CN103642844A - Method for preparing nano silver particles by reducing in bacillus thallus - Google Patents
Method for preparing nano silver particles by reducing in bacillus thallus Download PDFInfo
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- CN103642844A CN103642844A CN201310682242.9A CN201310682242A CN103642844A CN 103642844 A CN103642844 A CN 103642844A CN 201310682242 A CN201310682242 A CN 201310682242A CN 103642844 A CN103642844 A CN 103642844A
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Abstract
The invention relates to a method for preparing nano silver particles by reducing in bacillus thallus, relating to the field of preparation of nano silver materials, and aiming at solving the problems that the granularity is large and the particles are non-uniformly distributed in an existing method for preparing the nano silver by utilizing the bacteria. The method disclosed by the invention comprises the steps of I, preparing a culture liquid; II, adding an AgNO3 solution into the culture liquid, and culturing in a light-tight manner to obtain a bacterium solution; and III, centrifuging the bacterium solution, collecting thallus, ultrasonically crushing the thallus, and centrifuging to obtain the nano silver particles. The nano silver particles prepared by the method disclosed by the invention are narrow in granularity distribution and distributed in the range of 5nm to 15nm, and the prepared nano silver particles are unlikely to agglomerate, have good biological compatibility and are easy to collect and store and capable of realizing mass production. The prepared nano silver particles can be used for preparing different antibacterial agents and applied to manufacturing antibacterial ceramics, conventional fresh-keeping films and medical surgical dressing.
Description
Technical field
The present invention relates to nano silver material preparation field, be specifically related to by genus bacillus grown cultures in silver nitrate solution being prepared in thalline to the method for nano-Ag particles.
Background technology
Nanometer silver is a kind of typical nano material, because it has quantum size effect, small-size effect, surface effects and macro quanta tunnel effect, thereby present the unusual performance of the aspect such as optical, electrical, magnetic, thermodynamics and chemical reaction that is different from common material, be widely used in the fields such as chemical industry, medicine, antibacterial, metallurgical, Aeronautics and Astronautics, electronics, have broad application prospects.
Traditional preparation method of nano-Ag particles is mainly Physical and chemical method, and Physical synthesizing nano-silver energy consumption is high, and autohemagglutination easily occurs nano-Ag particles in depositing process; Chemical method chemical reagent used has harm to human body and environment to a certain extent, and some stablizers or dispersion agent even have carinogenicity.Therefore, have simple to operate, reaction temperature and, the biological reducing method of the advantage such as environmental protection just arises at the historic moment.
Research finds, bacterium, actinomycetes, fungi and plant etc. can be used for synthesis of nano ag material, and to have growth and breeding fast because of it for bacterium, and the features such as strong adaptability to environment, temperature, pressure and acidity are paid close attention to widely preparing aspect nanometer silver.At present, the method that bacterium is prepared nanometer silver has 3 kinds: adopt microorganism culture supernatant, adopt thalline soak solution, adopt the soak solution of thalline and silver nitrate solution.It is simple substance silver by silver ion reduction that these methods are all utilized the reducing substance in solution, and the nano-Ag particles particle diameter of preparation is large, skewness.And by being grown in the cultivation of the genus bacillus in silver nitrate solution, the method for preparing nano-Ag particles in thalline has no report.
Summary of the invention
The present invention seeks to solve method that current bacterium prepares nanometer silver and have the large and problem pockety of grain diameter, and a kind of method of utilizing reduction in genus bacillus body to prepare nano-Ag particles is provided.
Utilize the interior reduction of genus bacillus body to prepare the method for nano-Ag particles, carry out according to the following steps:
One, get 1 and connect collarium genus bacillus, be seeded in the Erlenmeyer flask that contains 200ml LB substratum, at 37 ℃, in the shaking culture case of 120r/min, cultivate 20-24h, obtain nutrient solution;
Two, in nutrient solution, add 5ml10mM AgNO
3solution, at 37 ℃, in the shaking culture case of 120r/min, lucifuge is cultivated 5-25 days, obtains bacterium liquid;
Three, bacterium liquid centrifugal 10min under the condition of 5000r/min, collects thalline, then ultrasonication, centrifugal 5min under the condition of 15000r/min again, remove cell debris, obtain the nano-Ag particles that particle diameter is 5～15nm, complete and utilize reduction in genus bacillus body to prepare nano-Ag particles;
Wherein in step 1, genus bacillus is bacillus amyloliquefaciens (Bacillus amyloliquefaciens) zxw01, the center preservation of Ta Yi China Committee for Culture Collection of Microorganisms common micro-organisms, deposit number is CGMCC No.8464, the preservation time is on November 11st, 2013, and preservation address is No. 3, No. 1, Beichen Lu, Chaoyang District, Beijing City institute.
