Natural product lime oil and silver nitrate compositions and uses thereof

Abstract

The present application belongs to the technical field of bacteriostatic and deodorant products, and specifically discloses a composition of natural product lime essential oil and silver nitrate. High-concentration lime essential oil alone has an inhibitory effect on Escherichia coli BW 25113, but the inhibitory effect of single AgNO3 group on Escherichia coli BW 25113 is not obvious. The present application combines natural product lime essential oil with silver nitrate, and finds that the bactericidal effect on Escherichia coli BW 25113 is remarkable. The synergistic effect of lime essential oil and silver nitrate can reduce the use of high-concentration lime essential oil, while ensuring the bactericidal effect. The composition of the present application can more efficiently play a bacteriostatic role and can replace the use of antibiotics. The above effect is also applicable to inhibiting Staphylococcus aureus and drug-resistant Escherichia coli. In addition, the deodorizing effect on ammonia water shows that the combination of lime essential oil and silver nitrate enhances the deodorizing effect, the use amount of the two components is low, it is safe and effective, the raw materials are easy to obtain, and it has a good application prospect in the preparation of bacteriostatic and deodorant products.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of bacteriostatic and deodorant products, and particularly discloses a composition of natural product lime essential oil and silver nitrate and application thereof. BACKGROUND

[0002] Lime essential oil is distilled from lime berries. The smell is quite strong, bitter and sweet, and yellowish. The main components of lime essential oil are terpinene, coriander oil alcohol, and pinol, which have the effects of anti-evil, antibacterial, and antiviral.

[0003] Some studies have shown that due to different mechanisms of different essential oil components in playing a bacteriostatic effect, the combined use of multiple essential oil components can play a synergistic effect, and antagonism may also occur. The development of composite preparations of essential oils combined with other types has become a major research direction. Silver ions can strongly attract the sulfhydryl group (-SH) on the protease in the bacterial body, rapidly combine with it, make the protease lose activity, and cause the death of bacteria. There are few studies on the synergistic effect of essential oils combined with compounds in bactericidal effect, and therefore, the study of compounds that can have a combined bactericidal effect with lime essential oil has good application prospects.

[0004] In addition, environments that need to be disinfected, such as livestock, factories, sewage treatment, landfills, hospitals, and the like, also face the demand for deodorization. However, the existing chemical deodorants have high use cost, large use amount, and high environmental risk, and therefore, the development of green, low-risk, and low-cost deodorants also has strong practical significance. SUMMARY

[0005] The purpose of the present application is to overcome the defects and deficiencies in the prior art, and to provide a combination of natural product lime essential oil and silver nitrate.

[0006] The second purpose of the present application is to provide the use of the above combination in the field of bacteriostasis and deodorization.

[0007] The purpose of the present application is achieved by the following technical solutions:

[0008] A bacteriostatic and deodorizing composition composed of lime essential oil and silver nitrate.

[0009] Preferably, in the essential oil compound, the concentration of lime essential oil is 1%-5%, and the concentration of silver nitrate is 0.25-0.5 μg / mL.

[0010] The present application also provides the use of the bacteriostatic and deodorizing composition in the preparation of bacteriostatic and deodorizing products.

[0011] Preferably, the bacteriostatic object of the product is foodborne bacteria, such as Escherichia coli or / and Staphylococcus aureus.

[0012] More preferably, the E. coli is drug-resistant E. coli.

[0013] Preferably, the deodorizing object of the product is ammonia or aqueous ammonia.

[0014] Preferably, the form of the product is solid, liquid, pump spray or semi-solid gel.

[0015] The application also provides a bacteriostatic and deodorizing product comprising the bacteriostatic and deodorizing composition.

[0016] Preferably, the solvent of the bacteriostatic and deodorizing product is water.

[0017] More preferably, the concentration of the lime essential oil is 1% to 5%. The above-mentioned deodorizing product preferably exists in the form of a solution, which can be directly sprayed for convenient use.

[0018] Compared with the prior art, the application has the following beneficial effects:

[0019] The application provides a combination of the natural product lime essential oil and silver nitrate, and it is found that the combination of lime essential oil and silver nitrate has a significant bactericidal effect on E. coli BW 25113. The synergy of lime essential oil and silver nitrate can reduce the use of high-concentration lime essential oil while ensuring the bactericidal effect. The above-mentioned effect is also applicable to the inhibition of Staphylococcus aureus and drug-resistant E. coli. The composition of the application has a more efficient bacteriostatic effect and can replace the use of antibiotics. The deodorizing effect on aqueous ammonia shows that the combination of lime essential oil and silver nitrate enhances the deodorizing effect, the use amount of the two components is low, it is safe and effective, the raw materials are easy to obtain, and the application has a good application prospect in the preparation of bacteriostatic and deodorizing products. BRIEF DESCRIPTION OF DRAWINGS

[0020] Figure 1 It is a statistical result graph of the antibacterial effect in Example 2.

[0021] Figure 2 It is a statistical result graph of the antibacterial effect in Example 3.

[0022] Figure 3 It is a statistical result graph of the antibacterial effect in Example 4. DETAILED DESCRIPTION

[0023] The specific embodiments of the application will be described below, which will elaborate and comprehensively describe the technical solutions of the application. It should be pointed out that the provided embodiments only represent a part of the application, not all. Based on the embodiments in the application, all other embodiments obtained by those skilled in the art without creative labor are within the protection scope of the application.

