CN102888428B - Method for synthesizing nano silver by utilizing Bacillus amyloliquefaciensBacillus amyloliquefaciens LSSE-62 - Google Patents
Method for synthesizing nano silver by utilizing Bacillus amyloliquefaciensBacillus amyloliquefaciens LSSE-62 Download PDFInfo
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Abstract
The invention relates to the field of microorganism and in particular relates to a method for synthesizing nano silver by utilizing Bacillus amyloliquefaciens LSSE-62. The method comprises the following steps of: 1) fermenting and cultivating the Bacillus amyloliquefaciens LSSE-62, and separating to obtain thallus and fermented supernate; and 2) taking the thallus and fermented supernate obtained in the step 1) as reaction matrix, adding AgNO3 solution to cause concentration of the reaction matrix in the obtained mixed solution to be 1-4mg/mL and concentration of AgNO3 to be 1-3mM, and reacting under the condition of sunniness or ultraviolet irradiation, so as to obtain nano silver particles, wherein the preservation number of the Bacillus amyloliquefaciens LSSE-62 is CGMCC No.4157. The synthetic method disclosed by the invention is safe and has no toxic or harm effect, synthesis speed is high, and reaction can be completed within 80-100min, so that the synthetic method is efficient, safe and simple.
Description
Technical field
The present invention relates to microorganism field, particularly, the present invention relates to a kind of method of utilizing bacillus amyloliquefaciens LSSE-62 synthesizing nano-silver.
Background technology
In recent years, metal nanoparticle more and more is applied to various fields, as electronics, medicine and biological technical field.Nanometer silver is as a kind of metal nanoparticle of uniqueness, and it,, in field applied researcies such as anti-biotic material, therapeutics, biomolecule detection and catalysis, has caused the concern of researcher.
Green Chemistry will seek development safety, environmental protection, energy-conservation green technology.The traditional chemical method was widely applied in synthesizing of nanometer silver, yet the toxicant that these methods relate to and hot conditions do not meet principle (the document 1:P.Quaresma that sees reference in detail, L.Soares, the L.Contar of Green Chemistry, A.Miranda, I.Osorio, P.A.Carvalho, R.Franco and E.Pereira, Green Chem., 2009,11,1889-1893).The biosynthesizing nanometer silver technology of development in recent years is considered to simply, gentle, eco-friendly synthetic method, as the method for preparing nanometer silver by bacterium, fungi, biomolecules, (document 2:K.B.Narayanan and N.Sakthivel in detail sees reference, Adv.Colloid Interface Sci., 2010,156,1-13).Albumen and enzyme are proved to be nanometer silver and synthesize and stable important matrix, and in the process of preparation, albumen and enzyme are as reductive agent and stablizer, by Ag
+be reduced to nanometer silver, however not clear for its detailed reaction mechanism.Owing to being easy to be operated and genetic modification, bacterium is considered to most promising nanometer silver synthetic substrate.Yet most of bacterium resultant velocity of report is all slow at present, the Silver Nitrate of reduction 1mM needs 24-120 hour, and this has limited its industrial applications greatly.Research finds, under visible ray (tungsten-iodine lamp) illuminate condition, enterobacteria (bacterium as white as kerekou pneumonia) but quickly synthesizing nano silver, yet this class bacterium is common pathogenic bacteria, there is safety issue, do not meet principle (A.R.Shahverdi, the S.Minaeian of Green Chemistry, H.R.Shahverdi, H.Jamalifar and A.-A.Nohi, Process Biochem., 2007,42,919-923).Therefore, the application attempts carrying out synthesizing nano-silver by food-grade microorganisms (bacillus amyloliquefaciens LSSE-62) first.
Summary of the invention
The object of the present invention is to provide a kind of method of utilizing bacillus amyloliquefaciens LSSE-62 synthesizing nano-silver.
According to the method for utilizing bacillus amyloliquefaciens LSSE-62 synthesizing nano-silver of the present invention, said method comprising the steps of:
1) bacillus amyloliquefaciens LSSE-62 is carried out to fermentation culture, and separation obtains thalline and fermented supernatant fluid;
2) get thalline in step 1) or supernatant liquor as response matrix, add AgNO
3solution, in the mixing solutions that makes to obtain, response matrix concentration is 1～4mg/mL, AgNO
3concentration is 1～3mM, under solar radiation or UV-irradiation condition, reacts, and obtains nano-Ag particles;
Wherein,
Described bacillus amyloliquefaciens LSSE-62 deposit number is CGMCC No.4157.
