CN113907094B - Ultraviolet sterilization synergist-France cypress essential oil and application thereof - Google Patents

Abstract

The invention belongs to the technical field of ultraviolet sterilization, and discloses an ultraviolet sterilization synergist and a method for sterilizing it in combination with ultraviolet rays. This study is based on the weak bactericidal effect of A-band ultraviolet (UVA) and French Cypress essential oil (the bactericidal amount is 0.5log-1.5log when used alone), and the combined effect has obvious synergistic bactericidal effect. Compared with the traditional ultraviolet sterilization technology, the present invention can kill bacteria more efficiently and reduce the influence of the quality of the ultraviolet lamp on the disinfection effect.

Description

A kind of ultraviolet bactericidal synergist French cypress essential oil and its application

technical field

The invention relates to the technical field of ultraviolet sterilization, more specifically, to an ultraviolet sterilization synergist French cypress essential oil and its application.

Background technique

Ultraviolet germicidal technology is a convenient method with no chemical residues and low impact on the environment, and is often used to disinfect gases, liquids and solid surfaces. Ultraviolet light is a general term for radiation with a wavelength of 10nm-400nm in the electromagnetic spectrum. It is invisible light with a higher frequency than blue-violet light. Ultraviolet rays are classified into UVA, UVB, UVC and UVD. Ultraviolet lamp sterilization is used in various places due to its low cost, convenient use, and no drug resistance. In recent years, due to the extensive use of antibiotics, bacterial drug resistance has become more and more serious. With the emergence of multi-drug resistant bacteria, the effect of treating infections caused by bacteria has gradually weakened. There are more and more applications on the Internet, and the research on the sterilization effect of ultraviolet rays is also getting more and more in-depth.

The principle of ultraviolet sterilization technology is to use ultraviolet radiation of appropriate wavelength to destroy the molecular structure of DNA (deoxyribonucleic acid) or RNA (ribonucleic acid) in the cells of organisms, causing the death of growing cells and/or regenerative cells to achieve the effect of sterilization. Ultraviolet sterilization technology is based on modern epidemic prevention science, medicine, and photodynamics. It uses specially designed high-efficiency, high-intensity, and long-life ultraviolet rays to irradiate the surface of objects to directly kill various bacteria, viruses, parasites, and algae on the surface of objects. pathogen. At the same time, it can kill bacteria without developing antibiotic resistance, so it will not lead to increased bacterial resistance. However, low-energy ultraviolet light itself is not enough to achieve a very good sterilization effect, and the sterilization time is long and cannot completely kill bacteria.

French Cypress (Cypress) essential oil is distilled from fresh leaves and cones. Colorless or very light yellow, with a woody and balsamic attractive amber odor. The main components of cypress essential oil are fenene, cedrol, cedar camphor, etc., which have antibacterial, insecticidal, and sedative effects. In order to enhance the ultraviolet bactericidal effect, it is an essential step to study the synergist of ultraviolet sterilizing effect. However, the reported synergistic effects of essential oils on ultraviolet rays are still relatively small, and it is urgent to add new ultraviolet bactericidal synergists to increase the bactericidal effect of ultraviolet rays on various occasions.

Contents of the invention

The technical problem to be solved by the present invention is to overcome the above-mentioned defects existing in the prior art, and to provide the application of French cypress essential oil as an ultraviolet bactericidal synergist.

The second object of the present invention is to provide a method of using French cypress essential oil in combination with ultraviolet light to sterilize.

The purpose of the present invention is achieved through the following technical solutions:

Application of French cypress essential oil as a synergist for ultraviolet germicidal treatment.

The research of the present invention finds that the combination of French cypress essential oil and ultraviolet rays can significantly enhance the ultraviolet bactericidal ability, so it can be used as a synergist for ultraviolet sterilization.

The invention also provides an ultraviolet bactericidal synergist, which includes French cypress essential oil.

The present invention also provides a method for sterilizing ultraviolet rays combined with French cypress essential oil, the irradiation time of ultraviolet light is 30 minutes, and the concentration of French cypress essential oil is 1%.

Preferably, in the above sterilization method, the ultraviolet intensity is 2.4-3.0mW/cm ² .

Preferably, in the above sterilization method, preheating is required for 30 minutes before ultraviolet irradiation.

Preferably, the method for above-mentioned sterilization comprises the following steps:

(1) Mix the bacterial liquid with 1% French cypress essential oil and place it in a six-hole plate, set the UV irradiation conditions: the wavelength range is 300-460nm, the power is 18W, the distance from the six-hole plate to the UV lamp is 8cm, and the ultraviolet intensity 2.4-3.0mW/cm ² ;

(2) Preheat the UV box for 20-40 minutes, put the six-hole plate into the UV box and irradiate for 25-35 minutes.

