CN112450229B - Plant essential oil type ultraviolet sterilization synergist and ultraviolet combined sterilization method thereof - Google Patents
Plant essential oil type ultraviolet sterilization synergist and ultraviolet combined sterilization method thereof Download PDFInfo
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- 244000203593 Piper nigrum Species 0.000 claims description 23
- 235000008184 Piper nigrum Nutrition 0.000 claims description 23
- 235000013614 black pepper Nutrition 0.000 claims description 23
- 238000002156 mixing Methods 0.000 claims description 14
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- 238000009281 ultraviolet germicidal irradiation Methods 0.000 claims description 3
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N65/00—Biocides, pest repellants or attractants, or plant growth regulators containing material from algae, lichens, bryophyta, multi-cellular fungi or plants, or extracts thereof
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
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- A01N65/00—Biocides, pest repellants or attractants, or plant growth regulators containing material from algae, lichens, bryophyta, multi-cellular fungi or plants, or extracts thereof
- A01N65/06—Coniferophyta [gymnosperms], e.g. cypress
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N65/00—Biocides, pest repellants or attractants, or plant growth regulators containing material from algae, lichens, bryophyta, multi-cellular fungi or plants, or extracts thereof
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- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N65/00—Biocides, pest repellants or attractants, or plant growth regulators containing material from algae, lichens, bryophyta, multi-cellular fungi or plants, or extracts thereof
- A01N65/08—Magnoliopsida [dicotyledons]
- A01N65/28—Myrtaceae [Myrtle family], e.g. teatree or clove
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L2/00—Methods or apparatus for disinfecting or sterilising materials or objects other than foodstuffs or contact lenses; Accessories therefor
- A61L2/02—Methods or apparatus for disinfecting or sterilising materials or objects other than foodstuffs or contact lenses; Accessories therefor using physical phenomena
- A61L2/08—Radiation
- A61L2/10—Ultraviolet radiation
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Abstract
The invention belongs to the technical field of ultraviolet sterilization, and discloses a plant essential oil type ultraviolet sterilization synergist and a method for sterilizing by combining ultraviolet. The research is based on that the A-band Ultraviolet (UVA) and the plant essential oil have weak sterilization effects, and the synergistic sterilization effect is obvious after the combined action of the Ultraviolet (UVA) and the plant essential oil. Compared with the traditional ultraviolet sterilization technology, the ultraviolet sterilization device can kill bacteria more efficiently and reduce the influence of the quality of the ultraviolet lamp on the sterilization effect.
Description
Technical Field
The invention relates to the technical field of ultraviolet sterilization, in particular to an ultraviolet sterilization synergist for plant essential oil and a method for sterilizing by combining ultraviolet.
Background
The uv sterilization technique is a convenient method with no chemical residue and little environmental impact, and is commonly used to disinfect gas, liquid and solid surfaces. Ultraviolet radiation is a generic term for radiation in the electromagnetic spectrum having a wavelength of 400nm to 10nm and does not cause human vision. It is invisible light having a higher frequency than bluish violet light, and ultraviolet rays are classified into UVA, UVB, UVC, and UVD. The ultraviolet lamp sterilization is used in various places due to the advantages of low cost, convenient use, no drug resistance and the like. In recent years, bacterial drug resistance is getting worse due to the use of a large amount of antibiotics, and the effect of treating infection caused by germs is gradually weakened along with the appearance of multi-drug resistant bacteria, so that the research on non-antibiotic sterilization technology is more and more applied in clinic, and the research on ultraviolet sterilization effect is more and more intensive.
The principle of the ultraviolet sterilization technology is that ultraviolet radiation with proper wavelength is utilized to destroy the molecular structure of DNA (deoxyribonucleic acid) or RNA (ribonucleic acid) in organism cells to cause death of growing cells and/or regenerating cells, thereby achieving the sterilization effect. The ultraviolet sterilization technology is based on modern epidemic prevention science, medicine and photodynamic, and specially designed ultraviolet with high efficiency, high strength and long service life is adopted to irradiate the surface of an object to directly kill various pathogens such as bacteria, viruses, parasites, algae and the like on the surface of the object. Meanwhile, bacteria can be killed under the condition of not generating antibiotic drug resistance, so that fine drug resistance is not improved. However, low-energy ultraviolet light by itself is not sufficient to achieve a very good germicidal effect, the germicidal time is long and the bacteria cannot be completely killed.
In order to enhance the ultraviolet sterilization effect, the research of the ultraviolet sterilization effect synergist is an essential step. However, the currently reported essential oil still has less ultraviolet sterilization synergistic effect, and a new ultraviolet sterilization synergistic agent needs to be supplemented urgently to increase the sterilization effect of ultraviolet rays in various occasions.
Disclosure of Invention
The invention aims to overcome the defects in the prior art and provide the application of the plant essential oil as the ultraviolet sterilization synergist.
