CN111184024A - Method for preparing nano-silver composite bacteriostatic agent by using thyme leaf extracting solution - Google Patents

Method for preparing nano-silver composite bacteriostatic agent by using thyme leaf extracting solution Download PDF

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CN111184024A
CN111184024A CN202010036632.9A CN202010036632A CN111184024A CN 111184024 A CN111184024 A CN 111184024A CN 202010036632 A CN202010036632 A CN 202010036632A CN 111184024 A CN111184024 A CN 111184024A
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nano
silver
thyme
bacteriostatic agent
pva
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王磊
宿红艳
李玉洁
刘晓玲
郝朋
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Ludong University
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Ludong University
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    • AHUMAN NECESSITIES
    • A01AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01NPRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N59/00Biocides, pest repellants or attractants, or plant growth regulators containing elements or inorganic compounds
    • A01N59/16Heavy metals; Compounds thereof
    • AHUMAN NECESSITIES
    • A01AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01NPRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N25/00Biocides, pest repellants or attractants, or plant growth regulators, characterised by their forms, or by their non-active ingredients or by their methods of application, e.g. seed treatment or sequential application; Substances for reducing the noxious effect of the active ingredients to organisms other than pests
    • A01N25/22Biocides, pest repellants or attractants, or plant growth regulators, characterised by their forms, or by their non-active ingredients or by their methods of application, e.g. seed treatment or sequential application; Substances for reducing the noxious effect of the active ingredients to organisms other than pests containing ingredients stabilising the active ingredients

Abstract

The invention relates to a preparation method of a thyme leaf extracting solution and a biosynthetic nano-silver composite bacteriostatic agent, belonging to the technical field of biological nano. The method comprises the following steps: 1. cleaning and drying the thyme leaves; 2. weighing 1.0g of thyme leaf, cutting the leaf into pieces, adding 20mL of extractant, shaking at 37 deg.C and 200rpm for 1-8h, filtering, centrifuging at 12000rpm for 10min, collecting supernatant to obtain thyme leaf extractive solution, and storing at 4 deg.C; 3. adding PVA and silver nitrate powder into 40mL of ultrapure water, stirring and mixing for 5-10min, adding 1-10mL of thyme leaf extracting solution, fixing the volume to 50mL, and magnetically stirring for 1-100min in a constant-temperature water bath at 40-100 ℃ to obtain the biosynthetic nano silver/PVA composite bacteriostatic agent. According to the invention, the nano silver biosynthesized by thyme leaves can be loaded on PVA, so that the stability of silver nanoparticles can be greatly enhanced, and the antibacterial activity of the nano silver is also obviously improved compared with that of PVA loaded chemically synthesized nano silver.

Description

Method for preparing nano-silver composite bacteriostatic agent by using thyme leaf extracting solution
Technical Field
The invention relates to a preparation method of a thyme leaf extracting solution and a biosynthetic nano-silver composite bacteriostatic agent, belonging to the technical field of biological nano.
Background
The nano Silver (Silver nanoparticles, AgNPs) is a metallic Silver simple substance with the particle size of 10-100nm prepared by adopting a nano technology. The unique surface effect and quantum size effect of the nano silver particles enable the sterilization effect to generate qualitative leap, and the extremely small amount of nano silver can kill hundreds of pathogenic microorganisms such as escherichia coli, staphylococcus aureus, gonococcus, chlamydia trachomatis and the like within a few minutes, has the characteristics of wide antibacterial spectrum, strong antibacterial activity, lasting antibacterial property, no drug resistance and the like, becomes a substitute of traditional bacteriostatic agents such as antibiotics and the like, and has wide development and application prospects. Has been widely applied to the fields of coating, textile, plastic and rubber products, ceramic and glass products, medicine, medical appliances and the like as a new antibacterial material.