According to the method for utilizing bacillus amyloliquefaciens LSSE-62 synthesizing nano-silver of the present invention, described step 2) in, add AgNO
3solution after, then drip and to contain Cl
-solution, make Cl in mixing solutions
-concentration be 0.5～3mM.
According to the method for utilizing bacillus amyloliquefaciens LSSE-62 synthesizing nano-silver of the present invention, in described step 2) in, Cl contained
-solution comprise: NaCl, KCl or CaCl
2.
According to the method for utilizing bacillus amyloliquefaciens LSSE-62 synthesizing nano-silver of the present invention, described step 2) in sunlit intensity be: 10000～100000lx; Ultraviolet ray intensity is 300lx.
According to the method for utilizing bacillus amyloliquefaciens LSSE-62 synthesizing nano-silver of the present invention, bacillus amyloliquefaciens LSSE-62 in described step 1), depositary institution is Chinese common micro-organisms culture presevation management committee's common micro-organisms center (CGMCC), deposit number is CGMCC No.4157, preservation date on September 14th, 2010.
According to one embodiment of the invention, what described method was concrete comprises the following steps:
A, fermentation culture
Bacillus amyloliquefaciens LSSE-62 is inoculated in LB liquid seed culture medium (containing peptone 10g/L, yeast powder 5g/L, NaCl10g/L, pH7.2), and 37 ℃, 180rpm cultivates 12h, according to 3%(v/w) inoculum size, add in the LB liquid nutrient medium, 37 ℃, 200rpm cultivates 24h; The centrifugal collection thalline of 10000rpm, supernatant liquor is as the nanometer silver synthetic substrate; Thalline centrifuge washing twice, ultrasonic disruption, centrifugal removal cell debris, cell extract is as the nanometer silver synthetic substrate.
B, nanometer silver synthesize
Synthetic substrate comprises cell extract and/or fermented supernatant fluid, and reaction system is: substrate concn is 1-4mg/mL, AgNO
3final concentration is 1-3mM, and the NaCl final concentration is 0.5-3mM, under solar radiation or UV-irradiation condition, and reaction 100min.
The detection of c, nanometer silver
Can detect the synthetic of nanometer silver by ultraviolet-visible spectrophotometry and colour-change, characterize shape, size and the crystal formation of nanometer silver by transmission electron microscope, energy dispersion X-ray spectrum (EDS) and X-ray diffraction (XRD).Detect residual silver ions by inductive coupling plasma emission spectrograph (ICP-OES).
Utilize at present the method for preparing nanometer silver by bacterium, fungi, biomolecules, because its simple, gentle characteristics receive publicity, but the kind of the bacterium of realizing this purpose that can be used at present, fungi, biomolecules is but very limited, and exist transformation time long, there is the shortcomings such as toxic side effect, and bacillus amyloliquefaciens LSSE-62 of the present invention is food-grade microorganisms, and the generated time of nanometer silver is shortened to the 2h left and right by existing 24h.In concrete building-up process, the condition of reaction has significant impact to effect of the present invention, it is embodied in the selection of illumination condition, adopts in the present invention in the situation of bacillus amyloliquefaciens LSSE-62 as matrix, and sunlight and UV-light have an important impact to of the present invention equally.In addition, not only can adopt in the present invention the thalline synthesizing nano-silver of bacillus amyloliquefaciens LSSE-62, in the fermentation culture process at this thalline, the secretory product that supernatant liquor contains bacillus amyloliquefaciens LSSE-62 contains Cl simultaneously
-ion, therefore, find also efficient synthesizing nano-silver of fermented supernatant fluid in the present invention.
The method of bacillus amyloliquefaciens LSSE-62 synthesizing nano-silver of passing through provided by the present invention, take bacillus amyloliquefaciens as response matrix, safe and reliable; Cell extract and fermented supernatant fluid by bacillus amyloliquefaciens prepare nano-Ag particles, can complete reaction in 80～100min, and resultant velocity is fast; Synthetic nanometer silver is the nanocrystal of size distribution at 4-40nm; The nano-Ag particles solution height is stable, at room temperature can keep not occurring in 2 months obvious gathering; Nanometer silver has efficient anti-microbial activity to gram-positive microorganism and Gram-negative bacteria.
