A kind of sterilization method based on gold nano grain collaboration high voltage electric field plasma
Technical field
The present invention relates to a kind of sterilization methods based on gold nano grain collaboration high voltage electric field plasma, belong to nanometer
Material sterilization technology field.
Background technique
In recent years, with the successive exposure of various regions food safety malignant event, it is total that foodborne bacterial pathogens have become the whole world
Same problems faced.These pathogens are easy to the contaminated food products during food processing and production, transport, storage etc., cause to eat
Product secrete noxious material while rotten, to cause the diseases such as hemorrhagic enteritis, hemolytic uremic syndrome, septicemia.Cause
How this, effectively inhibit the pathogen even killed in food to have become the hot spot studied at present.Gold nano grain is a kind of ruler
The very little nano material between 1-100nm, due to its unique skin effect, small-size effect, quantum effect, macroscopic quantum tunnel
Channel effect etc. makes it show the properties such as special light, electricity, thermal and magnetic, to show certain bactericidal activity.High voltage electric field
Plasma sterilization is a kind of novel cold sterilization technology, generated gas ionization using the strong electric field that high voltage between plate generates etc.
Gas ions, plasma discharge generate active oxygen a small bundle of straw, etc. for silkworms to spin cocoons on such as: hydroxyl radical free radical, single line too oxygen and ozone can destroy the thin of bacterium
After birth, cell wall etc., to reach bactericidal effect.The technology is applied to the fields such as water, air cleaning earliest, starts recent years
Applied to the cold sterilization research of food.
Although gold nano grain and high voltage electric field plasma have bactericidal effect, both methods all exists certain
Disadvantage.Compared to silver nano-grain, the bacteriostatic activity of gold nano grain is low, and exclusive use is not able to satisfy the requirement actually sterilized,
And the gold nano grain of high concentration is in bacterium solution and its unstable, is easy to happen coagulation, loses fungistatic effect;High voltage electric field etc. from
Daughter sterilization technology presently, there are the problem of be: 1) must controlled atmosphere sealed package can be only achieved preferable bactericidal effect, operate numerous
It is trivial, it is at high cost;2) it needs using higher voltage strength, but since equipment is affected by environment big, it is past under rainy day wet condition
It is past to be unable to reach required high pressure;3) under so high voltage strength, equipment uses meeting so that electrode and insulation for a long time
The temperature of plate increases, so that equipment can not work normally.Based on the above reasons, so that the technology is unable to satisfy actual production at present
It needs.
Summary of the invention
The purpose of the present invention is provide in view of the above technical problems it is a kind of based on gold nano grain collaboration high voltage electric field etc. from
The sterilization method of daughter.This method comprehensively utilizes the synergistic effect of two kinds of method for disinfection, does not need controlled atmosphere sealed package, sterilizes
Time is short, low using voltage, it is only necessary to which a small amount of gold nano grain can reach good bactericidal effect, and treatment process medium temperature
Significant change will not occur for degree, provide important technical support for the regulation of fresh food logistics fresh-keeping packaging security quality.
The purpose of the present invention can be achieved through the following technical solutions:
A kind of sterilization method based on gold nano grain collaboration high voltage electric field plasma, this method includes following step
It is rapid:
1) preparation of PVP functional gold: nanogold particle is mixed with aqueous povidone solution, system
It is standby to obtain the PVP solution containing nanogold;
2) the PVP solution obtained in step 1) containing nanogold is added in bacterium solution, is sufficiently mixed culture;
3) mixed liquor after step 2) culture is placed between two electrodes of plasma producing apparatus, in height
Plasma sterilization processing is carried out under the conditions of piezoelectric field.
The partial size of gold nano grain is 10~100nm in the step of above-mentioned method for disinfection (1).As preferred: in step (1)
The partial size of gold nano grain is 10~50nm.
In the step of above-mentioned method for disinfection (1) in aqueous povidone solution polyvinylpyrrolidone mass concentration
For 0.1-10%.As preferred: the mass concentration of polyvinylpyrrolidone is 0.5-3% in aqueous povidone solution.
The concentration of nanogold particle is in PVP solution in the step of above-mentioned method for disinfection (1) in step (1) containing nanogold
1~100nmol/L;It is preferred that: the concentration of nanogold particle is 30~80nmol/L in the PVP solution containing nanogold.