The present invention by having prepared nano-Ag particles to being grown in the cultivation of the genus bacillus in silver nitrate solution in thalline, the interior synthetic nanometer silver narrow diameter distribution of genus bacillus thalline and difficult reunion, in building-up process, do not adopt any tensio-active agent, protective material and linking agent; Prepare nanometer silver with traditional physics, chemical process and have that cost is high, complicated operation has the problems such as harm to compare with chemical reagent used with environment to human body, the invention provides a kind of nanometer silver preparation method with low cost, simple to operate and eco-friendly, and the nano-Ag particles narrow diameter distribution of preparation, be distributed in 5～15nm, be difficult for reuniting, there is good biocompatibility, be easy to Collection and preservation and the production of can magnifying.
In the present invention, utilize the nano-Ag particles of reduction preparation in genus bacillus body, under the identical condition of nanometer silver concentration, action time is longer, and bacteriostasis rate is higher, therefore can be used in the various antiseptic-germicides of preparation, be applied to manufacture anti-bacteria ceramic, conventional preservative film, medical dressing etc.
The nano-Ag particles of preparing in the present invention, can substitute super fine silver powder and do electric slurry, and the conductive resin of making, can save silver powder 50%, and with this conductive resin welding metal and pottery, coatingsurface is very smooth; Also can be used in and prepare electrode, can increase the direct contact area of electrode and liquid or gas, improve the efficiency of battery, be conducive to the miniaturization of battery.Can also using nano-Ag particles as the solid support material of enzyme, realize the Direct electron transfer of enzyme, improve the sensitivity of biosensor.
Accompanying drawing explanation
Fig. 1 is the transmission electron microscope figure of the nano-Ag particles prepared in embodiment;
Fig. 2 is the x-ray diffraction pattern of the nano-Ag particles prepared in embodiment.
Embodiment
Technical solution of the present invention is not limited to following cited embodiment, also comprises the arbitrary combination between each embodiment.
Embodiment one: present embodiment utilizes the interior reduction of genus bacillus body to prepare the method for nano-Ag particles, carries out according to the following steps:
One, get 1 and connect collarium genus bacillus, be seeded in the Erlenmeyer flask that contains 200ml LB substratum, at 37 ℃, in the shaking culture case of 120r/min, cultivate 20-24h, obtain nutrient solution;
Two, in nutrient solution, add 5ml10mM AgNO
3solution, at 37 ℃, in the shaking culture case of 120r/min, lucifuge is cultivated 25 days, obtains bacterium liquid;
Three, bacterium liquid centrifugal 10min under the condition of 5000r/min, collects thalline, then ultrasonication, centrifugal 5min under the condition of 15000r/min again, remove cell debris, obtain the nano-Ag particles that particle diameter is 5～15nm, complete and utilize reduction in genus bacillus body to prepare nano-Ag particles;
Wherein in step 1, genus bacillus is bacillus amyloliquefaciens (Bacillus amyloliquefaciens) zxw01, the center preservation of Ta Yi China Committee for Culture Collection of Microorganisms common micro-organisms, deposit number is CGMCC No.8464, the preservation time is on November 11st, 2013, and preservation address is No. 3, No. 1, Beichen Lu, Chaoyang District, Beijing City institute.