The second purpose of the invention is to provide a method for sterilizing by combining plant essential oil with ultraviolet.
The purpose of the invention is realized by the following technical scheme:
the application of the plant essential oil as the ultraviolet sterilization synergist is characterized in that the plant essential oil comprises tea tree essential oil, black pepper essential oil, European fir essential oil and Eucalyptus leaf essential oil.
The research of the invention finds that the combination of plant essential oil and ultraviolet can obviously enhance the ultraviolet sterilization capability, so the plant essential oil can be used as a synergist for ultraviolet sterilization.
The invention also provides an ultraviolet sterilization synergist which comprises the plant essential oil.
The invention also provides a method for sterilizing by combining plant essential oil with ultraviolet, which is characterized in that the plant essential oil and external irradiation are used for sterilizing, and the irradiation time of the ultraviolet is 30min, wherein, when the plant essential oil is tea tree essential oil, the concentration of the plant essential oil is 0.25 percent, when the plant essential oil is black pepper essential oil, the concentration of the black pepper essential oil is 1 percent, when the plant essential oil is European fir essential oil, the concentration of the European fir essential oil is 1 percent, and when the plant essential oil is Eucalyptus leaf essential oil, the concentration of the Eucalyptus leaf essential oil is 0.25 percent.
Preferably, in the method for sterilizing, the ultraviolet intensity is 2.4-3.0mW/cm2。
Preferably, in the sterilization method, the ultraviolet irradiation is performed for 30min before preheating.
Preferably, the method of sterilizing comprises the steps of:
(1) mixing the bacterial liquid and the plant essential oil, placing the mixture in a six-hole plate, and setting ultraviolet irradiation conditions: wavelength range of 300-460nm, power of 18W, distance from the six-hole plate to the ultraviolet lamp tube of 8cm, and ultraviolet intensity of 2.4-3.0mW/cm2；
(2) Preheating the ultraviolet box for 20-40min, and placing the six-hole plate in the ultraviolet box for irradiating for 25-35 min.
More preferably, the final concentration of the bacterial liquid in the step (1) is 106CFU/mL。
Compared with the prior art, the invention has the following beneficial effects:
the invention provides a synergist which has a synergistic effect with ultraviolet, and the research is based on that the sterilizing effect of Ultraviolet (UVA) in the A wave band and plant essential oil is weak (the sterilizing amount is 0.5log-1.5log when the ultraviolet and plant essential oil act independently), and the synergist has an obvious synergistic sterilizing effect after combined action. Compared with the traditional ultraviolet sterilization technology, the ultraviolet sterilization device can kill bacteria more efficiently and reduce the influence of the quality of the ultraviolet lamp on the sterilization effect.
Detailed Description
The following further describes the embodiments of the present invention. It should be noted that the description of the embodiments is provided to help understanding of the present invention, but the present invention is not limited thereto. In addition, the technical features involved in the embodiments of the present invention described below may be combined with each other as long as they do not conflict with each other.
The test methods used in the following experimental examples are all conventional methods unless otherwise specified; the materials, reagents and the like used are, unless otherwise specified, commercially available reagents and materials.
The ultraviolet lamp used was brand PHILIPS TL-D; wattage: 18W; voltage: 220V; wavelength: (UVA)300-469 nm; tube diameter of the lamp tube: 25 mm; length: 60 cm. The tea tree essential oil, black pepper essential oil, fir essential oil and eucalyptus leaf essential oil are of Satya brand (Italy).
The judgment principle is as follows: for example, the bacterial quantity reduced by the experimental group is reduced by 3 log values on the basis of the bacterial quantity reduction of the independent ultraviolet treatment and the independent essential oil treatment, namely the essential oil and the ultraviolet have the synergistic bactericidal effect.
Example 1 germicidal Effect of different UV irradiation times on ATCC25922 Strain
1. Experimental materials:
(1) the ultraviolet lamp used for the test is PHILIPS TL-D brand; wattage: 18W; voltage: 220V; wavelength: (UVA)300-469 nm; tube diameter of the lamp tube: 25 mm; length: 60 cm.
(2) Test medium: the MH agar medium and the MacConkey agar medium (purchased from Guangdong Huancao Microscience Co., Ltd.) which were sterilized by autoclaving were cooled to 40 ℃, 20mL of the MH agar medium and the MacConkey agar medium were put into a sterile petri dish using a pipette, and naturally dried for 30min to obtain the MH agar medium and the MacConkey agar medium.
Coli standard strain ATCC25922 (laboratory collection).
2. Preparation work before the test:
(1) starting an ultraviolet lamp and continuously irradiating for 30 minutes for preheating;
(2) the E.coli standard strain ATCC25922 was cultured on MacconyKa medium to a suitable size.