More preferably, the final concentration of the bacterial solution in step (1) of the above sterilization method is 10 ⁶ CFU/mL.

Compared with the prior art, the present invention has the following beneficial effects:

The present invention provides a kind of synergist that has had synergistic effect with ultraviolet, this research is based on A band ultraviolet (UVA) and French cypress essential oil bactericidal effect weak (bactericidal amount is at 0.5log-1.5log when acting alone), its After combined action, it has obvious synergistic bactericidal effect. Compared with the traditional ultraviolet sterilization technology, the present invention can kill bacteria more efficiently and reduce the influence of ultraviolet lamp quality on disinfection effect.

Detailed ways

Specific embodiments of the present invention will be further described below. It should be noted here that the descriptions of these embodiments are used to help understand the present invention, but are not intended to limit the present invention. In addition, the technical features involved in the various embodiments of the present invention described below may be combined with each other as long as they do not constitute a conflict with each other.

The test methods used in the following experimental examples are conventional methods unless otherwise specified; the materials and reagents used are commercially available reagents and materials unless otherwise specified.

The ultraviolet lamp used is PHILIPS TL-D brand; wattage: 18W; voltage: 220V; wavelength: (UVA) 300-469nm; tube diameter: 25mm; length: 60cm. The French cypress essential oil is Satya brand (Italy).

Judgment principle: If the bacterial count of the experimental group is reduced by 2 logarithms on the basis of the reduction of the bacterial count of the single ultraviolet treatment and the single essential oil treatment, it means that the essential oil and ultraviolet light have a synergistic bactericidal effect.

The bactericidal effect of embodiment 1 different ultraviolet irradiation time to ATCC 25922 bacterial strain

1. Experimental materials:

(1) The ultraviolet lamp used in the test is PHILIPS TL-D brand; wattage: 18W; voltage: 220V; wavelength: (UVA) 300-469nm; tube diameter: 25mm; length: 60cm.

(2) Test medium: Cool the autoclaved MH agar medium and MacConkey agar medium (purchased from Guangdong Huankai Microbiology Technology Co., Ltd.) to 55°C, and use a pipette gun to take 20mL to sterile culture Dish and let it dry naturally for 30 minutes to prepare MH agar medium and MacConkey agar medium.

Escherichia coli standard strain ATCC25922 (preserved in the laboratory).

2. Preparations before the test:

(1) Turn on the ultraviolet lamp and continue to irradiate for 30 minutes to preheat;

(2) The Escherichia coli standard strain ATCC25922 was cultured to a suitable size on MacConkey medium.

3. Evaluation experiment of ultraviolet sterilizing effect:

(1) Inoculate a single colony of Escherichia coli ATCC 25922 into a centrifuge tube containing 4 mL of MH broth, place it in a shaker at 37 degrees at 180 rpm and incubate for 4 hours, then take out the centrifuge tube;

(2) Put the centrifuge tube into the centrifuge, centrifuge at 5000 rpm for 8 minutes, pour off the supernatant, add an equal volume of normal saline to resuspend, and perform gradient dilution to make the final bacterial volume 10 ⁶ CFU/mL;

(3) Add 1mL of bacterial solution into the six-well plate;

(4) A blank control group and an ultraviolet irradiation treatment group were set up, wherein the ultraviolet irradiation treatment group was divided into three groups, which were respectively irradiated for 15 min, 30 min and 60 min.

(5) After the ultraviolet irradiation is over, draw 100 μL of the bacterial solution and add it to a 2mL centrifuge tube filled with 900 μL of 0.85% normal saline for gradient dilution. After dilution, draw 25 μL and drop it on the MH agar medium, and incubate in a 37-degree incubator for 16-18 hours. Counting was carried out, and the experimental results were subjected to statistical analysis after three biological repetitions.

The results are shown in Table 1. With the increase of irradiation time, the antibacterial effect of ultraviolet on bacteria increased. Among them, in the ultraviolet irradiation 15min group, the number of bacteria was not much different from that of the blank control group, indicating that short ultraviolet irradiation time could not affect the ATCC25922 produced obvious antibacterial effect. The number of bacteria in the ultraviolet irradiation group for 30 minutes began to decrease, indicating that ultraviolet light had a certain antibacterial effect on bacteria.

The bactericidal effect of table 1 ultraviolet irradiation time on ATCC 25922 strain

Embodiment 2 measures the MIC of different concentrations of French cypress essential oils to ATCC 25922 bacterial strains

1. Experimental materials:

(1) Test: The autoclaved MH broth was cooled for later use.

(2) Resin Azure: Macklin (Macklin) brand, MW is 251.17, and purity is 90%;

Escherichia coli standard strain ATCC25922 (preserved in the laboratory).