In present embodiment, genus bacillus used is bacillus amyloliquefaciens (Bacillus amyloliquefaciens) zxw01, the morphological specificity of bacterial strain: it is bacillus, long 1.5～1.8 μ m, wide 0.3～0.4 μ m, has central spore, atrichia, without pod membrane, growth bacterium colony is circular, and surface irregularity is opaque, and edge is irregular.The physiological and biochemical property of bacterial strain: it is gram-positive microorganism, catalase contact experiment is positive, casein hydrolysis, Starch Hydrolysis, gelatin hydrolysis, the hydrolysis of urea positive, acid is all produced in glucose, sucrose, seminose, fructose experiment, acid is not produced in D-wood sugar, L-arabinose, melibiose, maltose, sorbyl alcohol, inositol, N.F,USP MANNITOL, lactose, rhamnosyl experiment, nitrate reduction experiment, Fu Pu experiment, Citrate trianion experiment, the urobenzoic acid experiment positive, phenylalanine experiment is negative.
Embodiment two: present embodiment is different from embodiment one, at 37 ℃, cultivates 22h in the shaking culture case of 120r/min in step 1.Other is identical with embodiment one.
Adopt following examples to verify beneficial effect of the present invention:
Embodiment:
Utilize the interior reduction of genus bacillus body to prepare the method for nano-Ag particles, carry out according to the following steps:
One, get 1 and connect collarium genus bacillus, be seeded in the Erlenmeyer flask that contains 200ml LB substratum, at 37 ℃, in the shaking culture case of 120r/min, cultivate 22h, obtain nutrient solution;
Two, in nutrient solution, add 5ml10mM AgNO
3solution, at 37 ℃, in the shaking culture case of 120r/min, lucifuge is cultivated 25 days, obtains bacterium liquid;
Three, bacterium liquid centrifugal 10min under the condition of 5000r/min, collects thalline, then ultrasonication, centrifugal 5min under the condition of 15000r/min again, remove cell debris, obtain the nano-Ag particles that particle diameter is 5～15nm, complete and utilize reduction in genus bacillus body to prepare nano-Ag particles;
Wherein in step 1, genus bacillus is bacillus amyloliquefaciens (Bacillus amyloliquefaciens) zxw01, the center preservation of Ta Yi China Committee for Culture Collection of Microorganisms common micro-organisms, deposit number is CGMCC No.8464, the preservation time is on November 11st, 2013, and preservation address is No. 3, No. 1, Beichen Lu, Chaoyang District, Beijing City institute.
Carry out transmission electron microscope method analysis:
By the bacterium liquid centrifugal 10min under the condition of 5000r/min that cultivates 25 days, the genus bacillus thalline that collection obtains, through 2.5% glutaraldehyde (phosphoric acid buffer preparation) fixedly after 2h, in concentration is respectively 50%, 70%, 90%, 100% acetone, sample is dewatered.Then, sample is placed on 37 ℃ of baking ovens and spends the night.Sample is taken out from incubator with treating block machine finishing, expose tissue, then with treating block machine, the tissue exposing is accomplished to miter angle.The tissue block trimming after embedding, being cut into thickness is 0.5～1.0 μ m, maximum section area 2mm
2ultrathin section(ing).Section is positioned on the coated copper mesh of carbon.Once complete, with 3% acetic acid uranium, ultrathin section(ing) is dyeed, and with lead citrate, detect the ultrastructure of cell.Utilize TECNAIG2 instrument (Holland) under 60kV, to carry out imaging.As shown in Figure 1, it is spherical that this product particle is substantially, be evenly distributed, and median size 5nm, purity is high.
Carry out XRD analysis:
In step 2, lucifuge is cultivated and within 25 days, is obtained bacterium liquid, and centrifugal (7000g, 10 minutes) results, dry, and then use aseptic washing 3 times.Dry moisture for XRD analysis instrumental analysis.X-ray is 1.6KW(40KV, 40mM by power) copper X-x ray tube produce.At 2 θ, be to measure between 25-90 degree.As shown in Figure 2, gained spectrum 2 θ value peak values are at 38.45 °, and 44.48 °, 64.69 ° and 77.62 °, corresponding (111), (200), (220) and (311) face are observed the existence of silver-colored diffraction pattern.