3. Ultraviolet sterilization effect evaluation experiment:
(1) inoculating Escherichia coli ATCC25922, placing the single colony in a centrifuge tube filled with 4mL MH broth, putting the centrifuge tube into a 37-degree shaking table, incubating for 4 hours at 220 revolutions, and taking out the centrifuge tube;
(2) placing the centrifuge tube in a centrifuge, centrifuging for 8min at 5000 rpm, pouring out supernatant, adding equal volume of physiological saline for resuspension, and performing gradient dilution to obtain final bacterial load of 106CFU/mL；
(3) Adding 1mL of bacterial liquid into a six-hole plate;
(4) setting blank control group and ultraviolet irradiation treatment group, wherein the ultraviolet irradiation treatment group is divided into three groups, and irradiating for 15min, 30min and 60min respectively.
(5) After the ultraviolet irradiation is finished, 100 mu L of bacterial liquid is absorbed and added into a 2mL centrifuge tube filled with 900 mu L of 0.85% physiological saline for gradient dilution, 25 mu L of bacterial liquid is absorbed and dropped on an MH agar culture medium after dilution, the culture box with the temperature of 37 ℃ is incubated for 16-18h, counting is carried out, and the statistical analysis is carried out after the experimental result is repeated through three biology.
The results are shown in table 1, the bacteriostatic effect of ultraviolet on bacteria is increased with the increase of the irradiation time, wherein the difference between the number of bacteria in the group irradiated with ultraviolet for 15min and the number of bacteria in the blank control group is not large, which indicates that the short irradiation time of ultraviolet cannot produce a significant bacteriostatic effect on ATCC 25922. The number of bacteria in the group irradiated by ultraviolet for 30min begins to decrease, which indicates that the ultraviolet begins to have a certain bacteriostatic effect on the bacteria.
TABLE 1 germicidal Effect of UV irradiation time on ATCC25922 strains
Example 2 determination of MIC of different concentrations of essential oils against ATCC25922 Strain
1. Experimental materials:
(1) and (3) testing: autoclaved MH broth was cooled for use.
(2) Resin azure: macklin (Maclin) brand, MW 251.17, purity 90%;
coli standard strain ATCC25922 (laboratory collection).
2. Preparation work before the test:
(1) taking out the essential oil from the refrigerator, balancing to room temperature, and sucking part for later use;
(2) the E.coli standard strain ATCC25922 was cultured on MacconyKa agar medium to a suitable size.
(3) 0.2512g of resin azure powder was weighed into a centrifuge tube, and 10mL of pure water was added to dissolve the resin azure powder, and the concentration of the resin azure solution was 10 mM/L.
3. Evaluation experiment of bactericidal effect of different essential oils:
(1) inoculating Escherichia coli ATCC25922, placing single colony in a centrifuge tube filled with 4mL MH broth, putting the centrifuge tube in a 37-degree shaking table, incubating for 4 hours at 180 revolutions, and taking out the centrifuge tube;
(2) the incubated E.coli was diluted 100-fold using MH broth to about 106CFU/mL for standby;
(3) taking a sterile 96-well plate, adding 180 mu L of MH broth medium to the 1 st well, and adding 100 mu L of MH broth medium to the 2 nd-11 th well;
(4) adding 20 mu L of essential oil with the original concentration into the 1 st hole, sucking 100 mu L to the 2 nd hole after blowing and beating uniformly, and repeating the steps, sucking 100 mu L from the 10 th hole and discarding;
(5) adding 100 mu L of diluted bacterium liquid into the 1 st to 11 th holes, and adding 200 mu L of MH broth into the 12 th hole;
(6) repeating the steps (3) to (5) for three times;
(7) and putting the inoculated 96-well plate into a 37-degree incubator for incubation for 16-18h, sucking 10 mu L of 10mM/L resin azure with the concentration of 0.1mM/L, adding the resin azure into the hole, incubating for 2h, and reading the result.
The results are shown in table 2, the Mic value of the tea tree essential oil is 0.156%, bacteria are not inhibited from growing obviously under the tea tree essential oil concentration lower than the Mic value, but the effect time of the Mic is 16-18h, the tea tree essential oil with the concentration of 0.156% has weak effect, the effect time of the experiment is 30min, so that the essential oil has weak bactericidal effect within 30min, and the use concentration of 0.25% of the tea tree essential oil as the ultraviolet synergist is finally selected.
TABLE 2 MIC of tea tree essential oil against ATCC25922 strain and concentrations selected for the experiment
| Experimental strains | Tea tree essential oil Mic | Concentration for experiment |
| ATCC 25922 | 0.156％ | 0.25％ |
As shown in Table 3, the Mic value of the black pepper essential oil is greater than 1%, bacteria are not inhibited from growing obviously at the concentration of the black pepper essential oil lower than the Mic value, and the concentration of the black pepper essential oil of 1% is finally selected as the use concentration of the ultraviolet synergist in consideration of high use cost of the black pepper essential oil with high concentration and low use concentration which is usually diluted in practical application.