2. Preparations before the test:

(1) Take the French cypress essential oil out of the refrigerator and equilibrate it to room temperature, absorb some of it for later use;

(2) The Escherichia coli standard strain ATCC 25922 was cultured to a suitable size on MacConkey agar medium.

(3) Weigh 0.2512 g of resin azure powder into a centrifuge tube, add 10 mL of pure water to dissolve, and the concentration of resin azure solution is 10 mM/L.

3. Evaluation experiment of the bactericidal effect of French cypress essential oil:

(1) Inoculate a single colony of Escherichia coli ATCC 25922 into a centrifuge tube containing 4 mL of MH broth, place it in a shaker at 37 degrees at 180 rpm and incubate for 4 hours, then take out the centrifuge tube;

(2) Use MH broth to dilute the incubated E. coli to 100 times, about 10 ⁶ CFU/mL, and set aside;

(3) Take a sterile 96-well plate, add 180 μL MH broth medium to the first well, and add 100 μL MH broth medium to the 2-11 wells;

(4) Add 20 μL of the original concentration of essential oil to the first hole, pipette evenly, then pipette 100 μL to the second well, and so on, and then absorb 100 μL to the tenth hole and discard; at this time, the concentration of essential oil in each well is 10%, 5%, and 2.5%. , 1.25%, 0.625%, 0.3125%, 0.156%, 0.078%, 0.039%, 0.0195%.

(5) Add 100 μL of diluted bacterial solution to wells 1 to 10. At this time, the concentration of essential oil in each well is 5%, 2.5%, 1.25%, 0.625%, 0.3125%, 0.156%, 0.078%, 0.039%, 0.0195%, 0.01%, add 100 μL bacterial solution to the 11th well, add 200 μL MH broth to the 12th well;

(6) Steps (3) to (5) were repeated for three repetitions in parallel;

(7) Put the inoculated 96-well plate into a 37-degree incubator and incubate for 16-18 hours, pipette 10 μL of 10 mM/L resin azure into the wells, the concentration of resin azure is 0.1 mM/L, and after incubation for 2 hours, read Get the result.

The results are shown in Table 2. The MIC value of the French cypress essential oil is 0.313%, at which the growth of bacteria is inhibited. Finally, the concentration of 1% French cypress essential oil is selected as the concentration of the ultraviolet synergist.

Table 2 The MIC and experimental concentration of French cypress essential oil to ATCC 25922 strain

Experimental strain French Cypress Essential Oil Mic The concentration used in the experiment ATCC 25922 0.313% 1%

Example 3 The bactericidal effect of French cypress essential oil on ATCC 25922 bacterial strain after 30 minutes

1. Experimental materials:

(1) Experimental medium: cool the autoclaved MH agar medium and MacConkey agar medium (purchased from Guangdong Huankai Microbiology Technology Co., Ltd.) to 40°C, and use a pipette to take 20mL to sterile culture Dish and let it dry naturally for 30 minutes to prepare MH agar medium and MacConkey agar medium.

Escherichia coli standard strain ATCC25922 (preserved in the laboratory).

2. Preparations before the test:

(1) Take the French cypress essential oil out of the refrigerator and equilibrate it to room temperature, absorb some of it for later use;

(2) Cultivate Escherichia coli standard strain ATCC 25922 on MacConkey medium to a suitable size.

3. Evaluation experiment of the bactericidal effect of French cypress essential oil:

(1) Inoculate a single colony of Escherichia coli ATCC 25922 into a centrifuge tube containing 4 mL of MH broth, place it in a shaker at 37 degrees at 180 rpm and incubate for 4 hours, then take out the centrifuge tube;

(2) Put the centrifuge tube into the centrifuge, centrifuge at 5000 rpm for 8 minutes, pour off the supernatant, add an equal volume of normal saline to resuspend, and perform gradient dilution to make the final bacterial volume 10 ⁶ CFU/mL;

(3) Add 1 mL of the bacterial solution into a six-well plate, add 10 μL of the original concentration of Cypress essential oil and mix evenly so that the final concentration of the essential oil is 1%;

(4) Set control, blank control, 1% French cypress essential oil group were treated for 30min;

(5) Take 100 μL of the bacterial solution and add it to a 2ml centrifuge tube filled with 900 μL 0.85% normal saline for gradient dilution. After dilution, take 25 μL and drop it on the MH agar medium, incubate in a 37-degree incubator for 16-18 hours, and count the results. Statistical analysis was performed after three biological replicates.

The results are shown in Table 3. There was no significant difference between the number of bacteria in the blank control group and the group treated with French cypress essential oil for 30 minutes; indicating that 1% of French cypress essential oil treated ATCC 25922 bacteria for 30 minutes did not change the number of bacteria. It can be seen that 1% French cypress essential oil alone has no obvious antibacterial effect on ATCC 25922 bacteria.