Claims (2)
1. utilize reduction in genus bacillus body to prepare a method for nano-Ag particles, it is characterized in that it carries out according to the following steps:
One, get 1 and connect collarium genus bacillus, be seeded in the Erlenmeyer flask that contains 200ml LB substratum, at 37 ℃, in the shaking culture case of 120r/min, cultivate 20-24h, obtain nutrient solution;
Two, in nutrient solution, add 5ml10mM AgNO
3solution, at 37 ℃, in the shaking culture case of 120r/min, lucifuge is cultivated 25 days, obtains bacterium liquid;
Three, bacterium liquid centrifugal 10min under the condition of 5000r/min, collects thalline, then ultrasonication, centrifugal 5min under the condition of 15000r/min again, remove cell debris, obtain the nano-Ag particles that particle diameter is 5～15nm, complete and utilize reduction in genus bacillus body to prepare nano-Ag particles;
Wherein in step 1, genus bacillus is bacillus amyloliquefaciens (Bacillus amyloliquefaciens) zxw01, the center preservation of Ta Yi China Committee for Culture Collection of Microorganisms common micro-organisms, deposit number is CGMCC No.8464, the preservation time is on November 11st, 2013, and preservation address is No. 3, No. 1, Beichen Lu, Chaoyang District, Beijing City institute.
2. a kind of method of utilizing reduction in genus bacillus body to prepare nano-Ag particles according to claim 1, is characterized in that in step 1, at 37 ℃, in the shaking culture case of 120r/min, cultivating 22h.
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104357485A (en) * | 2014-11-14 | 2015-02-18 | 东北林业大学 | Method for preparing Ag/AgCl visible light catalyst by using sulfate reducing bacteria |
| CN107052360A (en) * | 2017-04-13 | 2017-08-18 | 湖南工业大学 | A kind of method that utilization spine spore mould prepares Nano Silver |
| CN107904262A (en) * | 2017-11-03 | 2018-04-13 | 浙江大学 | A kind of method that nano silver is prepared based on bacterial extract |
| CN111549076A (en) * | 2020-05-14 | 2020-08-18 | 东北林业大学 | Preparation method and application of fluorescent silver nanocluster probe |
| WO2024183090A1 (en) * | 2023-03-06 | 2024-09-12 | 江苏科技大学 | Method for synthesizing silver oxide nanomaterial using microorganism with assistance of low-frequency ultrasound |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102888428A (en) * | 2011-07-21 | 2013-01-23 | 中国科学院过程工程研究所 | Method for synthesizing nano silver by utilizing Bacillus amyloliquefaciensBacillus amyloliquefaciens LSSE-62 |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102888428A (en) * | 2011-07-21 | 2013-01-23 | 中国科学院过程工程研究所 | Method for synthesizing nano silver by utilizing Bacillus amyloliquefaciensBacillus amyloliquefaciens LSSE-62 |
Non-Patent Citations (1)
| Title |
|---|
| NALENTHIRAN PUGAZHENTHIRAN 等: "Microbial synthesis of silver nanoparticles by Bacillus sp.", 《J NANOPART RES》 * |
Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104357485A (en) * | 2014-11-14 | 2015-02-18 | 东北林业大学 | Method for preparing Ag/AgCl visible light catalyst by using sulfate reducing bacteria |
| 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 |
| CN107052360A (en) * | 2017-04-13 | 2017-08-18 | 湖南工业大学 | A kind of method that utilization spine spore mould prepares Nano Silver |
| CN107904262A (en) * | 2017-11-03 | 2018-04-13 | 浙江大学 | A kind of method that nano silver is prepared based on bacterial extract |
| CN107904262B (en) * | 2017-11-03 | 2021-04-13 | 浙江大学 | Method for preparing nano-silver based on bacterial extract |
| CN111549076A (en) * | 2020-05-14 | 2020-08-18 | 东北林业大学 | Preparation method and application of fluorescent silver nanocluster probe |
| CN111549076B (en) * | 2020-05-14 | 2023-04-28 | 东北林业大学 | Preparation method and application of fluorescent silver nanocluster probe |
| WO2024183090A1 (en) * | 2023-03-06 | 2024-09-12 | 江苏科技大学 | Method for synthesizing silver oxide nanomaterial using microorganism with assistance of low-frequency ultrasound |
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Application publication date: 20140319 |