Bacterium is Staphylococcus aureus bacterium, Escherichia coli, mouse typhus sramana in the bacterium solution of the step of above-mentioned method for disinfection (2)
Salmonella or bacillus subtilis.As preferred: bacterium is Staphylococcus aureus bacterium in the bacterium solution of step (2).
Incubation time in the step of above-mentioned method for disinfection (2) is 1~10h.As preferred: incubation time is 2~8h.
The voltage strength 10-40kv/cm of step 3) the plasma generation device of above-mentioned method for disinfection, the time of processing
For 20-200s.
Gold nano grain of the present invention can be commercial product, can also be prepared by disclosed literature method
It arrives, the method includes but is not limited to following method: ultrapure water and gold chloride is mixed and heated agitating solution to boiling, 5-
Rapidly join citric acid three sodium solution after 8min, solution from it is colourless become red after, stop heating, continue to stir 15min, be made
Obtain gold nano grain.
Beneficial effects of the present invention:
(1) present invention, which does not need controlled atmosphere sealed package i.e., can reach good bactericidal effect, and operation sequence is simple, at low cost
It is honest and clean.
(2) by the sterilization experiment to representative Gram-positive pathogenic bacterium staphylococcus aureus, as a result table
It is bright: to be handled through nanogold and plasma body cooperative treated bacterial number well below simple plasma or gold nano grain
Group.
(3) synergistic effect of two methods is utilized, bactericidal effect is good, and noresidue is generated without harmful by-products, avoids and adopts
With the safety issue of chemical bactericide.
(4) high voltage electric field pdp body synergic nano gold method for disinfection carries out at normal temperature, and short processing time uses electricity
It forces down, is a kind of efficient cold sterilization technology, can be used for food sterilization storage, Shelf-life.
The principle of the present invention:
The sterilizing mechanisms of existing patent of invention (CN 104126641, CN 104784722) are: the object that needs are sterilized
It is packed with a certain proportion of air seal, nano material is coated on the inside of packaging film, is generated using high voltage between plate powerful
Gas ionization is generated plasma by electric field, active oxygen a small bundle of straw, etc. for silkworms to spin cocoons on that plasma discharge generates such as: it is hydroxyl radical free radical, ozone, ultraviolet
Line etc. can destroy cell membrane and cell wall of bacterium etc.;On the other hand, ultraviolet light can be catalyzed TiO2Etc. nano materials generate it is antibacterial
Activity, to play the role of Synergistic biocidal.
Sterilizing mechanisms of the invention are: first for a period of time by nanogold particle and Bacteria Culture, entering nano particle
To inside bacterium, the high voltage electric field for recycling plasma to generate acts on intracellular gold nano grain, due to nanometer golden watch
Face is negatively charged, and under the intensity of extra electric field, surface charge starts concussion and is then formed about on gold nano grain surface
Gas ions, plasma discharge generates hydroxyl radical free radical etc., to play the role of sterilization.The present invention, which is that one kind is relatively straightforward, to be killed
Bacterium mode, compared with existing two patents, principle is different, cannot predict to obtain the party by existing two patents
Method.
Detailed description of the invention
Fig. 1 high voltage electric field plasma generating system.
Fig. 2 gold nano grain transmission electron microscope picture.
Specific embodiment
Below with reference to embodiment, the present invention will be further described, and but the scope of the present invention is not limited thereto:
High voltage electric field plasma generating system used in the embodiment of the present invention is as shown in Figure 1, the device includes voltage
Modulator, high voltage electric field generator, top electrode, lower electrode and insulation protective board, among upper/lower electrode place equipped with nanogold with
24 porocyte culture plates of bacterium mixed liquor are equipped with upper insulation protective board between the upper surface and top electrode of plate, the lower surface of plate is under
Lower insulation protective board is equipped between electrode.
Embodiment 1
The PVP solution that mass concentration is 0.5% is prepared, takes the gold nano grain 4mL that concentration is 8nM in centrifuge tube,
13000r/min after being centrifuged 10min, removes supernatant, and 2mL 0.5%PVP solution is added and is resuspended, 30min is reacted at room temperature, thus
Obtain the gold nano grain solution of PVP functionalization.Isometric golden yellow is added in the gold nano grain solution of PVP functionalization
Staphylococcus culture fluid, 37 DEG C of culture 2h, is put between two electrodes after culture and carries out corona treatment, at plasma
The time of reason is 20s, and the voltage strength of processing is 10kv/cm.