TABLE 3 MIC of black pepper essential oil against ATCC25922 strain and concentrations selected for the experiment
The results are shown in table 4, the Mic value of the European fir essential oil is 0.156%, bacteria are not obviously inhibited from growing under the concentration of the European fir essential oil lower than the Mic value, but the Mic action time is 16-18h, the European fir essential oil with the concentration of 0.156% has weak action effect, the action time of the experiment is 30min, in order to ensure that the essential oil has weak sterilization effect within 30min, the European fir essential oil with the concentration of 1% is finally selected as the use concentration of the ultraviolet synergist, and the European fir essential oil with the concentration of 1% is finally selected as the use concentration of the ultraviolet synergist.
TABLE 4 MIC of European fir essential oil against ATCC25922 strain and concentrations selected for the experiment
| Experimental strains | European fir essential oil Mic | Concentration for experiment |
| ATCC 25922 | 0.156％ | 1％ |
As shown in Table 5, the Mic value of Eucalyptus leaf essential oil is 0.313%, and at concentrations of Eucalyptus leaf essential oil lower than the Mic value, the bacteria were not significantly inhibited from growing, and the concentration of Eucalyptus leaf essential oil of 0.25% was finally selected as the UV enhancer, in view of the high cost of use of high concentration of Eucalyptus leaf essential oil and the fact that it is usually diluted to be used at low concentration in practical use.
TABLE 5 MIC of Eucalyptus leaf essential oil against ATCC25922 strain and concentrations selected for the experiment
| Experimental strains | Eucalyptus leaf essential oil Mic | Concentration for experiment |
| ATCC 25922 | 0.313％ | 0.25％ |
Example 3 fungicidal effect of different essential oils on ATCC25922 Strain after 30min
1. Experimental materials:
(1) test medium: the MH agar medium and the MacConkey agar medium (purchased from Guangdong Huancao Microscience Co., Ltd.) which were sterilized by autoclaving were cooled to 40 ℃, 20mL of the MH agar medium and the MacConkey agar medium were put into a sterile petri dish using a pipette, and naturally dried for 30min to obtain the MH agar medium and the MacConkey agar medium.
Coli standard strain ATCC25922 (laboratory collection).
2. Preparation work before the test:
(1) taking out tea tree essential oil/fructus Piperis essential oil/European fir essential oil/Eucalyptus leaf essential oil from refrigerator, balancing to room temperature, and sucking part;
(2) the E.coli standard strain ATCC25922 was cultured on MacconyKa medium to a suitable size.
3. Tea tree essential oil bactericidal effect evaluation experiment:
(1) inoculating Escherichia coli ATCC25922, placing single colony in a centrifuge tube filled with 4mL MH broth, putting the centrifuge tube in a 37-degree shaking table, incubating for 4 hours at 180 revolutions, and taking out the centrifuge tube;
(2) placing the centrifuge tube in a centrifuge, centrifuging for 8min at 5000 rpm, pouring out supernatant, adding equal volume of physiological saline for resuspension, and performing gradient dilution to obtain final bacterial load of 106CFU/mL；
(3) Preparing 1% DMSO (dimethyl sulfoxide) by using pure water, subpackaging in 2mL centrifuge tubes, wherein each tube is 200 mu L, then adding 200 mu L of the essential oil with the original concentration into the tube, blowing, uniformly mixing, and diluting by times to enable the concentration of the essential oil to be 25%;
(4) adding 1mL of bacterial liquid into a six-hole plate, adding 10 mu L of 25% tea tree essential oil, and uniformly mixing to obtain a final concentration of 0.25%;
(5) setting a control, processing with 0.25% tea tree essential oil for 30 min;
(6) and (3) sucking 100 mu L of bacterial liquid, adding the bacterial liquid into a 2ml centrifuge tube filled with 900 mu L of 0.85% physiological saline, carrying out gradient dilution, sucking 25 mu L of the diluted bacterial liquid, dripping the diluted bacterial liquid on an MH agar culture medium, incubating the bacterial liquid in a 37-degree incubator for 16-18h, counting, and carrying out statistical analysis after three biological repetitions of experimental results.
The results are shown in table 6, the number of bacteria in the blank control group is not obviously different from the number of bacteria in the group which acts on the tea tree essential oil for 30 min; it is stated that 0.25% tea tree essential oil did not change the number of bacteria after 30min treatment with ATCC25922 bacteria. The result shows that 0.25% tea tree essential oil has weak bactericidal effect on ATCC25922, so that the tea tree essential oil with the concentration of 0.25% is finally used for 30min in combination with ultraviolet as a final condition.