The method of bacillus amyloliquefaciens synthesizing nano-silver provided by the present invention, have stronger novelty and practicality, and its advantage is as follows:
(1) utilize first bacillus amyloliquefaciens synthesizing nano-silver particle, synthetic nanometer silver is the nanocrystal of size distribution at 4-40nm, the retainable high dispersing stability of nano-class silver colloidal solution, and gram-positive microorganism and Gram-negative bacteria are had to efficient anti-microbial activity.
(2) synthetic method safety non-toxic evil effect, resultant velocity is fast, can complete reaction in 80～100min, is a kind of efficient, safety, simple synthetic method.
The accompanying drawing explanation
The ultraviolet-visible absorption spectroscopy that Fig. 1 is synthesizing nano-silver under different cell extraction substrate concentrations;
The Zeta-potential that Fig. 2 is synthesizing nano-silver under different cell extraction substrate concentrations;
Fig. 3 adds the ultraviolet-visible absorption spectroscopy of different concns NaCl synthesizing nano-silver in cell extract;
Fig. 4 adds the Zeta-potential of different concns NaCl synthesizing nano-silver in cell extract;
The ultraviolet-visible absorption spectroscopy that Fig. 5 is cell extract synthesizing nano-silver under different sunlight intensity;
The ultraviolet-visible absorption spectroscopy that Fig. 6 is cell extract synthesizing nano-silver under sunlight, UV-light and indoor white light conditions;
The scanning electron microscope picture that Fig. 7 is the cell extract synthesizing nano-silver;
The energy dispersion X-ray spectrum EDS that Fig. 8 is the cell extract synthesizing nano-silver
The size distribution statistics that Fig. 9 is the cell extract synthesizing nano-silver;
The X ray diffracting spectrum that Figure 10 is the cell extract synthesizing nano-silver (XRD);
Figure 11 is to Bacillus subtillis and colibacillary growth inhibition ratio on the nanometer silver liquid medium within;
Figure 12 is the inhibition circle that nanometer silver produces Bacillus subtillis on solid plate;
Figure 13 is the inhibition circle that nanometer silver produces intestinal bacteria on solid plate;
The ultraviolet-visible absorption spectroscopy that Figure 14 is synthesizing nano-silver under different fermentations supernatant liquor concentration;
The scanning electron microscope picture that Figure 15 is the fermented supernatant fluid synthesizing nano-silver;
The energy dispersion X-ray spectrum EDS that Figure 16 is the fermented supernatant fluid synthesizing nano-silver;
The size distribution statistics that Figure 17 is the fermented supernatant fluid synthesizing nano-silver.
Bacillus amyloliquefaciens LSSE-62(Bacillus amyloliquefaciens) be stored in China Committee for Culture Collection of Microorganisms's common micro-organisms center (preservation centre address: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City Institute of Microorganism, Academia Sinica on September 14th, 2010, postcode: 100101), its deposit number is: CGMCC No.4157.
Embodiment
The present invention is described in detail by the following examples, but all embodiment do not form any restriction to the present invention.
A, fermentation culture
Bacillus amyloliquefaciens LSSE-62 is inoculated in LB liquid seed culture medium (containing peptone 10g/L, yeast powder 5g/L, NaCl10g/L, pH7.2), and 37 ℃, 180rpm cultivates 12h, according to 3%(v/w) inoculum size, add in the LB liquid nutrient medium, 37 ℃, 200rpm cultivates 24h; The centrifugal collection thalline of 10000rpm, thalline centrifuge washing twice, ultrasonic disruption, centrifugal removal cell debris, cell extract is as the nanometer silver synthetic substrate.
B, nanometer silver synthesize
Be 1,2,3, add the silver nitrate solution of 1mM in the cell extract of 4mg/mL to concentration, under solar radiation (70000lx) condition, reaction 100min.
The sign of c, nanometer silver
The synthetic of nanometer silver can absorb and characterize by colour-change and its strong visible region, Fig. 1 shows, the reaction system of not adding cell extract does not produce colour-change, do not have visible region to absorb yet, 1,2,3, under the cell extract concentration of 4mg/mL, generate orange redly, and have strong visible absorption to produce, prove synthesizing of nanometer silver.Detect the surface potential of nano-Ag particles simultaneously, Fig. 2 shows, the surperficial Zeta-potential absolute value of nanometer silver all is greater than 50mV, when nano grain surface Zeta-potential absolute value is greater than 50mV, the nanoparticles solution system can keep high stability, the nanometer silver of observation discovery synthesized is at room temperature preserved more than two months obvious agglomeration is not occurred, and has also confirmed its high stability.