Embodiment 2
Prepare the PVP solution that mass concentration is 1%;Take the gold nano grain 12mL that concentration is 8nM in centrifuge tube,
13000r/min after being centrifuged 10min, removes supernatant, and 3mL 1%PVP solution is added and is resuspended, 30min is reacted at room temperature, thus
To the gold nano grain solution of PVP functionalization.Isometric golden yellow Portugal is added in the gold nano grain solution of PVP functionalization
Grape coccus culture solution, 37 DEG C of culture 3h, is put between two electrodes after culture and carries out corona treatment, plasma treatment
Time be 80s, the voltage strength of processing is 20kv/cm.
Embodiment 3
Prepare the PVP solution that mass concentration is 1.5%;Take the gold nano grain 24mL that concentration is 8nM in centrifuge tube,
13000r/min after being centrifuged 10min, removes supernatant, and 3mL 1.5%PVP solution is added and is resuspended, 30min is reacted at room temperature, thus
Obtain the gold nano grain solution of PVP functionalization.Isometric golden yellow is added in the gold nano grain solution of PVP functionalization
Staphylococcus culture fluid, 37 DEG C of culture 4h, is put between two electrodes after culture and carries out corona treatment, at plasma
The time of reason is 200s, and the voltage strength of processing is 400kv/cm.
Contrast groups 1-1
In addition to not using plasma treatment, other conditions are the same as embodiment 1.
Contrast groups 1-2
1) 500 μ L physiological saline are taken to be added in the inoculum of 500 μ L, 37 DEG C of culture 4h.
2) it germy 24 orifice plate will be filled is put between two electrodes and carry out corona treatment, the time of plasma treatment
For 20s, the voltage strength of processing is 10kv/cm.
Contrast groups 1-3
In addition to strain uses the micrococcus lysodeikticus for known concentration instead, other conditions are the same as embodiment 1.
Contrast groups 1-4
In addition to strain uses the pseudomonas aeruginosa for known concentration instead, other conditions are the same as embodiment 1.
Contrast groups 1-5
In addition to process object is staphylococcus aureus, other treatment conditions are the same as application No. is 201410347682.3 patents
Middle embodiment 1.
Contrast groups 1-6
In addition to process object is staphylococcus aureus, other treatment conditions are the same as application No. is 201510182548.7 patents
Middle embodiment 1.
Bactericidal property detection:
Sample in Examples 1 to 3, the blank group and contrast groups bacteria colony count before sterilization processing is 8.0 ±
0.23log。
Treated that bacteria colony count subtracts by plasma sterilization for sample in Examples 1 to 3, blank group and contrast groups
It is as shown in the table for few value.
Bacteria colony count reduced value after 1 corona treatment of table
Group |
Clump count reduced value (log) |
Embodiment 1 |
7.62±0.23 |
Embodiment 2 |
7.83±0.12 |
Embodiment 3 |
7.55±3.7 |
Blank group |
0 |
Contrast groups 1-1 |
2.34±0.61 |
Contrast groups 1-2 |
3.05±0.83 |
Contrast groups 1-3 |
2.64±0.33 |
Contrast groups 1-4 |
3.04±0.15 |
Comparative example 1-5 |
3.15±0.25 |
Comparative example 1-6 |
3.48±0.71 |
As can be seen from the table, nanogold and (Examples 1 to 3) staphylococcus aureus in plasma body cooperative processing group
Clump count reduced value be far longer than blank group, simple nanogold processing group (contrast groups 1-1) and simple corona treatment group
(contrast groups 1-2), fungistatic effect are obvious.
As can be seen from the table, plasma treated, the nanogold of various concentration all has bactericidal effect.
By comparative example 1~3 and contrast groups (1-3,1-4) as can be seen that this method is to micrococcus lysodeikticus and verdigris
Pseudomonad does not have good bactericidal effect, apparent to the bactericidal effect of staphylococcus aureus instead.
By comparative example 1~3 and comparative example group (1-5,1-6) as can be seen that using there are two the methods of patent
There is no apparent bactericidal effect to staphylococcus aureus.
Embodiment 4
Prepare the PVP solution that mass concentration is 0.5%;Take the gold nano grain 4mL that concentration is 8nM in centrifuge tube,
13000r/min after being centrifuged 10min, removes supernatant, and 2mL 0.5%PVP solution is added and is resuspended, 30min is reacted at room temperature, thus
Obtain the gold nano grain solution of PVP functionalization.Isometric large intestine bar is added in the gold nano grain solution of PVP functionalization
Bacteria culture fluid, 37 DEG C of culture 2h, is put between two electrodes after culture and carries out corona treatment, plasma treatment when
Between be 20s, the voltage strength of processing is 10kv/cm.