Nano silver is generally prepared by physical and chemical methods, but the two methods have certain defects: the physical method has high requirements on equipment; in the chemical method, reducing agents such as sodium borohydride, hydrazine hydrate, dimethylformamide and the like, and stabilizing agents such as polyvinylpyrrolidone, sodium dodecyl sulfate and the like are required to be added in the preparation process, and the chemical substances cause great harm to the health of human beings and the environment. In addition, the silver nanoparticles prepared by the method are extremely easy to agglomerate and oxidize, so that the popularization of the silver nanoparticles in practical application is limited. In contrast, in recent years, attempts have been made to biosynthesize nano silver by using active substances contained in living bodies such as plants and microorganisms as a reducing agent and a stabilizer, and compared with the conventional nano silver preparation method, the biosynthesizing method has attracted attention because of the advantages of wide raw material sources, low price, mild reaction conditions, environmental friendliness and the like. The biological extract is rich in protein, lipid, polysaccharide and other biological macromolecules, can be combined with the nano material to endow the nano material with unique physicochemical properties and biological characteristics, so that the product is more stable and the bacteriostatic effect is more obvious. Although there are many reports of biosynthesized nano-silver bacteriostats at present, research shows that synthetic products of different biological materials have great difference, the forms and the stabilities of the products are different, and the sensitivities of different pathogenic bacteria to different biosynthesized nano-silver are also different.
Thyme (Thymus mongolicus Ronn), belonging to the genus Thymus of the family Labiatae, is named because of its strong floral aroma, and is also called thyme, such as thyme, thym. Thyme is native to the coast of the mediterranean sea and is widely cultivated as a spice commonly used for cooking, and more than 300 and 400 varieties exist around the world. The herba Thymi mainly contains volatile oil components such as thymol, carvacrol, linalool, geraniol and cymene, and flavone components such as taxifolin, baicalein, thymol glycoside, genkwanin and apigenin. Besides volatile oils and flavonoids, thyme also contains organic acids, amino acids, glycosides and many trace elements. Thyme can be used as medicine, and has effects of resisting oxidation, scavenging oxygen free radicals, resisting bacteria and antisepsis, dispelling pathogenic wind and relieving pain, relieving cough and eliminating phlegm, promoting digestion, and improving headache. Because the active substances in different plant resources have rich diversity and can endow different physicochemical properties and biological activity to the biological synthesis of the nano-silver, the utilization and screening of more biological materials to synthesize the novel nano-silver have important application value and practical significance.
Polyvinyl alcohol (PVA) is a biodegradable polymer material, and has the advantages of low price, convenient synthesis, safety, low toxicity and environmental protection, and in recent years, PVA is widely applied to the aspects of synthetic materials, emulsifiers, film coating agents, adhesives, pharmaceutical excipients and the like due to the advantages of good film forming property, high thermal stability and the like. The PVA is used as the nano silver carrier, so that the agglomeration of silver nano particles can be prevented, and the stability of the silver nano particles is greatly improved.
Although reports of PVA loaded with nano silver exist at present, research on PVA loaded biosynthesis of nano silver is still blank, and composite nano silver is often greatly different due to the fact that nano silver with different performances is loaded, and the antibacterial activity and stability of the composite nano silver are directly influenced.
Disclosure of Invention
The invention aims to provide a method for preparing a biosynthetic nano silver/PVA composite bacteriostatic agent by utilizing a thyme leaf extracting solution, which is synthesized by a one-step method, is simple to operate and has low production cost. The nano silver biosynthesized by the thyme leaves is loaded on the PVA, so that the stability of the silver nanoparticles can be greatly enhanced, and the antibacterial activity of the nano silver is also obviously improved compared with that of the PVA loaded chemically synthesized nano silver.
A method for preparing a nano-silver composite bacteriostatic agent by utilizing a thyme leaf extracting solution is characterized by comprising the following steps of:
1. pretreatment of materials
Cleaning and drying the thyme leaves;
the thyme leaf is washed clean by tap water and then washed by ultrapure water for 3 times;
and the drying temperature is controlled at 60 ℃, and the mixture is dried to constant weight.
2. Preparation of thyme leaf extract
Weighing 1.0g of thyme leaf, cutting the leaf into pieces, adding 20mL of extractant, shaking at 37 deg.C and 200rpm for 1-8h, filtering, centrifuging at 12000rpm for 10min, collecting supernatant to obtain thyme leaf extractive solution, and storing at 4 deg.C;
the filtration is carried out by adopting Whatman No.1 qualitative filter paper;
the extractant is any one of deionized water, 40% ethanol, 70% ethanol, 100% methanol and 100% diethyl ether;
3. preparing nano silver composite bacteriostatic agent
Adding a certain amount of PVA and silver nitrate powder into 40mL of ultrapure water, stirring and mixing for 5-10min, adding 1-10mL of thyme leaf extracting solution, fixing the volume to 50mL, and magnetically stirring for 1-100min in a constant-temperature water bath at 40-100 ℃ to obtain the biosynthetic nano-silver/PVA composite bacteriostatic agent;
the final concentration of the PVA in mass fraction is 0.01-50%, and the final concentration of the silver nitrate is 0.1-10 mM;
and (3) centrifuging the nano silver/PVA composite bacteriostatic agent at 12000rpm for 10min, resuspending the nano silver/PVA composite bacteriostatic agent with ultrapure water, and storing the bacteriostatic agent in a dark place.