A, fermentation culture
Bacillus amyloliquefaciens LSSE-62 is inoculated in LB liquid seed culture medium (containing peptone 10g/L, yeast powder 5g/L, NaCl10g/L, pH7.2), and 37 ℃, 180rpm cultivates 12h, according to 3%(v/w) inoculum size, add in the LB liquid nutrient medium, 37 ℃, 200rpm cultivates 24h; The centrifugal collection thalline of 10000rpm, thalline centrifuge washing twice, ultrasonic disruption, centrifugal removal cell debris, cell extract is as the nanometer silver synthetic substrate.
B, nanometer silver synthesize
The silver nitrate solution that adds 1mM in the cell extract that is 3mg/mL to concentration, add 0.5,1,2, the NaCl of 3mM, under solar radiation (70000lx) condition, and reaction 100min.
The sign of c, nanometer silver
The synthetic of nanometer silver can absorb and characterize by colour-change and its strong visible region, and accompanying drawing 3 shows, adds NaCl and can significantly strengthen the synthetic of nanometer silver.Fig. 4 demonstration, the surperficial Zeta-potential absolute value of nanometer silver all is greater than 50mV, and the nanoparticles solution system can keep high stability.
A, fermentation culture
Bacillus amyloliquefaciens LSSE-62 is inoculated in LB liquid seed culture medium (containing peptone 10g/L, yeast powder 5g/L, NaCl10g/L, pH7.2), and 37 ℃, 180rpm cultivates 12h, according to 3%(v/w) inoculum size, add in the LB liquid nutrient medium, 37 ℃, 200rpm cultivates 24h; The centrifugal collection thalline of 10000rpm, thalline centrifuge washing twice, ultrasonic disruption, centrifugal removal cell debris, cell extract is as the nanometer silver synthetic substrate.
B, nanometer silver synthesize
The silver nitrate solution that adds 1mM in the cell extract that is 4mg/mL to concentration, solar radiation (30000,40000,50000,70000lx) under the condition, the reaction 100min.
The sign of c, nanometer silver
The synthetic of nanometer silver can absorb and characterize by colour-change and its strong visible region, accompanying drawing 5 shows, can not synthesizing nano-silver under dark condition, sunlight strength affects the resultant velocity of nanometer silver, the nanoparticles solution system of synthesized can keep high stability, at room temperature deposits agglomeration does not occur in 2 months.
A, fermentation culture
Bacillus amyloliquefaciens LSSE-62 is inoculated in LB liquid seed culture medium (containing peptone 10g/L, yeast powder 5g/L, NaCl10g/L, pH7.2), and 37 ℃, 180rpm cultivates 12h, according to 3%(v/w) inoculum size, add in the LB liquid nutrient medium, 37 ℃, 200rpm cultivates 24h; The centrifugal collection thalline of 10000rpm, thalline centrifuge washing twice, ultrasonic disruption, centrifugal removal cell debris, cell extract is as the nanometer silver synthetic substrate.
B, nanometer silver synthesize
The silver nitrate solution that adds 1mM in the cell extract that is 4mg/mL to concentration, under solar light irradiation, UV-irradiation (300lx) and indoor white light (300lx) condition, reaction 100min.
The sign of c, nanometer silver
Accompanying drawing 6 demonstrations, during reaction 100min, but quickly synthesizing nano silver under the UV-irradiation condition compares the sunlight direct projection and do not have the significance variation, and under indoor white light conditions, resultant velocity is very slow, needs to continue to react 20 hours, just can reach maximum value.Therefore direct sunlight and uviolizing proposed by the invention is more efficient reaction conditions.Solar radiation not only can provide powerful intensity of illumination, and sun power is the renewable resources of clean environment firendly, and the present invention utilizes sunlight can save a large amount of electric energy that source of artificial light consumes.
A, fermentation culture
Bacillus amyloliquefaciens LSSE-62 is inoculated in LB liquid seed culture medium (containing peptone 10g/L, yeast powder 5g/L, NaCl10g/L, pH7.2), and 37 ℃, 180rpm cultivates 12h, according to 3%(v/w) inoculum size, add in the LB liquid nutrient medium, 37 ℃, 200rpm cultivates 24h; The centrifugal collection thalline of 10000rpm, thalline centrifuge washing twice, ultrasonic disruption, centrifugal removal cell debris, cell extract is as the nanometer silver synthetic substrate.
