CN110272922A - It is a kind of to utilize method of careless pseudomonad cell-free supernatants synthesizing nano-silver and products thereof and application - Google Patents
It is a kind of to utilize method of careless pseudomonad cell-free supernatants synthesizing nano-silver and products thereof and application Download PDFInfo
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Abstract
The invention discloses a kind of method using careless pseudomonad cell-free supernatants synthesizing nano-silver, includes the following steps: 1) to cultivate careless pseudomonad in NB culture medium, collect bacterial suspension；2) bacterial suspension is centrifuged, collects supernatant, obtains cell-free supernatants；3) AgNO is added in cell-free supernatants3Solution, obtained reaction solution obtain nano-Ag particles by centrifugation, washing, vacuum freeze drying.Nano-Ag particles stable structure produced by the present invention, good dispersion, with good bio-compatibility, there is stronger in vitro and in vivo antibacterial activity to Dickeya dadantii CZ1501, the nano metal bacteriostatic agent of Eco-friendly and good biocompatibility be can be made into for preventing and treating sweet potato stem rot, traditional chemical fungicide is alleviated to the burden of environment, there is good promotion prospect in agricultural production.
Description
Technical field
The invention belongs to green bio nano materials technical fields, and in particular to a kind of to utilize careless pseudomonad without thin
The method of born of the same parents' supernatant synthesizing nano-silver and its in application agriculturally.
Background technique
Nano material refers to its structural units within the scope of 1~100nm, because of its unique physicochemical property, as size is small, ratio
Surface area is big etc., has wide development potentiality in fields such as Science and Technologies.Wherein, nano silver (Silver
Nanoparticles, AgNPs) have stronger antibiotic property, to the Gram-negatives such as Escherichia coli, staphylococcus aureus with
Gram-positive bacterium has potential bacteriostatic activity.
The conventional method of synthesizing nano-silver mainly has physical method, chemical method, microbial method at present, utilizes physical method and chemistry
Method has simple possible, but energy demand is big in its synthesis process, it is at high cost, easily cause environmental pollution.In contrast, micro-
Biology has the advantages such as vitality is strong, wide adaptability, be easy to get, reproduction speed is fast, genetic modification facilitates, therefore closes frequently as biology
At the raw material of nano metal.
The Chinese invention patent application of Publication No. CN107904262A is disclosed a kind of prepared based on bacterial extract and received
Nano silver synthetics are made using D. radiodurans (Deinococcus radiodurans) in the method for meter Yin, then by nanometer
AgNO is added in silver-colored synthetics3It is reacted to obtain nano-Ag particles in solution.
The Chinese invention patent application of Publication No. CN105039419A discloses a kind of using Trichoderma asperellum
The method of (Trichoderma asperellum) synthesizing nano-silver, using addition silver ion and nitre in Trichoderma asperellum supernatant
Hydrochlorate is protected from light and cultivates up to nano silver, and obtained nano silver particle diameter distribution is uniform, and property is more excellent.
Bacterial supernatant contains extracellular polymeric (the Extracellular Polymeric of itself generation
Substances, EPS), EPS is secreted by microorganism, mainly bacterium in external some high scores under the conditions of certain environment
Sub- polymer, such as protein, nucleic acid, lipid, polysaccharide ingredient, play an important role in the formation of nano-Ag particles.But
Be can be used for disclosed in the prior art synthesizing nano-silver particle microbe species it is less, therefore, be based on the cell-free hair of bacterium
Ferment supernatant (CFCS) synthesizing nano-silver has very big development prospect.
In addition, Dickeya dadantii (D.dadantii) is the pathogen for causing sweet potato stem rot, the bacteriosis
Huge economic loss is brought in field and storage to sweet potato, worldwide causes great concern.In order to control the disease
Evil, many compounds such as chlorine dioxide glutaraldehyde have been applied in field trials than Zha Er oronain, Cefmepidum Chloride etc., but
Effect is unobvious.
Summary of the invention
The object of the present invention is to provide a kind of using careless pseudomonad supernatant as reducing agent synthesizing nano-silver
Method, the preparation process is environmentally protective, production cost is low, equipment is simple, and obtained nano-Ag particles size is small, good dispersion.
Another object of the present invention is to provide above-mentioned nano-Ag particles to prevent and treat the application in field sweet potato stem rot,
Above-mentioned nano-Ag particles to the pathogenic original Dickeya dadantii CZ1501 of Sweet Potato, root rot have it is stronger external and
Antibacterial activity in vivo can replace traditional chemical control method, environmentally friendly.