TABLE 6 fungicidal Effect of tea Tree essential oil on ATCC25922 Strain
4. Black pepper essential oil bactericidal effect evaluation experiment:
(1) inoculating Escherichia coli ATCC25922, placing single colony in a centrifuge tube filled with 4mL MH broth, putting the centrifuge tube in a 37-degree shaking table, incubating for 4 hours at 180 revolutions, and taking out the centrifuge tube;
(2) placing the centrifuge tube in a centrifuge, centrifuging for 8min at 5000 rpm, pouring out supernatant, adding equal volume of physiological saline for resuspension, and performing gradient dilution to obtain final bacterial load of 106CFU/mL；
(3) Adding 1mL of bacterial liquid into a six-hole plate, adding 10 mu L of black pepper essential oil with the original concentration, and uniformly mixing to ensure that the final concentration of the essential oil is 1%;
(4) setting contrast, blank contrast, and treating with 1% black pepper essential oil for 30 min;
(5) and (3) sucking 100 mu L of bacterial liquid, adding the bacterial liquid into a 2ml centrifuge tube filled with 900 mu L of 0.85% physiological saline, carrying out gradient dilution, sucking 25 mu L of the diluted bacterial liquid, dripping the diluted bacterial liquid on an MH agar culture medium, incubating the bacterial liquid in a 37-degree incubator for 16-18h, counting, and carrying out statistical analysis after three biological repetitions of experimental results.
The results are shown in table 7, the bacterial numbers of the blank control group and the bacterial number of the group which has the effect of the black pepper essential oil for 30min are not obviously distinguished; it is demonstrated that 1% black pepper essential oil has no change in bacterial count after 30min of bacterial treatment with ATCC 25922. The 1% black pepper essential oil has weak bactericidal effect on ATCC25922, so that the 1% black pepper essential oil is finally used for 30min in combination with ultraviolet as a final condition.
TABLE 7 fungicidal Effect of Black Pepper essential oil on ATCC25922 strains
5. Evaluation experiment of the sterilizing effect of the European fir essential oil:
(1) inoculating Escherichia coli ATCC25922, placing single colony in a centrifuge tube filled with 4mL MH broth, putting the centrifuge tube in a 37-degree shaking table, incubating for 4 hours at 180 revolutions, and taking out the centrifuge tube;
(2) placing the centrifuge tube in a centrifuge, centrifuging for 8min at 5000 rpm, pouring out supernatant, adding equal volume of physiological saline for resuspension, and performing gradient dilution to obtain final bacterial load of 106CFU/mL；
(3) Adding 1mL of bacterial liquid into a six-hole plate, adding 10 mu L of European fir essential oil with the original concentration, and uniformly mixing to ensure that the final concentration of the essential oil is 1%;
(4) setting comparison, blank comparison, and treating with 1% European fir essential oil for 30 min;
(5) and (3) sucking 100 mu L of bacterial liquid, adding the bacterial liquid into a 2ml centrifuge tube filled with 900 mu L of 0.85% physiological saline, carrying out gradient dilution, sucking 25 mu L of the diluted bacterial liquid, dripping the diluted bacterial liquid on an MH agar culture medium, incubating the bacterial liquid in a 37-degree incubator for 16-18h, counting, and carrying out statistical analysis after three biological repetitions of experimental results.
The results are shown in Table 8, the number of bacteria in the blank control group is not obviously different from the number of bacteria in the group which acts on the European fir essential oil for 30 min; it is demonstrated that 1% of European fir essential oil has no change in the number of bacteria after 30min treatment with ATCC25922 bacteria. The result shows that 1% of the European fir essential oil has weak sterilization effect on ATCC25922, so that the European fir essential oil with the concentration of 1% is finally used for 30min in combination with ultraviolet as the final condition.
TABLE 8 fungicidal Effect of European fir essential oil on ATCC25922 Strain
6. California leaf essential oil bactericidal effect evaluation experiment:
(1) inoculating Escherichia coli ATCC25922, placing single colony in a centrifuge tube filled with 4mL MH broth, putting the centrifuge tube in a 37-degree shaking table, incubating for 4 hours at 180 revolutions, and taking out the centrifuge tube;
(2) placing the centrifuge tube in a centrifuge, centrifuging for 8min at 5000 rpm, pouring out supernatant, adding equal volume of physiological saline for resuspension, and performing gradient dilution to obtain final bacterial load of 106CFU/mL；
(3) Preparing 1% DMSO (dimethyl sulfoxide) by using pure water, subpackaging in 2mL centrifuge tubes, wherein each tube is 200 mu L, then adding 200 mu L of the essential oil with the original concentration into the tube, blowing, uniformly mixing, and diluting by times to enable the concentration of the essential oil to be 25%;
(4) adding 1mL of bacterial liquid into six-hole plate, adding 10 μ L of 25% Eucalyptus leaf essential oil, and mixing well to obtain final concentration of 0.25%
(5) Setting a control, a blank control, and 0.25% Eucalyptus leaf essential oil group for 30 min;
(6) and (3) sucking 100 mu L of bacterial liquid, adding the bacterial liquid into a 2ml centrifuge tube filled with 900 mu L of 0.85% physiological saline, carrying out gradient dilution, sucking 25 mu L of the diluted bacterial liquid, dripping the diluted bacterial liquid on an MH agar culture medium, incubating the bacterial liquid in a 37-degree incubator for 16-18h, counting, and carrying out statistical analysis after three biological repetitions of experimental results.
