A composite alcohol disinfectant and its application

Abstract

The present invention belongs to the technical field of disinfection and sterilization, and discloses a novel alcohol synergist, including alcohol and lemon essential oil. Compared with alcohol disinfection alone, the present invention can kill bacteria more efficiently and reduce the alcohol concentration used. The combination of alcohol and essential oil provided by the present invention can significantly enhance the bactericidal effect of alcohol. The present invention uses Escherichia coli ATCC25922 and Staphylococcus aureus ATCC29213 as representative strains of Gram-negative bacteria and Gram-positive bacteria, and proves that lemon essential oil can be used as a bactericidal synergist for alcohol, thereby expanding the application of alcohol in disinfection and sterilization.

Description

Technical Field

[0001] The invention belongs to the technical field of disinfection and sterilization, and more specifically, relates to a novel alcohol enhancer and application thereof. Background Art

[0002] Alcohol is a colorless, toxic, volatile liquid chemical, also known as ethanol (Ethanol). It is a chemical widely used in industry and consumer products, and can also be used as an ingredient in beverages. In medicine and biology, alcohol has certain pharmacological effects because it can affect the human nervous system. However, alcohol can be highly toxic and harmful to the human body in high doses, so it needs to be used with caution. Alcohol has antibacterial and disinfectant properties, so it is widely used as a disinfectant for surgical and medical equipment. Common disinfectant alcohol is usually a 70% ethanol solution.

[0003] In addition, alcohol has some inevitable limitations: 1. Alcohol is irritating. If it is not properly contacted, it will damage the skin sebum, damage the conjunctiva, and may also cause allergies and other problems. 2. Alcohol is flammable. At room temperature and pressure, due to improper storage and operation, if it encounters open flames, high heat, etc., it is very likely to cause a fire. 3. In terms of the sterilization range, the sterilization spectrum of alcohol is limited and can only kill some viruses and bacteria (with envelopes). In order to enhance the bactericidal effect of alcohol, studying synergists is an essential step. However, there are still few related synergists reported so far. The development of alcohol disinfectant synergists can greatly expand the application of alcohol in the field of disinfection. Summary of the invention

[0004] In order to solve the above problems in the prior art, the present invention provides a novel alcohol synergist. Alcohol is one of the most commonly used disinfectants at present. The combination of alcohol and essential oil provided by the present invention can significantly enhance the bactericidal effect of alcohol. The present invention uses Escherichia coli ATCC25922 and Staphylococcus aureus ATCC29213 as representative strains of Gram-negative bacteria and Gram-positive bacteria, and proves that lemon essential oil can be used as a bactericidal synergist for alcohol.

[0005] To achieve the above object, the present invention provides the following technical solutions:

[0006] A disinfectant enhancer composed of alcohol and lemon essential oil. Lemon essential oil is usually extracted from the peel of lemon (Citrus medicavar.sarcodactylis) by cold pressing. The main ingredients include limonene, hesperidin, methyl hesperidin, etc. Lemon essential oil is widely used in aromatherapy, which has the effects of improving mood, relieving anxiety and relieving stress. It can also be used in skin care to help clean oil, balance oil secretion, and make the skin fresh and bright. Lemon essential oil has a wide range of applications in aromatherapy and natural care, but it must be used in accordance with safe use guidelines. Commonly found in fragrance products.

[0007] Preferably, the concentration (volume fraction) of alcohol (i.e. ethanol) is 5%-75%. As a specific solution, alcohol and water are mutually soluble, and the volume fraction concentration of alcohol in water is 5%-75%.

[0008] Preferably, the lemon essential oil is dissolved in a solvent capable of dissolving the lemon essential oil, and the concentration (volume fraction) of the lemon essential oil is 0.1%-5%.

[0009] Compared with the prior art, the present invention has the following beneficial effects:

[0010] The combination of alcohol and essential oil provided by the present invention can significantly enhance the bactericidal effect of alcohol. The present invention uses Escherichia coli ATCC25922 and Staphylococcus aureus ATCC29213 as representative strains of Gram-negative bacteria and Gram-positive bacteria, and proves that lemon essential oil can be used as a bactericidal synergist for alcohol, expanding the application of alcohol in disinfection and sterilization. BRIEF DESCRIPTION OF THE DRAWINGS

[0011] Figure 1 It is the bactericidal effect of lemon essential oil combined with alcohol on Escherichia coli ATCC25922.