Embodiment 5
Prepare the PVP solution that mass concentration is 1%;Take the gold nano grain 12mL that concentration is 8nM in centrifuge tube,
13000r/min after being centrifuged 10min, removes supernatant, and 3mL 1%PVP solution is added and is resuspended, 30min is reacted at room temperature, thus
To the gold nano grain solution of PVP functionalization.Isometric Escherichia coli are added in the gold nano grain solution of PVP functionalization
Culture solution, 37 DEG C of culture 3h, is put between two electrodes after culture and carries out corona treatment, the time of plasma treatment
For 80s, the voltage strength of processing is 20kv/cm.
Embodiment 6
Prepare the PVP solution that mass concentration is 1.5%;Take the gold nano grain 24mL that concentration is 8nM in centrifuge tube,
13000r/min after being centrifuged 10min, removes supernatant, and 3mL 1.5%PVP solution is added and is resuspended, 30min is reacted at room temperature, thus
Obtain the gold nano grain solution of PVP functionalization.Isometric large intestine bar is added in the gold nano grain solution of PVP functionalization
Bacteria culture fluid, 37 DEG C of culture 4h, is put between two electrodes after culture and carries out corona treatment, plasma treatment when
Between be 200s, the voltage strength of processing is 400kv/cm.
Contrast groups 2-1
In addition to not using plasma treatment, other conditions are the same as embodiment 4.
Contrast groups 2-2
1) 500 μ L physiological saline are taken to be added in the E. coli broth of 500 μ L, 37 DEG C of culture 4h.
2) it germy 24 orifice plate will be filled is put between two electrodes and carry out corona treatment, the time of plasma treatment
For 20s, the voltage strength of processing is 10kv/cm.
Contrast groups 2-3
In addition to strain uses the micrococcus lysodeikticus for known concentration instead, other conditions are the same as embodiment 4.
Contrast groups 2-4
In addition to strain uses the pseudomonas aeruginosa for known concentration instead, other conditions are the same as embodiment 4.
Contrast groups 2-5
In addition to process object is Escherichia coli, other treatment conditions are the same as application No. is implement in 201410347682.3 patents
Example 1.
Contrast groups 2-6
In addition to process object is Escherichia coli, other treatment conditions are the same as application No. is implement in 201510182548.7 patents
Example 1.
Bactericidal property detection:
Sample in embodiment 4~6, the blank group and contrast groups bacteria colony count before sterilization processing is 8.0 ±
0.23log。
Treated that bacteria colony count subtracts by plasma sterilization for sample in embodiment 4~6, blank group and contrast groups
It is as shown in the table for few value.
Bacteria colony count reduced value after 2 corona treatment of table
Group |
Clump count reduced value (log) |
Embodiment 4 |
7.53±0.15 |
Embodiment 5 |
7.36±1.81 |
Embodiment 6 |
7.81±0.94 |
Blank group |
0 |
Contrast groups 2-1 |
2.06±0.92 |
Contrast groups 2-2 |
2.95±0.54 |
Contrast groups 2-3 |
2.81±0.57 |
Contrast groups 2-4 |
3.01±0.41 |
Comparative example 2-5 |
3.28±0.91 |
Comparative example 2-6 |
3.52±0.62 |
As can be seen from the table, the bacterium colony of nanogold and (embodiment 4~6) Escherichia coli in plasma body cooperative processing group
Number reduced value is far longer than blank group, simple nanogold processing group (contrast groups 2-1) and simple corona treatment group (contrast groups
2-2), fungistatic effect is obvious.
As can be seen from the table, plasma treated, the nanogold of various concentration all has bactericidal effect.
By comparative example 4~6 and contrast groups (2-3,2-4) as can be seen that this method is to micrococcus lysodeikticus and verdigris
Pseudomonad does not have good bactericidal effect, apparent to the bactericidal effect of Escherichia coli instead.
By comparative example 4~6 and comparative example group (2-5,2-6) as can be seen that using there are two the methods of patent
There is no apparent bactericidal effect to Escherichia coli.