The results are shown in Table 9, the number of bacteria in the blank control group is not obviously distinguished from the number of bacteria in the group which has the effect of the eucalyptus leaf essential oil for 30 min; it is demonstrated that 0.25% Eucalyptus leaf essential oil has no change in bacterial count after 30min treatment with ATCC25922 bacteria. The result shows that the 0.25% eucalyptus leaf essential oil has weak bactericidal effect on ATCC25922, so the 0.25% eucalyptus leaf essential oil is finally used for 30min in combination with ultraviolet as the final condition.
TABLE 9 fungicidal Effect of Eucalyptus leaf essential oil on ATCC25922 Strain
Example 4 evaluation of the killing effect of UV and UV potentiators on E.coli ATCC25922
1. Experimental materials:
(1) the ultraviolet lamp used for the test is PHILIPS TL-D brand; wattage: 18W; voltage: 220V; wavelength: (UVA)300-469 nm; tube diameter of the lamp tube: 25 mm; length: 60 cm.
(2) Test medium: the MH agar medium and the MacConkey agar medium (purchased from Guangdong Huancao Microscience Co., Ltd.) which were sterilized by autoclaving were cooled to 40 ℃, 20mL of the MH agar medium and the MacConkey agar medium were put into a sterile petri dish using a pipette, and naturally dried for 30min to obtain the MH agar medium and the MacConkey agar medium.
Coli standard strain ATCC25922 (laboratory collection).
2. Preparation work before the test:
(1) starting an ultraviolet lamp and continuously irradiating for 30 minutes for preheating;
(2) taking out tea tree essential oil/fructus Piperis essential oil/European fir essential oil/Eucalyptus leaf essential oil from refrigerator, balancing to room temperature, and sucking part;
(3) the E.coli standard strain ATCC25922 was cultured on MacconyKa agar medium to a suitable size.
3. Tea tree essential oil and ultraviolet synergist combined effect evaluation experiment:
(1) inoculating Escherichia coli ATCC25922, placing single colony in a centrifuge tube filled with 4mL MH broth, putting the centrifuge tube in a 37-degree shaking table, incubating for 4 hours at 180 revolutions, and taking out the centrifuge tube;
(2) placing the centrifuge tube in a centrifuge, centrifuging for 8min at 5000 rpm, pouring out supernatant, adding equal volume of physiological saline for resuspension, and performing gradient dilution to obtain final bacterial load of 106CFU/mL；
(3) Preparing 1% DMSO (dimethyl sulfoxide) by using pure water, subpackaging in 2mL centrifuge tubes, wherein each tube is 200 mu L, then adding 200 mu L of the essential oil with the original concentration into the tube, blowing, uniformly mixing, and diluting by times to enable the concentration of the essential oil to be 25%;
(4) adding 1mL of the bacterial liquid into a six-hole plate, adding 10 μ L of 25% tea tree essential oil, and mixing to obtain a final concentration of 0.25%
(5) Setting contrast, blank contrast, treating with 0.25% essential oil for shading, placing into ultraviolet irradiation box, and irradiating with ultraviolet for 30 min;
(6) after the ultraviolet irradiation is finished, 100 mu L of bacterial liquid is absorbed and added into a 2ml centrifuge tube filled with 900 mu L of 0.1% physiological saline for gradient dilution, 25 mu L of bacterial liquid is absorbed and dropped on an MH agar culture medium after dilution, the culture box with the temperature of 37 ℃ is incubated for 16-18h, counting is carried out, and the statistical analysis is carried out after the experimental result is repeated through three biology.
In this experiment, a growth control group, an ultraviolet control group, an essential oil control group, and an ultraviolet plus essential oil test group were set, and the detection was performed according to the method of example 1.
The results are shown in Table 10, and the blank control group shows that the bacteria grow normally, which indicates that the Escherichia coli ATCC25922 can grow normally under the experimental conditions; under the irradiation of an ultraviolet lamp for 30min, the Escherichia coli ATCC25922 is not obviously reduced compared with a blank control group, which shows that the inhibition effect of the ultraviolet lamp on the Escherichia coli ATCC25922 is not obvious; the same results also show that 0.25% tea tree essential oil has no significant inhibitory effect on escherichia coli ATCC 25922; the combined action result of 30min shows that the synergistic effect of the ultraviolet rays and 0.25% of tea tree essential oil has obvious sterilization effect on escherichia coli ATCC 25922.
