution;
(2) mixing the burdock root extract in the step (1) with AgNO3And (3) mixing the solutions, reacting at 20-30 ℃ in the dark to obtain a mixed solution, and centrifuging, washing and vacuum freeze-drying the mixed solution to obtain the nano-silver particles.
2. The method for preparing nano-silver particles by using the burdock root extracting solution according to claim 1, wherein the mass ratio of the burdock root powder to the deionized water is 1: 100-200.
3. The method for preparing nano silver particles by using burdock root extracting solution according to claim 1, wherein in the step (2), AgNO is adopted3The concentration of the solution is 0.5-2 mM, and the burdock root extract and AgNO are3The volume ratio of the solution is 1-2: 7.
4. The method for preparing nano-silver particles by using the burdock root extracting solution as claimed in claim 1, wherein in the step (2), the burdock root extracting solution and AgNO are used3Solution mixingAnd (3) immediately placing the mixture in a dark environment at 20-30 ℃ for reaction, and stopping the reaction when the color of the mixed solution is changed from reddish brown to dark brown.
5. The method for preparing nano silver particles by using the burdock root extracting solution according to claim 1, wherein in the step (2), continuous stirring is required in a light-shielding reaction process, the stirring speed is 200-1000 rpm/min, and the stirring time is 2-3 h.
6. The method for preparing nano silver particles by using the burdock root extracting solution according to claim 1, wherein in the step (2), the centrifugation conditions are as follows: the rotation speed is 10000-14000 rpm, and the time is 10-15 min.
7. The nano-silver particles prepared by the method for preparing the nano-silver particles by using the burdock root extracting solution according to any one of claims 1 to 6, wherein the particle size of the nano-silver particles is 5 to 50 nm.
8. The use of the nano-silver particles according to claim 7 as an agricultural fungicide in the control of bacterial blight of rice.
9. The application of the nano-silver particles as the agricultural bactericide in preventing and controlling bacterial blight of rice is characterized in that the application method comprises the following steps: and dissolving the nano silver particles in water to obtain AgNPs solution, and uniformly spraying the AgNPs solution on the rice seedlings.
10. The application of the nano-silver particles as the agricultural bactericide in preventing and treating the bacterial blight of rice as claimed in claim 8, wherein the concentration of the AgNPs solution is 5-20 μ g/ml.
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