The method for biologically synthesizing the nano-silver composite bacteriostatic agent by the thyme leaf extract has the following beneficial effects:
1. the production cost is low, the synthesis steps are simple, the synthesis can be completed by only one step, the synthesis speed is high, and the environment is protected;
2. on the basis that the dispersibility and stability of nano-silver are improved by biomolecules contained in thyme such as amino acid, protein, glucoside and the like, the agglomeration and oxidation of nano-silver particles are further prevented by a PVA carrier, the synthesized product solution has high stability, can be kept away from light at 4 ℃ for 6 months without obvious aggregation, and can still maintain high antibacterial activity after being treated at 100 ℃ for 1 hour;
3. the synthesized product has higher antibacterial activity to gram-positive bacteria and gram-negative bacteria, and the antibacterial activity is obviously improved compared with the chemically synthesized nano silver/PVA.
Drawings
FIG. 1: the ultraviolet-visible absorption spectrum of the biosynthetic nano silver/PVA composite bacteriostatic agent prepared in the embodiment 1 of the invention;
FIG. 2: the ultraviolet-visible absorption spectrum of the biosynthetic nano silver/PVA composite bacteriostatic agent prepared in the embodiment 2 of the invention;
FIG. 3: a transmission electron microscope image of the biosynthetic nano silver/PVA composite bacteriostatic agent prepared at 80 ℃ in the embodiment 2 of the invention;
FIG. 4: an experimental graph of the bacteriostatic circle of the biosynthetic nano silver/PVA composite bacteriostatic agent prepared at 80 ℃ in the embodiment 2 of the invention on pseudomonas aeruginosa;
FIG. 5: and (3) testing the heat stability of the antibacterial preparation.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Example 1
In this embodiment, the nano-silver composite bacteriostatic agent is biosynthesized by the thyme leaf extract, and the preparation method is as follows:
1. collecting thyme leaf, washing with tap water, washing with ultrapure water for 3 times, drying at 60 deg.C, weighing 1.0g thyme leaf, cutting leaf into pieces, adding 20mL 40% ethanol, shaking at 37 deg.C and 200rpm for 2 hr, filtering with Whatman No.1 qualitative filter paper, centrifuging at 12000rpm for 10min, collecting supernatant to obtain thyme leaf extractive solution, and storing at 4 deg.C for use.
2. Adding 400mg of PVA and 50mg of silver nitrate into 40mL of ultrapure water, stirring and mixing for 10min, adding 1mL of the thyme leaf extracting solution, diluting to 50mL of volume, magnetically stirring in a constant-temperature water bath at 90 ℃ for respectively reacting for 0, 5, 10, 15 and 30min to obtain the biological synthesis nano silver/PVA composite bacteriostatic agent, centrifuging the prepared nano silver/PVA composite bacteriostatic agent at 12000rpm for 10min, resuspending the ultrapure water, and storing in a dark place.
3. Visible-ultraviolet absorption spectrum of biosynthetic nano silver/PVA composite bacteriostatic agent
The synthesis of nano silver can be characterized by strong visible region absorption in a visible-ultraviolet absorption spectrogram. As shown in figure 1, no absorption peak exists at the beginning of the reaction, after the reaction is carried out for 5min, the composite material begins to generate a characteristic absorption peak of the nano-silver particles at the position with the wavelength of 413nm, the absorption peak is gradually enhanced along with the prolonging of the reaction time, and a strong absorption peak of the nano-silver appears at the position of 420nm after 30min, which proves that the thyme leaf extract can successfully mediate the synthesis of the nano-silver.