TABLE 10 Bactericidal Effect of UV and tea tree essential oil combination on ATCC25922 Strain
4. Effect evaluation experiment of black pepper essential oil combined with ultraviolet synergist:
(1) inoculating Escherichia coli ATCC25922, placing single colony in a centrifuge tube filled with 4mL MH broth, putting the centrifuge tube in a 37-degree shaking table, incubating for 4 hours at 180 revolutions, and taking out the centrifuge tube;
(2) placing the centrifuge tube into a centrifuge,centrifuging for 8min at 5000 rpm, pouring out supernatant, adding equal volume of physiological saline for resuspension, and performing gradient dilution to obtain final bacterial load of 106CFU/mL；
(3) Adding 1mL of bacterial liquid into a six-hole plate, adding 10 mu L of black pepper essential oil with the original concentration, and uniformly mixing;
(4) setting contrast, blank contrast, treating with 1% essential oil for shading, placing into ultraviolet irradiation box, and irradiating with ultraviolet for 30 min;
(5) after the ultraviolet irradiation is finished, 100 mu L of bacterial liquid is absorbed and added into a 2ml centrifuge tube filled with 900 mu L of 0.1% physiological saline for gradient dilution, 25 mu L of bacterial liquid is absorbed and dropped on an MH agar culture medium after dilution, the culture box with the temperature of 37 ℃ is incubated for 16-18h, counting is carried out, and the statistical analysis is carried out after the experimental result is repeated through three biology.
In this experiment, a growth control group, an ultraviolet control group, an essential oil control group, and an ultraviolet plus essential oil test group were set, and the detection was performed according to the method of example 1.
The results are shown in Table 11, where bacteria grew normally in the blank control group, indicating that E.coli ATCC25922 grew normally under the experimental conditions; under the irradiation of an ultraviolet lamp for 30min, the Escherichia coli ATCC25922 is not obviously reduced compared with a blank control group, which shows that the inhibition effect of the ultraviolet lamp on the Escherichia coli ATCC25922 is not obvious; the same results also show that 1% black pepper essential oil has no obvious inhibition effect on escherichia coli ATCC 25922; the combined action result of 30min shows that the synergistic effect of the ultraviolet rays and the 1% black pepper essential oil has obvious sterilization effect on escherichia coli ATCC 25922.
TABLE 11 combination of UV and Black Pepper essential oil fungicidal effect on ATCC25922 strains
5. Effect evaluation experiment of combination of European fir essential oil and ultraviolet synergist:
(1) inoculating Escherichia coli ATCC25922, placing single colony in a centrifuge tube filled with 4mL MH broth, putting the centrifuge tube in a 37-degree shaking table, incubating for 4 hours at 180 revolutions, and taking out the centrifuge tube;
(2) placing the centrifuge tube in a centrifuge, centrifuging for 8min at 5000 rpm, pouring out supernatant, adding equal volume of physiological saline for resuspension, and performing gradient dilution to obtain final bacterial load of 106CFU/mL；
(3) Adding 1mL of bacterial liquid into a six-hole plate, adding 10 mu L of European fir essential oil with the original concentration, and uniformly mixing;
(4) setting contrast, blank contrast, treating with 1% essential oil for shading, placing into ultraviolet irradiation box, and irradiating with ultraviolet for 30 min;
(5) after the ultraviolet irradiation is finished, 100 mu L of bacterial liquid is absorbed and added into a 2ml centrifuge tube filled with 900 mu L of 0.1% physiological saline for gradient dilution, 25 mu L of bacterial liquid is absorbed and dropped on an MH agar culture medium after dilution, the culture box with the temperature of 37 ℃ is incubated for 16-18h, counting is carried out, and the statistical analysis is carried out after the experimental result is repeated through three biology.
In this experiment, a growth control group, an ultraviolet control group, an essential oil control group, and an ultraviolet plus essential oil test group were set, and the detection was performed according to the method of example 1.
The results are shown in Table 12, where bacteria grew normally in the blank control group, indicating that E.coli ATCC25922 grew normally under the experimental conditions; under the irradiation of an ultraviolet lamp for 30min, the Escherichia coli ATCC25922 is not obviously reduced compared with a blank control group, which shows that the inhibition effect of the ultraviolet lamp on the Escherichia coli ATCC25922 is not obvious; the same results also show that 1% of european fir essential oil has no significant inhibitory effect on escherichia coli ATCC 25922; the combined action result of 30min shows that the synergistic effect of the ultraviolet rays and the 1 percent of European fir essential oil has obvious bactericidal effect on Escherichia coli ATCC 25922.