The purpose of the present invention is achieved through the following technical solutions:
A method of using careless pseudomonad supernatant synthesizing nano-silver, include the following steps:
(1) careless pseudomonad (Pseudomonas poae) is cultivated in NB culture medium, collects bacterial suspension；
(2) bacterial suspension is centrifuged, collects supernatant, obtains cell-free supernatants (CFCS)；
(3) AgNO is added in cell-free supernatants3Solution, obtained reaction solution is by centrifugation, washing, vacuum freeze drying
Obtain nano-Ag particles.
The present invention is using careless pseudomonad cell-free supernatants as reducing agent and AgNO3Solution mixing has been made average
Partial size is small, stable structure, can effectively inhibit the active nano silver of sweet potato Pathogen, further improve the antibacterial effect of nano silver
Fruit can effectively prevent field sweet potato stem rot.
In step (1), NB culture medium is made of 1% tryptone, 1% sucrose, 0.3% peptone, 0.1% yeast powder,
10g tryptone, 10g sucrose, 3g peptone, 1g yeast powder need to be weighed by preparing 1L NB culture medium.
In step (2) and (3), in the bacterial suspension centrifuging and taking supernatant and reaction solution centrifuging and taking precipitation process, turn
Speed is 8000~10000rpm, and 10~20min of centrifugation time, centrifugation time is long, revolving speed will cause energy waste fastly；Centrifugation time
Short, revolving speed will lead to impure in supernatant slowly, influence nano particle purity.
In step (3), the AgNO3The concentration of solution is 0.5~2mM, and concentration is too low to be not enough to synthesize nano particle, dense
The high stability and combined coefficient for then not only influencing product structure is spent, and will cause the unnecessary waste of raw material.
In step (3), the bacterial supernatant and AgNO3The volume ratio of solution is 2~3:10, and metal oxide solution is used
The excessively high material resources that will lead to are measured to waste, and too low be equivalent to of dosage reduces raw material input amount, eventually reduces combined coefficient.
In step (3), the mixed reaction solution, which is placed in 30~32 DEG C of shaking tables, be protected from light oscillation, the color of liquid to be mixed
Reaction is terminated from the light yellow dark brown that becomes.
The invention also discloses a kind of nano silver according to made from above-mentioned preparation method, the partial size of the nano silver is 20~
100nm。
The nano silver can be attached to bacterial cell surface, destroy cellular morphology, increase cell-permeant by cell wall
Property, this is because nano silver can aoxidize caused by plasma membrane surfaces albumen.Experiment shows novel nano Argent grain prepared by the present invention
Stable structure, good dispersion have good bio-compatibility, have stronger body to Dickeya dadantii CZ1501
Outer and antibacterial activity in vivo.
The invention also discloses application of the above-mentioned nano silver in prevention and treatment sweet potato stem rot.This is because what the present invention obtained
Nano-Ag particles have stronger in vitro and in vivo to the pathogenic original Dickeya dadantii CZ1501 of Sweet Potato, root rot
Antibacterial activity, therefore can be made into the nano metal bacteriostatic agent of Eco-friendly and good biocompatibility, it can not only play very well
Control efficiency, also reduce traditional chemical fungicide to the burden of environment, have in agricultural production and promote well
Prospect.
Compared with the prior art, the present invention has the following beneficial effects:
(1) synthetic method of the present invention is simple, cheap, safe and harmless, belongs to green synthesis process, is suitble to extensive raw
It produces；
(2) present invention utilizes careless pseudomonad supernatant and AgNO for the first time3Solution mixing, be made average grain diameter it is small, knot
Structure is stablized, and the active nano silver of sweet potato Pathogen can be effectively inhibited；
(3) nano-Ag particles produced by the present invention can be made into nano metal bacteriostatic agent for preventing and treating sweet potato stem rot, this is anti-
It is environmentally protective to control method, does not pollute the environment, potential hazard will not be brought to human body.