Example 2
In this embodiment, the nano-silver composite bacteriostatic agent is biosynthesized by the thyme leaf extract, and the preparation method is as follows:
1. collecting thyme leaf, washing with tap water, washing with ultrapure water for 3 times, and oven drying at 60 deg.C. Weighing 1.0g of thyme leaf, cutting the leaf into pieces, adding 20mL of 70% ethanol, shaking at 37 deg.C and 200rpm for 1h, filtering with Whatman No.1 qualitative filter paper, centrifuging at 12000rpm for 10min, collecting supernatant to obtain thyme leaf extractive solution, and storing at 4 deg.C;
2. adding 250mg of PVA and 20mg of silver nitrate into 40mL of ultrapure water, stirring and mixing for 5min, adding 1mL of the thyme leaf extracting solution, diluting to 50mL of constant volume, magnetically stirring the mixed solution at constant temperatures of 50, 60, 70 and 80 ℃ respectively, reacting for 20min to obtain the biological synthesis nano silver/PVA composite bacteriostatic agent, centrifuging the prepared nano silver/PVA composite bacteriostatic agent at 12000rpm for 10min, resuspending the ultrapure water, and storing in a dark place.
3. Visible-ultraviolet absorption spectrum of the biosynthetic nano silver/PVA composite bacteriostatic agent. The synthesis of nano silver can be characterized by strong visible region absorption in a visible-ultraviolet absorption spectrogram. As shown in figure 2, the characteristic absorption peak of the nano-silver is not generated under the synthesis condition of 50 ℃, the characteristic absorption peak of the nano-silver starts to be generated at 424nm at 60 ℃, the peak height of the absorption peak is gradually strengthened along with the increase of the reaction temperature, the absorption peak is subjected to blue shift, and the characteristic absorption peak is generated at 419nm when the temperature is increased to 80 ℃, and the half-peak width is narrowed. Therefore, the thyme leaf extract can mediate the biosynthesis of the nano-silver bacteriostatic agent, the synthesis efficiency of the nano-silver is gradually enhanced along with the increase of the reaction temperature, the synthesized particle size is gradually reduced, and the particle size is more uniform.
4. And (4) observing the biosynthetic nano silver/PVA composite bacteriostatic agent by using a transmission electron microscope. 5-10 mul of the composite nano-silver bacteriostatic agent solution sample prepared at 80 ℃ in the step 2 is dripped on a copper net, dried at room temperature and observed by a transmission electron microscope under the voltage of 80-300 kV. As shown in figure 3, the nano silver particles biosynthesized from thyme are spherical, have relatively uniform particle size, are mostly about 30nm and are distributed on the surface of PVA in a monodispersed form, which indicates that the prepared nano silver composite material is in an ideal state.
Comparative example 1
Comparing the nano-silver/PVA composite bacteriostatic agent prepared at 80 ℃ in the embodiment 2 of the invention with the conventional chemically synthesized nano-silver/PVA composite bacteriostatic agent:
1) chemically synthesized nano silver/PVA composite bacteriostatic agent
Adding 250mg of PVA and 20mg of silver nitrate into 40mL of ultrapure water, stirring and mixing for 10min, adding sodium borohydride (final concentration is 2mM), fixing the volume to 50mL, magnetically stirring the mixed solution at the constant temperature of 80 ℃, reacting for 20min to obtain the chemically synthesized nano silver/PVA composite bacteriostatic agent, centrifuging the prepared nano silver/PVA composite bacteriostatic agent at 12000rpm for 10min, resuspending the ultrapure water, and storing in a dark place.
2) Preparation of LB Medium
Preparing an LB liquid culture medium: 5g of yeast extract, 10g of peptone, 5g of sodium chloride and ultra-pure water are added to reach the constant volume of 1000mL, and the pH value is adjusted to 7.2. The LB solid medium is prepared by adding 1.5-2% agar to the culture solution prepared above. Placing the above two culture mediums in an autoclave, and autoclaving at 120 deg.C and 0.1MPa for 20 min. Cooling the sterilized liquid culture medium to room temperature, and storing in a refrigerator at 4 ℃; and the solid culture medium is subpackaged in a sterilized culture dish in a clean bench before solidification, cooled to room temperature, and stored in a refrigerator at 4 ℃ after solidification.