TABLE 12 Bactericidal Effect of UV and European fir essential oil combination on ATCC25922 Strain
6. The effect evaluation experiment of the eucalyptus leaf essential oil combined with the ultraviolet synergist comprises the following steps:
(1) inoculating Escherichia coli ATCC25922, placing the single colony in a centrifugal tube filled with 4mLMH broth, putting the centrifugal tube in a 37-degree shaking table, incubating for 4 hours at 180 revolutions, and taking out the centrifugal tube;
(2) placing the centrifuge tube in a centrifuge, centrifuging for 8min at 5000 rpm, pouring out supernatant, adding equal volume of physiological saline for resuspension, and performing gradient dilution to obtain final bacterial load of 106CFU/mL；
(3) Preparing 1% DMSO (dimethyl sulfoxide) by using pure water, subpackaging in 2mL centrifuge tubes, wherein each tube is 200 mu L, then adding 200 mu L of the essential oil with the original concentration into the tube, blowing, uniformly mixing, and diluting by times to enable the concentration of the essential oil to be 25%;
(4) adding 1mL of bacterial liquid into six-hole plate, adding 10 μ L of 25% Eucalyptus leaf essential oil, and mixing well to obtain final concentration of 0.25%
(5) Setting contrast, blank contrast, treating with 0.25% essential oil for shading, placing into ultraviolet irradiation box, and irradiating with ultraviolet for 30 min;
(6) after the ultraviolet irradiation is finished, 100 mu L of bacterial liquid is absorbed and added into a 2ml centrifuge tube filled with 900 mu L of 0.1% physiological saline for gradient dilution, 25 mu L of bacterial liquid is absorbed and dropped on an MH agar culture medium after dilution, the culture box with the temperature of 37 ℃ is incubated for 16-18h, counting is carried out, and the experimental result is subjected to statistical analysis after three biological repetitions.
In this experiment, a growth control group, an ultraviolet control group, an essential oil control group, and an ultraviolet plus essential oil test group were set, and the detection was performed according to the method of example 1.
The results are shown in Table 13, where the bacteria grew normally in the blank control group, indicating that E.coli ATCC25922 grew normally under the experimental conditions; under the irradiation of an ultraviolet lamp for 30min, the Escherichia coli ATCC25922 is not obviously reduced compared with a blank control group, which shows that the inhibition effect of the ultraviolet lamp on the Escherichia coli ATCC25922 is not obvious; the same results also show that 0.25% of eucalyptus leaf essential oil has no significant inhibitory effect on escherichia coli ATCC 25922; the combined action result of 30min shows that the synergistic effect of the ultraviolet rays and the eucalyptus leaf essential oil of 0.25 percent has obvious bactericidal effect on escherichia coli ATCC 25922.
TABLE 13 fungicidal Effect of UV and Eucalyptus leaf essential oil combination on ATCC25922 Strain
The embodiments of the present invention have been described in detail, but the present invention is not limited to the described embodiments. It will be apparent to those skilled in the art that various changes, modifications, substitutions and alterations can be made in these embodiments without departing from the principles and spirit of the invention, and the scope of protection is still within the scope of the invention.
Claims (4)
1. A method for sterilizing by ultraviolet combined plant essential oil is characterized in that plant essential oil and ultraviolet irradiation are used for sterilizing, wherein the plant essential oil is selected from one of tea tree essential oil, black pepper essential oil, European fir essential oil and Eucalyptus globulus L.essential oil; the irradiation time of the ultraviolet is 30min, and the ultraviolet intensity is 2.4-3.0mW/cm2(ii) a Wherein, when the plant essential oil is tea tree essential oil, the concentration of the plant essential oil is 0.25%; when the plant essential oil is black pepper essential oil, the concentration of the black pepper essential oil is 1%; when the plant essential oil is the European fir essential oil, the concentration of the European fir essential oil is 1%; when the plant essential oil is Eucalyptus leaf essential oil, the concentration of Eucalyptus leaf essential oil is 0.25%.
2. The method of claim 1, wherein the pre-heating is performed for 30min before the UV irradiation.
3. The method for sterilizing by combining ultraviolet light with plant essential oil according to claim 2, which comprises the following steps:
(1) mixing the bacterial liquid and the plant essential oil, placing the mixture in a six-hole plate, and setting ultraviolet irradiation conditions: wavelength range 300-18W, the distance from the six-hole plate to the ultraviolet lamp tube is 8cm, and the ultraviolet intensity is 2.4-3.0mW/cm2；
(2) Preheating the ultraviolet box for 20-40min, and placing the six-hole plate in the ultraviolet box for irradiating for 25-35 min.
4. The method for sterilizing by combining ultraviolet and plant essential oil according to claim 3, wherein the final concentration of the bacterial liquid in the step (1) is 106CFU/mL。
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