Detailed description of the invention
Fig. 1 is the color change photo of synthesizing nano-silver particle in embodiment 1；
Fig. 2 is nano-Ag particles UV-Vis figure in embodiment 1；
Fig. 3 is the FTIR figure of nano-Ag particles in embodiment 1；
Fig. 4 is the TEM figure of nano-Ag particles in embodiment 1；
Fig. 5 is the XRD diagram of nano-Ag particles in embodiment 1；
Fig. 6 is the SEM-EDS figure of nano-Ag particles in embodiment 1；
Fig. 7 is that various concentration Nano silver solution is made to the growth effect figure of sweet potato Pathogen in nano silver in embodiment 1；
Fig. 8 is that the biomembrane that various concentration Nano silver solution forms sweet potato Pathogen is made in nano silver in embodiment 1
Influence diagram；
Fig. 9 is that various concentration Nano silver solution is made on the influence of the inhibition zone of sweet potato Pathogen in nano silver in embodiment 1
Figure；
Figure 10 is that nano-Ag particles that concentration is 2mg/ml are made to sweet potato Pathogen cell wall in nano silver in embodiment 1
Influence diagram；
Figure 11 is that various concentration Nano silver solution is made to the internal antibacterial effect of sweet potato Pathogen in nano silver in embodiment 1
Fruit figure.
Specific embodiment
Combined with specific embodiments below, the present invention is furture elucidated.
Bacterial strain used in the following example 1~3 is careless pseudomonad (Pseudomonas poae), is purchased from positioned at Beijing
The China General Microbiological culture presevation administrative center (CGMCC) of the institute 3 of Chaoyang District North Star West Road 1, deposit number are as follows:
CGMCC No.18734。
Embodiment 1
(1) Bacteria Culture: careless pseudomonad single bacterium drops down onto 5ml Tube propagation base on picking NA agar plate, 30 DEG C,
It after 200rpm overnight shaking culture, is forwarded in NB fluid nutrient medium according to 1% ratio, cultivation temperature is 30 DEG C, oscillation rate
200rpm is incubated overnight；
(2) collection of supernatant: the bacterial suspension that step (1) is obtained is centrifuged 10min simultaneously according to revolving speed 8000rpm
Supernatant is collected, cell-free supernatants (CFCS) is obtained；
(3) preparation of nano silver: the bacterial supernatant and 1mM AgNO for taking step (2) to obtain3Solution is filled according to 1:3 ratio
Divide mixing, be put into 200rpm shaking table and be protected from light oscillation 48h, then obtained reaction solution is passed through into 8000rpm, be centrifuged 15min,
And be washed with distilled water 3 times, vacuum freeze drying obtains nano-Ag particles.
Embodiment 2
(1) Bacteria Culture: careless pseudomonad single bacterium drops down onto 5ml Tube propagation base on picking NA agar plate, 30 DEG C,
It after 200rpm overnight shaking culture, is forwarded in NB fluid nutrient medium according to 1% ratio, cultivation temperature is 30 DEG C, oscillation rate
200rpm is incubated overnight；
(2) bacterial suspension for obtaining step (1) is centrifuged 10min and collects supernatant, obtain according to revolving speed 10000rpm
Cell-free supernatants (CFCS)；
(3) bacterial supernatant and 0.5mM AgNO for taking step (2) to obtain3Solution is sufficiently mixed according to 1:5 ratio, is put into
It carries out being protected from light oscillation 48h in 200rpm shaking table.Obtained reaction solution is passed through into 8000rpm again, is centrifuged 10min, and use distilled water
Washing 3 times, vacuum freeze drying obtains nano-Ag particles.
Embodiment 3
(1) Bacteria Culture: careless pseudomonad single bacterium drops down onto 5ml Tube propagation base on picking NA agar plate, 30 DEG C,
It after 200rpm overnight shaking culture, is forwarded in NB fluid nutrient medium according to 1% ratio, cultivation temperature is 30 DEG C, oscillation rate
200rpm is incubated overnight；
(2) bacterial suspension for obtaining step (1) is centrifuged 15min and collects supernatant, obtain according to revolving speed 9000rpm
Cell-free supernatants (CFCS)；
(3) bacterial supernatant and 1.5mM AgNO for taking step (2) to obtain3Solution is sufficiently mixed according to 1:4 ratio, is put into
It carries out being protected from light oscillation 48h in 200rpm shaking table.Obtained reaction solution is passed through into 10000rpm again, is centrifuged 15min, and use distilled water
Washing 3 times, vacuum freeze drying obtains nano-Ag particles.
The performance and fungistatic effect of the nano-Ag particles mediated in embodiment 1 using careless pseudomonad are examined
It surveys, obtained result is as follows:
(1) performance characterization of nano silver
Fig. 1 is the color change photo of nano silver in preparation process, and liquid is before careless pseudomonad supernatant is added in discovery
Milky is added supernatant and is protected from light oscillation, and as the time increases, liquid color is gradually deepened, and is rufous after 48h.