[0012] Figure 2 It is the bactericidal effect of lemon essential oil combined with alcohol on Staphylococcus aureus ATCC29213. DETAILED DESCRIPTION

[0013] The technical scheme of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention and the accompanying drawings. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments in the present invention, all other embodiments obtained by those of ordinary skill in the art without making creative work are within the scope of protection of the present invention. If the specific conditions are not specified in the embodiments, they are prepared according to the conditions recommended by the normal conditions or the manufacturers. If the manufacturer is not specified in the reagents or instruments used, they are all conventional products that can be purchased commercially.

[0014] Example 1 Determination of the MIC of lemon essential oil and alcohol against ATCC 25922 and ATCC 29213 strains

[0015] 1. Experimental materials:

[0016] (1) Alcohol, lemon essential oil

[0017] Experimental: The autoclaved MH broth was cooled and set aside.

[0018] (2) Resin Azure: Macklin brand, MW 251.17, purity 90%;

[0019] (3) Escherichia coli standard strains ATCC25922 and ATCC29213 (laboratory collection).

[0020] 2. Preparation before the test:

[0021] (1) Take the lemon essential oil out of the refrigerator and equilibrate it to room temperature, and absorb part of it for later use;

[0022] (2) Escherichia coli standard strains ATCC 25922 and ATCC 29213 were cultured on LB agar medium until they reached an appropriate size.

[0023] (3) Weigh 0.2512 g of resin azure powder into a centrifuge tube and add 10 mL of pure water to dissolve it. The concentration of the resin azure solution is 10 mM / L.

[0024] 3. Lemon essential oil / alcohol bactericidal effect evaluation experiment:

[0025] (1) Inoculate a single colony of ATCC 25922 or ATCC 29213 in a centrifuge tube containing 4 mL of MH broth, place in a shaker at 37°C at 180 rpm for 4 hours, and then remove the centrifuge tube;

[0026] (2) Use MH broth to dilute the incubated ATCC25922 and ATCC29213 100 times, about 10 6 CFU / mL, reserve;

[0027] (3) Take a sterile 96-well plate, add 180 μL of MH broth medium to well 1, and add 100 μL of MH broth medium to wells 2-11;

[0028] (4) Add 20 μL of original concentration of lemon essential oil / alcohol to the first well, blow and mix evenly, then pipette 100 μL into the second well, and so on, pipette 100 μL into the tenth well and discard; at this time, the concentrations of lemon essential oil / alcohol in each well are 10%, 5%, 2.5%, 1.25%, 0.625%, 0.3125%, 0.156%, 0.078%, 0.039%, and 0.0195%, respectively.

[0029] (5) Add 100 μL of diluted bacterial solution to wells 1 to 10. At this time, the concentrations of lemon essential oil / alcohol in each well are 5%, 2.5%, 1.25%, 0.625%, 0.3125%, 0.156%, 0.078%, 0.039%, 0.0195%, and 0.01%, respectively. Add 100 μL of bacterial solution to well 11, and add 200 μL of MH broth to well 12;

[0030] (6) Repeat steps (3) to (5) three times in parallel;

[0031] (7) Place the inoculated 96-well plate in a 37°C incubator and incubate for 16-18 hours. Then, pipette 10 μL of 10 mM / L resin azure into the wells. The resin azure concentration is 0.1 mM / L. After incubation for 2 hours, read the results.

[0032] The results are shown in Table 2. The MIC value of lemon essential oil for ATCC25922 is 0.62%, and the MIC value for ATCC29213 is 0.31%. At this concentration, the growth of bacteria is inhibited. The MIC value of alcohol for ATCC25922 is 10%, and the MIC value for ATCC29213 is 10%. At this concentration, the growth of bacteria is inhibited. Since the action time of alcohol and lemon essential oil is short, 0.5% and 1% lemon essential oil and 10% alcohol are selected for combination.

[0033] Table 1 MIC of lemon essential oil and alcohol against ATCC 25922 and ATCC 29213 strains

[0034]

[0035] Example 2: Bactericidal effect of lemon essential oil / alcohol on ATCC 25922 and ATCC 29213 strains after 1 hour of action

[0036] 1. Experimental materials:

[0037] (1) Alcohol and lemon essential oil.