Table 3 The bactericidal effect of French cypress essential oil on ATCC 25922 strain

Example 4 UV and UV synergist to Escherichia coli ATCC 25922 killing effect evaluation

1. Experimental materials:

(1) The ultraviolet lamp used in the test is PHILIPS TL-D brand; wattage: 18W; voltage: 220V; wavelength: (UVA) 300-469nm; tube diameter: 25mm; length: 60cm.

(2) Test medium: Cool the autoclaved MH agar medium and MacConkey agar medium (purchased from Guangdong Huankai Microbiology Technology Co., Ltd.) to 40°C, and use a pipette gun to take 20mL to sterile culture Dish and let it dry naturally for 30 minutes to prepare MH agar medium and MacConkey agar medium.

Escherichia coli standard strain ATCC25922 (preserved in the laboratory).

2. Preparations before the test:

(1) Turn on the ultraviolet lamp and continue to irradiate for 30 minutes to preheat;

(2) Take out the French cypress essential oil from the refrigerator and equilibrate it to room temperature, absorb some of it for later use;

(3) The Escherichia coli standard strain ATCC25922 was cultured to an appropriate size on MacConkey agar medium.

3. Effect evaluation experiment of ultraviolet synergist:

(1) Inoculate a single colony of Escherichia coli ATCC 25922 into a centrifuge tube containing 4 mL of MH broth, place it in a shaker at 37 degrees and incubate at 180 rpm for 4 hours, then take out the centrifuge tube;

(2) Put the centrifuge tube into the centrifuge, centrifuge at 5000 rpm for 8 minutes, pour off the supernatant, add an equal volume of normal saline to resuspend, and perform gradient dilution to make the final bacterial volume 10 ⁶ CFU/mL;

(3) Add 1 mL of the bacterial solution into a six-well plate, add 10 μL of the original concentration of French cypress essential oil and mix well;

(4) Set up control, blank control, 1% essential oil treatment and shading, put them into the ultraviolet irradiation box, and the combined action group and the ultraviolet irradiation treatment group were irradiated with ultraviolet light for 30 minutes;

(5) After the ultraviolet irradiation is over, draw 100 μL of the bacterial solution and add it to a 2ml centrifuge tube filled with 900 μL of 0.85% normal saline for gradient dilution. After dilution, draw 25 μL and drop it on the MH agar medium, and incubate in a 37-degree incubator for 16-18 hours. Counting was carried out, and the experimental results were subjected to statistical analysis after three biological repetitions.

In this experiment, a growth control group, a UV control group, an essential oil control group, and a UV plus essential oil test group were set up, and the detection was carried out according to the method in Example 1.

The results are shown in Table 4. The bacteria in the blank control group grew normally, indicating that Escherichia coli ATCC25922 could grow normally under the experimental conditions; under the irradiation of ultraviolet lamp for 30 minutes, Escherichia coli ATCC 25922 did not significantly decrease compared with the blank control group, indicating that the ultraviolet light The inhibitory effect on Escherichia coli ATCC 25922 is not obvious; the same result also shows that 1% French cypress essential oil has no obvious inhibitory effect on Escherichia coli ATCC 25922; the combined effect of 30min results shows that the synergistic effect of ultraviolet rays and 1% French cypress essential oil The bactericidal effect on Escherichia coli ATCC 25922 is remarkable.

Table 4 The bactericidal effect of ultraviolet and French cypress essential oil combined on ATCC 25922 strain

The embodiments of the present invention have been described in detail above, but the present invention is not limited to the described embodiments. For those skilled in the art, without departing from the principle and spirit of the present invention, various changes, modifications, substitutions and modifications to these embodiments still fall within the protection scope of the present invention.

Claims (4)

1. A method for sterilizing by ultraviolet combined with French cypress essential oil, characterized in that the irradiation time of ultraviolet is 25-35min, the concentration of French cypress essential oil is 1%, and the ultraviolet intensity is 2.4-3.0 mW/cm ² . 2. the method for sterilizing by ultraviolet combined with French cypress essential oil according to claim 1, is characterized in that, need preheating 20-40min before carrying out ultraviolet irradiation. 3. the method for sterilizing by ultraviolet combined French cypress essential oil according to claim 2, is characterized in that, comprises the following steps: (1) Mix the bacterial liquid with 1% French cypress essential oil and place it in a six-hole plate, set the UV irradiation conditions: the wavelength range is 300-460 nm, the power is 18W, the distance from the six-hole plate to the UV lamp is 8cm, and the ultraviolet light Intensity of 2.4-3.0 mW/cm ² ; (2) Preheat the UV box for 30 minutes, put the six-well plate into the UV box and irradiate for 30 minutes. 4 . The method for sterilizing with ultraviolet light combined with French cypress essential oil according to claim 3 , wherein the final concentration of the bacterial solution in step (1) is 10 ⁶ CFU/mL.