Fig. 2 is to obtain UV-Vis using UV spectrophotometer measuring nano-Ag particles to scheme, and is presented in 420nm or so single
One, the surface plasma body resonant vibration peak of Qiang Erkuan, it was demonstrated that the formation and its stability of nano silver, and in many researchs, it is spherical
The initial token peak value of nano silver particles confirms synthesizing spherical nano silver between 410~450nm indirectly.
Fig. 3 is to be schemed using the FTIR of careless pseudomonad supernatant synthesizing nano-silver, is confirmed by the presence of amide group
Protein present in supernatant may be the reason of causing silver nano-grain quickly to restore and increase stability.
Fig. 4 is that the TEM of synthesizing nano-silver schemes, it can be seen that synthesizing nano-silver is relatively uniform spheric granules object.
Fig. 5 is the XRD diagram of nano-Ag particles, and XRD is a kind of for measuring the various compounds of crystallinity, qualitative detection, determining
Property differentiate the chemical species of crystalline material, size, the analytical technology of shape and structure, the position at peak shows depositing for nano-Ag particles
In average grain diameter 49.5nm.
Fig. 6 is to be schemed using the SEM-EDS of careless pseudomonad supernatant synthesizing nano-silver, carries out surface imaging using SEM, uses
In partial size, particle diameter distribution, the shape of nano material and the surface topography of analysis nano particle, using EDS to the state of element into
Row qualitative and quantitative analysis.The spherical shape of nano particle is consistent with images of transmissive electron microscope result as the result is shown, is evenly distributed, soilless sticking
Phenomenon, average grain diameter are 21.8~99.4nm.Further, since the matrix of the scanning electron microscope is made using tinfoil, EDS spectrogram
Middle there is Ag element and ensure that the presence of nano-Ag particles.
(2) In Vitro Bacteriostasis effect of the various concentration nano silver to sweet potato Pathogen
By the nano silver powder prepared in embodiment 1 be configured to respectively concentration be 0.012mg/ml, 0.025mg/ml,
0.05mg/ml solution, by measuring stand density, biofilm formation ability, the antibacterial circle diameter, cellular morphology variation of bacterium,
It illustrates the antifungal mechanism of synthesizing nano-silver and assesses the bacteriostasis property of various concentration nano silver.
Fig. 7 is growth effect figure of the various concentration Nano silver solution to sweet potato Pathogen, by Fig. 7 A it is found that individually thin
Bacterium cell free fermentation supernatant (CFCS) has no bacteriostasis, and concentration 0.012mg/ml, 0.025mg/ml and 0.05mg/ml
Nano silver can cause OD600 value to decline 65.5%, 70.8% and 90.0% respectively compared with control group, individually add 0.05mg/ml
AgNO3Also 63.5% can be reduced, this shows that nano silver antibacterial capacity of water is substantially proportional to its concentration, and Fig. 7 B is also demonstrated that conjunction
At nano silver compared to independent bacterial supernatant have stronger fungistatic effect.
In addition, research shows that middle biomembrane can protect pathogen from the influence of extraneous adverse environment, phytopathogen
Travelling activity grown with it, the formation of virulence and biomembrane it is related, facilitate bacterial pathogens invasion and be colonized host plant,
Therefore biofilm formation ability and motility size can be shown that the antibacterial activity of nano silver.
If Fig. 8 is shown, the nano silver of concentration 0.012mg/ml, 0.025mg/ml and 0.05mg/ml can lead to its extinction
Value declines 46.28%, 58.52%, 76.90% respectively, individually adds 0.05mg/ml AgNO compared to control group3Also have one
Determine fungistatic effect, is since its own is with strong oxidizing property.
Fig. 9 the result shows that, control group diameter be 2.3cm, 0.05mg/ml AgNO3Bacterium diameter is set to be reduced to 1.1cm,
The nano silver of 0.012mg/ml, 0.025mg/ml and 0.05mg/ml make respectively bacterium diameter reduce to 1.08cm, 1.02cm,
0.9cm.Fig. 8 explanation same as Fig. 9 is better to pathogen biofilm development inhibitory effect the result shows that nanometer silver concentration is higher.
Figure 10 is to be influenced using careless pseudomonad supernatant synthesizing nano-silver on sweet potato Pathogen cell wall in embodiment 1.