B, nanometer silver synthesize
The silver nitrate solution that adds 1mM in the cell extract that is 3mg/mL to concentration, add the NaCl of 2mM, under solar radiation (70000lx) condition, and reaction 80min.
The sign of c, nanometer silver
Accompanying drawing 7 shows, the scanning electron microscope demonstration, and synthetic nanometer silver be take trilateral as main, also has a small amount of trilateral.Energy dispersion X-ray spectrum (EDS) analyze to show that absorption peak (3keV) that typical nanocrystalline silver appears in the nano particle of synthesized (Fig. 8).The particle diameter statistical study shows that median size reaches 14.6nm (Fig. 9).X-ray diffraction (XRD) analyze to show that 2 θ values of the nanometer silver collection of illustrative plates of synthesized are 37.95 °, 45.95 °, and 64.22 ° and 76.74 ° (Figure 10), 111 of respectively corresponding simple substance silver crystal, 200,220 and 311 feature pedigrees, prove in the nanometer silver of synthesized and contain simple substance silver crystal.Synthesized nanometer silver Zeta-potential reaches-70.84, can keep the dispersion stabilization of height, under room temperature, places agglomeration does not occur in two months.Detect residual silver ions by inductive coupling plasma emission spectrograph (ICP-OES), negatively charged ion almost all is reduced simultaneously, and transformation efficiency reaches 98.23%, and in the safe microorganisms of hitherto reported, bacterial strain resultant velocity of the present invention is the fastest.
A, fermentation culture
Bacillus amyloliquefaciens LSSE-62 is inoculated in LB liquid seed culture medium (containing peptone 10g/L, yeast powder 5g/L, NaCl10g/L, pH7.2), and 37 ℃, 180rpm cultivates 12h, according to 3%(v/w) inoculum size, add in the LB liquid nutrient medium, 37 ℃, 200rpm cultivates 24h; The centrifugal collection thalline of 10000rpm, fermented supernatant fluid is as the nanometer silver synthetic substrate.
B, nanometer silver synthesize
Be 1,2, add the silver nitrate solution of 1mM in the fermented supernatant fluid of 3mg/mL to concentration, under solar radiation (70000lx) condition, reaction 80min.
The sign of c, nanometer silver
Accompanying drawing 14 shows, 1,2, under the cell extract concentration of 3mg/mL, reaction system generates orange red, and has strong visible absorption to produce, and prove synthesizing of nanometer silver.
A, fermentation culture
Bacillus amyloliquefaciens LSSE-62 is inoculated in LB liquid seed culture medium (containing peptone 10g/L, yeast powder 5g/L, NaCl10g/L, pH7.2), and 37 ℃, 180rpm cultivates 12h, according to 3%(v/w) inoculum size, add in the LB liquid nutrient medium, 37 ℃, 200rpm cultivates 24h; The centrifugal collection thalline of 10000rpm, fermented supernatant fluid is as the nanometer silver synthetic substrate.
B, nanometer silver synthesize
The silver nitrate solution that adds 1mM in the fermented supernatant fluid that is 2mg/mL to concentration, under solar radiation (70000lx) condition, reaction 80min.
The sign of c, nanometer silver
The scanning electron microscope demonstration, synthetic nanometer silver be take circle as main, also has a small amount of trilateral (accompanying drawing 15).EDS confirms that absorption peak (3keV) that typical nanocrystalline silver appears in the nano particle of synthesized (Figure 16).Statistical study shows that median size reaches 18.78nm (Figure 17).
The antibacterial effect evaluation of the nanometer silver that embodiment 8 prepares
The present invention has investigated the antibacterial effect of nanometer silver to gram-positive microorganism (Bacillus subtillis) and Gram-negative bacteria (intestinal bacteria), as Figure 11 shows in the nano-Ag particles liquid medium within of embodiment 3 synthesizeds, Bacillus subtillis and intestinal bacteria are had to significant antibacterial effect, on solid medium, nano-Ag particles has shown obvious inhibition zone, and gram-positive microorganism (Bacillus subtillis) and Gram-negative bacteria (intestinal bacteria) are also had to significant antibacterial effect (as Figure 12 and 13).Compare intestinal bacteria, Bacillus subtillis is more responsive to nanometer silver.In addition, embodiment 1～2, and 4～6 nanometer silvers that prepare have significant antibacterial effect to gram-positive microorganism (Bacillus subtillis) and Gram-negative bacteria (intestinal bacteria) equally.