3) Zone of inhibition experiment
Selecting typical clinical drug-resistant pathogenic bacteria pseudomonas aeruginosa as an indicator bacterium, and detecting the bacteriostatic activity of an active substance by adopting a cup and dish method to carry out a bacteriostatic circle experiment, wherein the method comprises the following specific steps: culturing Pseudomonas aeruginosa in LB liquid culture medium at 37 deg.C with shaking to logarithmic growth stage, and diluting with normal saline to 1 × 106And (3) uniformly coating 100 mu L of CFU/mL on a solid LB culture medium, empirically placing 4 Oxford cups (the outer diameter of each cup is 8mm) on the culture medium, and respectively adding 20 mu L of solutions to be detected. The result is shown in fig. 4, wherein 1 is the chemically synthesized nano silver/PVA composite bacteriostatic agent; 2 is the thyme biosynthesis nano silver/PVA composite bacteriostatic agent prepared at 80 ℃ in example 2; 3 is thyme extract; 4 is a 0.5% PVA solution. As can be seen from the figure, thyme extract and PVA which are used as synthetic raw materials have no bacteriostatic action; the biological synthesis nano silver/PVA composite bacteriostatic agent generates an obvious bacteriostatic circle for clinical drug-resistant pathogenic bacteria, and the bacteriostatic circle is larger than that generated by the chemical biological synthesis nano silver/PVA composite bacteriostatic agent. The biological synthesis nano silver particles are loaded on the PVA surface, so that the nano silver has larger specific surface area, the aggregation of silver nano particles is prevented, and the stability and the bacteriostatic effect of the silver nano particles are greatly improved.
4) Experiment of minimum inhibitory concentration
Common clinical drug-resistant pathogenic bacterium pseudomonas aeruginosa is used as a test bacterium, and a two-fold dilution method is adopted to determine the minimum inhibition of the biosynthetic composite nano-silver bacteriostatic agent and the chemical synthesis composite nano-silver bacteriostatic agentBacterial concentration (MIC). Meanwhile, a biological synthesized nano silver simple substance prepared without adding a carrier PVA in the preparation process of the two composite nano silver bacteriostats and a chemically synthesized nano silver simple substance are selected as a reference, and the bacteriostasis activities of the two simple substances are compared. The specific operation is as follows: diluting the pseudomonas aeruginosa bacterial suspension cultured to logarithmic growth period to 1 × 106CFU/mL, split into 8 test tubes, each tube with 1mL bacterial liquid. Diluting four solutions to be tested into 8 concentration gradients in a two-fold gradient manner, respectively adding 1mL of the four solutions to the test tube containing the bacterial liquid, respectively adding 2mL of LB liquid culture solution and 1 × 10 solution to two test tubes6CFU/mL of Pseudomonas aeruginosa suspension served as a negative control group and a positive control group. And culturing at 37 ℃ and 150rpm for 24h, observing the experimental result, and taking the concentration of the nano-silver solution corresponding to the test tube with the largest dilution multiple and clarity as the MIC of the solution. From table 1, it can be seen that the MICs of the 4 bacteriostatic agents tested are: the biosynthesis composite nano silver is 11.9 mu g/ml; chemically synthesizing composite nano silver 25.0 mu g/ml; the biosynthesis of pure nano silver is 50.0 mu g/ml; the chemical synthesis of pure nano silver is 100.0 mu g/ml. Therefore, the biological synthesized nano silver/PVA can achieve the effect of inhibiting the growth of drug-resistant pathogenic bacteria in a smaller dosage.
TABLE 1 determination of the Minimum Inhibitory Concentration (MIC) of nanosilver formulations for the Pseudomonas aeruginosa test
Note: "+" indicates turbidity, and "-" indicates no turbidity
Comparative example 2
Evaluation of stability
1) Evaluation of thermal stability
The sodium borohydride chemically synthesized nano silver/PVA composite bacteriostatic agent prepared in the comparative example 1, the thyme biologically synthesized nano silver/PVA composite bacteriostatic agent synthesized at 80 ℃ in the example 2, the thyme biologically synthesized pure nano silver and the sodium borohydride chemically synthesized pure nano silver are respectively heated in a water bath at 100 ℃ for 1 hour, and then pseudomonas aeruginosa is used as a test bacterium to perform a bacteriostatic circle experiment to evaluate the influence of the heating treatment on the bacteriostatic activity of the bacteriostatic agent. The experimental result is shown in figure 5, wherein 1 is the nano silver/PVA composite bacteriostatic agent chemically synthesized by sodium borohydride; 2 is thyme biosynthesis nano silver/PVA composite bacteriostatic agent; 3, synthesizing pure nano silver by thyme; 4, chemically synthesizing pure nano silver by using sodium borohydride; 5 is a positive control of the biological synthesis nano silver/PVA composite bacteriostatic agent without heat treatment; 6 is PVA solution negative control. It can be seen from the figure that the biosynthetic nano silver/PVA composite bacteriostatic agent without high-temperature treatment can generate an obvious bacteriostatic circle for pseudomonas aeruginosa, the negative PVA solution does not generate the bacteriostatic circle, the composite nano silver bacteriostatic agent and the biosynthetic pure nano silver bacteriostatic agent have better thermal stability, and still maintain higher bacteriostatic activity after high-temperature treatment, while the chemically synthesized pure nano silver bacteriostatic circle is relatively smaller. The thyme biosynthetic nano silver/PVA composite bacteriostatic agent has the largest bacteriostatic circle and the best thermal stability among the four materials subjected to high-temperature treatment.