[0038] (2) Experimental culture medium: The autoclaved LB agar medium (purchased from Guangdong Huankai Microbiological Technology Co., Ltd.) was cooled to 55°C, 20 mL was taken into a sterile culture dish using a pipette, and naturally dried for 30 min to prepare LB agar medium and MacConkey agar medium.

[0039] (3) Escherichia coli standard strains ATCC25922 and ATCC29213 (laboratory preserved or commercially available).

[0040] 2. Preparation before the test:

[0041] (1) The standard strain of Escherichia coli ATCC25922 and the standard strain of Staphylococcus aureus were cultured on LB agar medium until they reached an appropriate size.

[0042] (2) Dilute lemon essential oil to 5% and 10% concentrations with 1% DMSO and set aside.

[0043] 3. Lemon essential oil / alcohol bactericidal effect evaluation experiment:

[0044] (1) Inoculate a single colony of ATCC 25922 or ATCC 29213 in a centrifuge tube containing 4 mL of MH broth, place in a shaker at 37°C at 180 rpm for 4 hours, and then remove the centrifuge tube;

[0045] (2) Place the centrifuge tube in a centrifuge and centrifuge at 5000 rpm for 8 min. Pour off the supernatant and add an equal volume of saline to resuspend the tube so that the final bacterial count is approximately 10 8 CFU / mL;

[0046] (3) Set up different treatment groups, mix well and add to 6-well plates and incubate at 37°C for 1 h

[0047] ①Blank control group: 900 μL bacterial solution + 100 μL normal saline;

[0048] ② Alcohol treatment group: 900 μL bacterial solution + 100 μL original concentration alcohol;

[0049] ③ Lemon essential oil treatment group: 900 μL bacterial solution + 100 μL of the above diluted 5% / 10% lemon essential oil.

[0050] (4) After the incubation, 100 μL of bacterial solution was added to a 2 mL centrifuge tube containing 900 μL of 0.85% saline for gradient dilution. After dilution, 25 μL was dropped on MH agar medium and incubated in a 37°C incubator for 16-18 h. The cells were counted and the experimental results were statistically analyzed after three biological replicates.

[0051] (5) The results are shown in Table 2. The number of bacteria in the selected concentration of lemon essential oil and alcohol treatment group is not much different from that in the blank control group, indicating that the selected concentration of lemon essential oil and alcohol cannot produce a significant antibacterial effect on ATCC25922. The results are shown in Table 3. The number of bacteria in the selected concentration of lemon essential oil and alcohol treatment group is not much different from that in the blank control group, indicating that the selected concentration of lemon essential oil and alcohol cannot produce a significant antibacterial effect on ATCC29213.

[0052] Table 2 Bactericidal effect of lemon essential oil and alcohol on ATCC 25922 strain

[0053]

[0054] Table 3 Bactericidal effect of lemon essential oil and alcohol on ATCC29213 strain

[0055]

[0056]

[0057] Example 3: Bactericidal effect of lemon essential oil combined with alcohol for 1 h on ATCC 25922 and ATCC 29213 strains

[0058] 1. Experimental materials:

[0059] (1) Alcohol and lemon essential oil.

[0060] (2) Experimental culture medium: The autoclaved LB agar medium (purchased from Guangdong Huankai Microbiological Technology Co., Ltd.) was cooled to 55°C, 20 mL was taken into a sterile culture dish using a pipette, and naturally dried for 30 min to prepare LB agar medium and MacConkey agar medium.

[0061] (3) Escherichia coli standard strains ATCC25922 and ATCC29213 (laboratory preserved or commercially available).

[0062] 2. Preparation before the test:

[0063] (1) The standard strain of Escherichia coli ATCC25922 and the standard strain of Staphylococcus aureus were cultured on LB agar medium until they reached an appropriate size.

[0064] (2) Dilute lemon essential oil to 5% and 10% concentrations with 1% DMSO and set aside.