Figure 10 A is shown as normal, complete Dickeya dadantii CZ1501 bacterium cellular morphology, compared with negative control, from difference
Visual field TEM figure (Figure 10 B, C) illustrates that in the presence of 0.2mg/ml nano silver, bacterial strain CZ1501 cellular morphology significantly changes,
Matrix distribution is inconsistent, illustrates that nano silver can cause bacterial strain CZ1501 intercellular substance to outflow.
(3) internal fungistatic effect of the various concentration nano silver to sweet potato Pathogen
The nano silver synthesized in embodiment 1 is configured to various concentration Nano silver solution.Such as Figure 11, connect on Sweet Potato block
Kind of CZ1501 bacterial strain and after hot-house culture for 24 hours, there is the scab of diameter 3.47cm in sweet potato stem tuber, individually at bacterial supernatant
Be inoculated with after reason sweet potato and with bacterium solution, lesion diameter with not use supernatant processing result very nearly the same.But works as using concentration and be
Lesion size is obviously reduced after the processing of 0.012mg/ml, 0.025mg/ml and 0.05mg/ml nano silver, and diameter is respectively
1.80cm,1.48cm,1.10cm.0.05mg/ml AgNO is used alone3When processing, Lesion size about 1.90cm, with addition
The result of 0.012mg/ml nano silver processing is close.
It can be seen that bacterial supernatant is unobvious to the inhibiting effect of sweet potato Pathogen, but the nano silver of various concentration
It so that sweet potato stem tuber lesion diameter is reduced 50.0% or more, it is very big to illustrate that nano silver has in terms of controlling sweet potato stem rot
Potentiality.This data is consistent with the result of AgNPs antibacterial activity in vitro, shows that the inhibition of sweet potato stem rot may at least partly return
Cause is in nano silver to the external direct antibacterial activity of D.dadantii bacterial strain CZ1501.
Comparative example
In addition the present invention has also screened a variety of environmental bacterias, comprising: bacillus amyloliquefaciens (Bacillus
Amiloliquefaciens), Methylotrophic bacillus (Bacillus methylotrophicus), bacillus subtilis
(Bacillus subtilis), Paenibacillus polymyxa (Paenibacillus polymyxa), specific bacterial strain type such as following table
Shown in 1.
Bacterial strain in table 1 is utilized into 1 the method synthesizing nano-silver of embodiment, discovery cannot prepare nano silver, lead to
Bactericidal test is crossed, finds it to Dickeya dadantii CZ1501 also without inhibitory effect.
1 different bacterium of table prepares nano silver to Dickeya dadantii CZ1501 inhibitory effect
Claims (8)
1. a kind of method using careless pseudomonad cell-free supernatants synthesizing nano-silver, includes the following steps:
(1) careless pseudomonad is cultivated in NB culture medium, collects bacterial suspension；
(2) bacterial suspension is centrifuged, collects supernatant, obtains cell-free supernatants；
(3) AgNO is added in cell-free supernatants3Solution, obtained reaction solution are obtained by centrifugation, washing, vacuum freeze drying
Nano-Ag particles.
2. the method according to claim 1, wherein in step (1), by careless pseudomonad coated plate activation culture,
The amplification culture of NB culture medium, condition of culture are forwarded to 1%~3% switching amount again are as follows: revolving speed is 180~220rpm, temperature
It is 30~32 DEG C.
3. the method according to claim 1, wherein in step (1), the formula of the NB culture medium are as follows: 1% pancreas
Peptone, 1% sucrose, 0.3% peptone, 0.1% yeast powder.
4. the method according to claim 1, wherein in step (2) and step (3), centrifugation rate 8000~
10000rpm, centrifugation time are 10~20min.
5. the method according to claim 1, wherein in step (3), the AgNO3The concentration of solution be 0.5~
2mM, cell-free supernatants and AgNO3The volume ratio of solution is 2~3:10.
6. the method according to claim 1, wherein in step (3), the cell-free supernatants and AgNO3Solution
It after mixing, is placed in 30~32 DEG C of shaking tables and be protected from light oscillation, the color of liquid to be mixed becomes dark brown reaction knot from light yellow
Beam.
7. nano silver made from a kind of method of any one according to claim 1~6, which is characterized in that the partial size of the nano silver
For 20~100nm.
8. a kind of application of nano silver according to claim 7 in prevention and treatment sweet potato stem rot.
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| CN110804089A (en) * | 2019-10-18 | 2020-02-18 | 淮阴工学院 | Nano-silver synthetic protein derived from spherical lysine bacillus 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 |
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