Claims (3)
1. a method of utilizing bacillus amyloliquefaciens LSSE-62 synthesizing nano-silver, is characterized in that, said method comprising the steps of:
1) bacillus amyloliquefaciens LSSE-62 is carried out to fermentation culture, and separation obtains thalline and fermented supernatant fluid;
2) thalline of getting in step 1) obtains cell extract through fragmentation, using the supernatant liquor in cell extract or step 1) as response matrix, adds AgNO
3solution, in the mixing solutions that makes to obtain, response matrix concentration is 1～4mg/mL, AgNO
3concentration is 1～3mM, under solar radiation or UV-irradiation condition, reacts, and obtains nano-Ag particles;
Wherein, when cell extract during as response matrix, step 2) add AgNO
3solution after, then drip and to contain Cl
-solution;
Wherein, described sunlit intensity is 10000～100000lx, and UV-irradiation intensity is 300lx;
Wherein, described bacillus amyloliquefaciens LSSE-62 deposit number is CGMCC No.4157.
2. the method for utilizing bacillus amyloliquefaciens LSSE-62 synthesizing nano-silver according to claim 1, is characterized in that, described step 2) in, during as response matrix, add AgNO when cell extract
3solution after, then drip and to contain Cl
-solution, make Cl in mixing solutions
-concentration be 0.5～3mM.
3. the method for utilizing bacillus amyloliquefaciens LSSE-62 synthesizing nano-silver according to claim 1, is characterized in that, in described step 2) in, during as response matrix, contain Cl when cell extract
-solution comprise: NaCl, KCl or CaCl
2.
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| CN103146758A (en) * | 2013-02-22 | 2013-06-12 | 北京科技大学 | Method for preparing nano silver particles through microbial surface reduction |
| CN103642844A (en) * | 2013-12-13 | 2014-03-19 | 东北林业大学 | Method for preparing nano silver particles by reducing in bacillus thallus |
| CN103627664B (en) * | 2013-12-13 | 2016-02-03 | 东北林业大学 | One strain can reduce the bacillus amyloliquefaciens preparing nanometer silver |
| CN103710390B (en) * | 2013-12-18 | 2015-09-09 | 广东省微生物研究所 | A kind of simple method utilizing ferment product to prepare nanometer silver |
| CN104357485B (en) * | 2014-11-14 | 2017-08-25 | 东北林业大学 | A kind of method that utilization sulfate reducing bacteria prepares Ag/AgCl visible light catalysts |
| CN105039419B (en) * | 2015-06-29 | 2017-10-17 | 山东师范大学 | A kind of method of utilization Trichoderma asperellum synthesizing nano-silver |
| CN105562710A (en) * | 2016-01-06 | 2016-05-11 | 华中农业大学 | Method for biosynthesizing nano-silver through photoinduction of imperata leaf extract |
| CN107354175B (en) * | 2017-06-29 | 2021-05-18 | 华南理工大学 | Silver nano material and biological preparation method and application thereof |
| CN107653267A (en) * | 2017-07-26 | 2018-02-02 | 华南理工大学 | A kind of silver nano material and its biological preparation method and application |
| CN109097405A (en) * | 2018-09-04 | 2018-12-28 | 四川农业大学 | A kind of bulbus fritillariae cirrhosae endogenetic fungus mediates biological synthesis method and the application of nano silver |
| CN110272922B (en) * | 2019-03-14 | 2021-09-21 | 浙江大学 | Method for synthesizing nano-silver by utilizing pseudomonas praecox cell-free supernatant, product and application thereof |
| CN110804089B (en) * | 2019-10-18 | 2022-03-11 | 淮阴工学院 | Nano-silver synthetic protein derived from spherical lysine bacillus and application thereof |
| CN113966747B (en) * | 2021-06-30 | 2022-04-29 | 中国热带农业科学院环境与植物保护研究所 | Nano-silver composite bacillus agent for preventing and treating syzygium samarangense fruit rot and anthracnose of syzygium samarangense fruit and preparation method thereof |
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