[0065] 3. Experiment on the evaluation of the combined bactericidal effect of lemon essential oil and alcohol:

[0066] (1) Inoculate a single colony of ATCC 25922 or ATCC 29213 in a centrifuge tube containing 4 mL of MH broth, place in a shaker at 37°C at 180 rpm for 4 hours, and then remove the centrifuge tube;

[0067] (2) Place the centrifuge tube in a centrifuge and centrifuge at 5000 rpm for 8 min. Pour off the supernatant and add an equal volume of saline to resuspend the tube so that the final bacterial count is approximately 10 8 CFU / mL;

[0068] (3) Set up different treatment groups, mix well and add to 6-well plates and incubate at 37°C for 1 h;

[0069] ①Blank control group: 800 μL bacterial solution + 200 μL normal saline;

[0070] ② Alcohol treatment group: 800 μL bacterial solution + 100 μL normal saline + 100 μL original concentration alcohol;

[0071] ③ 0.5% lemon essential oil treatment group: 800 μL bacterial solution + 100 μL normal saline + 100 μL of the above diluted 5% lemon essential oil;

[0072] ④1% lemon essential oil treatment group: 800 μL bacterial solution + 100 μL normal saline + 100 μL of the above diluted 10% lemon essential oil;

[0073] ⑤ Alcohol + 0.5% lemon essential oil treatment group: 800 μL bacterial solution + 100 μL original concentration alcohol + 100 μL of the above diluted 5% lemon essential oil;

[0074] ⑥ Alcohol + 1% lemon essential oil treatment group: 800 μL bacterial solution + 100 μL original concentration alcohol + 100 μL of the above diluted 10% lemon essential oil.

[0075] (4) After the incubation, 100 μL of bacterial solution was added to a 2 mL centrifuge tube containing 900 μL of 0.85% saline for gradient dilution. After dilution, 25 μL was dropped on MH agar medium and incubated in a 37°C incubator for 16-18 h. The cells were counted and the experimental results were statistically analyzed after three biological replicates.

[0076] Lemon essential oil and alcohol had weak antibacterial effects. The use of 10% alcohol alone reduced the bacterial count by 0.13 log compared with the control group. The use of 0.5% and 1% lemon essential oil alone reduced the bacterial count by 0.27 log and 1.9 log compared with the control group. The combination of the two had a significant synergistic antibacterial effect. Figure 1, the combination of 10% alcohol and 1% lemon essential oil can completely kill Escherichia coli ATCC 25922, and the combination of 10% alcohol and 0.5% lemon essential oil can reduce the amount of Escherichia coli ATCC 25922 by 2.63 log compared with the use of either alone; as shown in Table 5 and Figure 2 , when the concentration of 10% alcohol and 0.5% and 1% lemon essential oil are combined, the sterilization effect on Staphylococcus aureus ATCC29213 is significantly improved compared with when used alone, and the bacterial count decreases by 2.011log and 3.74log. Compared with traditional alcohol disinfection, the present invention can kill bacteria more efficiently and reduce the alcohol concentration used.

[0077] Table 4 Bactericidal effect of alcohol and lemon essential oil on Escherichia coli ATCC 25922

[0078]

[0079] Table 5 Bactericidal effect of alcohol and lemon essential oil on Staphylococcus aureus ATCC29213

[0080]

[0081] The above embodiments of the present invention are merely examples for clearly illustrating the technical solution of the present invention, and are not intended to limit the specific implementation methods of the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the claims of the present invention shall be included in the protection scope of the claims of the present invention.

Claims

1. A disinfection synergist, characterized in that: The disinfection enhancer is composed of alcohol and lemon essential oil; the alcohol and water are miscible, the volume fraction concentration of the alcohol is 10%; the volume fraction concentration of the lemon essential oil is 0.1%-5%.

2. Use of the disinfection enhancer according to claim 1 in the preparation of products in the field of disinfection and purification.

3. The use according to claim 2, characterized in that: Disinfection and purification are used to disinfect Gram-negative bacteria and / or Gram-positive bacteria.

4. The use according to claim 3, characterized in that: The Gram-negative bacteria are Escherichia coli, and the Gram-positive bacteria are Staphylococcus aureus.

5. A combined sterilization method, characterized in that: The disinfection enhancer according to claim 1 is mixed with the bacterial solution and incubated at a temperature of 36.8°C to 37.5°C.

6. The combined sterilization method according to claim 5, characterized in that: The bacterial solution is Escherichia coli and Staphylococcus aureus, and the concentration of the bacterial solution is 10 8 CFU / mL.

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