Embodiment 7
Prepare the PVP solution that mass concentration is 0.5%;Take the gold nano grain 4mL that concentration is 8nM in centrifuge tube,
13000r/min after being centrifuged 10min, removes supernatant, and 2mL 0.5%PVP solution is added and is resuspended, 30min is reacted at room temperature, thus
Obtain the gold nano grain solution of PVP functionalization.Isometric mouse typhus is added in the gold nano grain solution of PVP functionalization
Salmonella culture solution, 37 DEG C of culture 2h, is put between two electrodes after culture and carries out corona treatment, at plasma
The time of reason is 20s, and the voltage strength of processing is 10kv/cm.
Embodiment 8
Prepare the PVP solution that mass concentration is 1%;Take the gold nano grain 12mL that concentration is 8nM in centrifuge tube,
13000r/min after being centrifuged 10min, removes supernatant, and 3mL 1%PVP solution is added and is resuspended, 30min is reacted at room temperature, thus
To the gold nano grain solution of PVP functionalization.It is husky that isometric mouse typhus is added in the gold nano grain solution of PVP functionalization
Door Salmonella culture solution, 37 DEG C of culture 3h are put between two electrodes after culture and carry out corona treatment, plasma treatment
Time be 80s, the voltage strength of processing is 20kv/cm.
Embodiment 9
Prepare the PVP solution that mass concentration is 1.5%;Take the gold nano grain 24mL that concentration is 8nM in centrifuge tube,
13000r/min after being centrifuged 10min, removes supernatant, and 3mL 1.5%PVP solution is added and is resuspended, 30min is reacted at room temperature, thus
Obtain the gold nano grain solution of PVP functionalization.Isometric mouse typhus is added in the gold nano grain solution of PVP functionalization
Salmonella culture solution, 37 DEG C of culture 4h, is put between two electrodes after culture and carries out corona treatment, at plasma
The time of reason is 200s, and the voltage strength of processing is 400kv/cm.
Contrast groups 3-1
In addition to not using plasma treatment, other conditions are the same as embodiment 4.
Contrast groups 3-2
1) 500 μ L physiological saline are taken to be added in the salmonella typhimurium culture solution of 500 μ L, 37 DEG C of culture 4h.
2) it germy 24 orifice plate will be filled is put between two electrodes and carry out corona treatment, the time of plasma treatment
For 20s, the voltage strength of processing is 10kv/cm.
Contrast groups 3-3
In addition to strain uses the micrococcus lysodeikticus for known concentration instead, other conditions are the same as embodiment 4.
Contrast groups 3-4
In addition to strain uses the pseudomonas aeruginosa for known concentration instead, other conditions are the same as embodiment 4.
Contrast groups 3-5
In addition to process object is salmonella typhimurium, other treatment conditions are the same as application No. is 201410347682.3 patents
Middle embodiment 1.
Contrast groups 3-6
In addition to process object is salmonella typhimurium, other treatment conditions are the same as application No. is 201510182548.7 patents
Middle embodiment 1.
Bactericidal property detection:
Sample in embodiment 7~9, the blank group and contrast groups bacteria colony count before sterilization processing is 8.0 ±
0.23log。
Treated that bacteria colony count subtracts by plasma sterilization for sample in embodiment 7~9, blank group and contrast groups
It is as shown in the table for few value.
Bacteria colony count reduced value after 3 corona treatment of table
Group |
Clump count reduced value (log) |
Embodiment 7 |
7.06±0.25 |
Embodiment 8 |
7.89±0.86 |
Embodiment 9 |
7.52±0.48 |
Blank group |
0 |
Contrast groups 3-1 |
2.12±0.74 |
Contrast groups 3-2 |
2.51±0.74 |
Contrast groups 3-3 |
2.69±0.39 |
Contrast groups 3-4 |
3.09±0.73 |
Comparative example 3-5 |
3.31±0.48 |
Comparative example 3-6 |
3.64±0.83 |
As can be seen from the table, nanogold and (embodiment 7~9) salmonella typhimurium in plasma body cooperative processing group
Clump count reduced value be far longer than blank group, simple nanogold processing group (3-1) and simple corona treatment group (3-2),
Fungistatic effect is obvious.
As can be seen from the table, plasma treated, the nanogold of various concentration all has bactericidal effect.
By comparative example 7~9 and contrast groups (3-3,3-4) as can be seen that this method is to micrococcus lysodeikticus and verdigris
Pseudomonad does not have good bactericidal effect, apparent to the bactericidal effect of salmonella typhimurium instead.
By comparative example 7~9 and comparative example group (3-5,3-6) as can be seen that using there are two the methods of patent
There is no apparent bactericidal effect to salmonella typhimurium.