2) Evaluation of Long-term stability
The method comprises the following steps of placing sodium borohydride chemically synthesized nano silver/PVA composite bacteriostatic agent, thyme biologically synthesized nano silver/PVA composite bacteriostatic agent prepared at 80 ℃ in example 2, thyme biologically synthesized pure nano silver and sodium borohydride chemically synthesized pure nano silver at 4 ℃ in a dark place, and regularly carrying out appearance observation, wherein the chemical synthesized pure nano silver has the phenomena of turbid solution and precipitation separation after being placed for 10 days; the chemically synthesized nano silver/PVA composite bacteriostatic agent and the biologically synthesized pure nano silver respectively have precipitation phenomena after 1 month and 3 months; and the solution of the thyme biosynthesis nano silver/PVA composite bacteriostatic agent is still clear after being placed for 6 months, and no precipitation phenomenon occurs. The inhibition zone of the solution which is precipitated on the pathogenic bacteria to be tested is obviously reduced, and the solution which is not precipitated still has an obvious inhibition zone on the pathogenic bacteria to be tested. Therefore, the high stability of the composite biological synthesis nano silver bacteriostatic agent prepared by the invention is beneficial to keeping the bacteriostatic activity of the bacteriostatic agent, and the bacteriostatic agent is suitable for being developed and utilized as a commercial bacteriostatic agent.
Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that changes, modifications, substitutions and alterations can be made in these embodiments without departing from the principles and spirit of the invention, the scope of which is defined in the appended claims and their equivalents.

Claims (7)

1. A method for preparing a nano-silver composite bacteriostatic agent by utilizing a thyme leaf extracting solution is characterized by comprising the following steps of:
1) pretreatment of materials
Cleaning and drying the thyme leaves;
2) preparation of thyme leaf extract
Weighing 1.0g of thyme leaf, cutting the leaf into pieces, adding 20mL of extractant, shaking at 37 deg.C and 200rpm for 1-8h, filtering, centrifuging at 12000rpm for 10min, collecting supernatant to obtain thyme leaf extractive solution, and storing at 4 deg.C;
3) preparing the nano-silver composite bacteriostatic agent
Adding PVA and silver nitrate powder into 40mL of ultrapure water, stirring and mixing for 5-10min, adding 1-10mL of thyme leaf extracting solution, fixing the volume to 50mL, and magnetically stirring for 1-100min in a constant-temperature water bath at 40-100 ℃ to obtain the biosynthetic nano silver/PVA composite bacteriostatic agent.
2. The method for preparing nano-silver composite bacteriostatic agent by using thyme leaf extracting solution according to claim 1, wherein in step 1), thyme leaves are washed clean with tap water and then washed with ultrapure water for 3 times.
3. The method for preparing the nano-silver composite bacteriostatic agent by utilizing the thyme leaf extracting solution according to claim 1, wherein the drying temperature in the step 1) is controlled at 60 ℃ and the nano-silver composite bacteriostatic agent is dried to constant weight.
4. The method for preparing nano-silver composite bacteriostatic agent by using thyme leaf extracting solution according to claim 1, wherein the filtration in the step 2) is performed by Whatman No.1 qualitative filter paper.
5. The method for preparing nano-silver composite bacteriostatic agent by using thyme leaf extracting solution according to claim 1, wherein the extracting agent in the step 2) is any one of deionized water, 40% ethanol, 70% ethanol, 100% methanol and 100% diethyl ether.
6. The method for preparing nano-silver composite bacteriostatic agent by using the thyme leaf extracting solution as claimed in claim 1, wherein the final concentration of PVA in the step 3) is 0.01-50% by mass fraction, and the final concentration of silver nitrate is 0.1-10 mM.
7. The method for preparing nano-silver composite bacteriostatic agent by using thyme leaf extracting solution according to claim 1, characterized in that the nano-silver/PVA composite bacteriostatic agent is centrifuged at 12000rpm for 10min in the step 3), and then is resuspended in ultrapure water and kept away from light.
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