[0024] The test method used in the embodiments of the present application is a conventional method unless otherwise specified; the materials, reagents, etc. used are commercially available reagents and materials unless otherwise specified, and the commercially available raw materials are used after removing insoluble impurities, etc. by general filtration methods.

[0025] The lime essential oil (LM) used is a product of Guangzhou Wenyuan Essential Oil Co., Ltd.; the silver nitrate (AgNO3) is a brand of Macklin, MW 169.87.

[0026] Determination principle: if the reduced bacterial amount of the experimental group is reduced by more than 0.5 log value on the basis of the bacterial amount reduction of the lime essential oil treatment alone and the silver nitrate treatment alone, it indicates that the lime essential oil and the silver nitrate have a synergistic antibacterial effect.

[0027] Example 1 MIC determination of lime essential oil and silver nitrate on Escherichia coli BW 25113 and Staphylococcus aureus ATCC 29213

[0028] 1. Experimental materials:

[0029] (1) Test: cool the sterilized MH broth for standby use.

[0030] (2) Lime essential oil: Guangzhou Wenyuan Essential Oil Co., Ltd.; silver nitrate: Macklin brand, MW 169.87.

[0031] Test strains: Escherichia coli wild strain BW 25113; Staphylococcus aureus quality control strain ATCC 29213.

[0032] 2. Preparation work before test:

[0033] (1) Subculture Escherichia coli wild strain BW 25113 and Staphylococcus aureus quality control strain ATCC 29213 on agar medium, and culture to an appropriate size.

[0034] (2) Weigh 0.1 g of silver nitrate powder in a centrifuge tube, add 10 mL of sterile water to dissolve, the silver nitrate concentration is 10 mg / mL, vortex to mix and filter, the filtrate is physically sterilized through a filter membrane with a diameter of 0.22 μm, and is divided into sterilized centrifuge tubes and stored in a -20℃ freezer.

[0035] 3. MIC determination experiment:

[0036] (1) Inoculate Escherichia coli BW 25113 and Staphylococcus aureus ATCC 29213 in a centrifuge tube containing 4 mL of MH broth, and place it in a 37℃ shaking bed at 180 rpm for 4 h, then take out the centrifuge tube;

[0037] (2) Dilute the incubated bacteria to 100 times with MH broth, about 106 CFU / mL, standby;

[0038] (3) Take a sterile 96-well plate, add 180 μL of MH broth medium to the first well, and add 100 μL of MH broth medium to the second to twelfth wells;

[0039] (4) Add 20 μL of lime essential oil or 20 μL of silver nitrate (640 μg / mL) to the first well, mix well, and then transfer 100 μL to the second well, and so on, and discard 100 μL from the tenth well;

[0040] (5) Add 100 μL of the diluted bacterial solution to the first to eleventh wells, and add 200 μL of MH broth to the twelfth well;

[0041] (6) The final concentration of lime essential oil in the first to tenth wells of the 96-well plate is 5%, 2.5%, 1.25%, 0.625%, 0.3125%, 0.156%, 0.078%, 0.039%, 0.0195%, and 0.00975%, respectively, the eleventh well is the growth control, and the twelfth well is the blank control;

[0042] (7) The final concentration of silver nitrate in the first to tenth wells of the 96-well plate is 32, 16, 8, 4, 2, 1, 0.5, 0.25, 0.125, and 0.0625 μg / mL, respectively, the eleventh well is the growth control, and the twelfth well is the blank control;

[0043] (8) Set up three rows of repeats for each drug;

[0044] (9) Place the inoculated 96-well plate in a 37°C incubator for 16-18 h, and read the results.

[0045] The results are shown in Table 1, the MIC value of lime essential oil for BW 25113 is >5%, and the MIC value of silver nitrate is 2 μg / mL; the MIC value of lime essential oil for ATCC29213 is >5%, and the MIC value of silver nitrate is 2 μg / mL.

[0046] Table 1 MIC of lime essential oil and silver nitrate against Escherichia coli and Staphylococcus aureus

[0047]

[0048] Example 2 Evaluation of the combined bactericidal effect of lime essential oil and silver nitrate on Escherichia coli BW 25113

[0049] 1. Experimental materials:

[0050] (1) Test: Cool the high-pressure sterilized LB broth, PBS buffer, and ultrapure water for standby use.

[0051] (2) Lime essential oil: Guangzhou Yuyuan Essential Oil Co., Ltd.; Silver nitrate: Macklin brand, MW 169.87

[0052] Test strain: Escherichia coli wild strain BW 25113 (laboratory preservation).

[0053] 2. Preparation before the test:

[0054] (1) The wild strain of Escherichia coli BW 25113 was subcultured on agar medium and cultured to an appropriate size.

[0055] (2) 0.1 g of silver nitrate powder was weighed on a precision balance in a centrifuge tube, 10 mL of sterile water was added for dissolution, the concentration of silver nitrate was 10 mg / mL, vortex mixing was performed, and filtration was performed. The filtrate was physically sterilized through a filter membrane with a diameter of 0.22 μm, and was divided into sterilized centrifuge tubes and stored in a -20°C refrigerator.

[0056] 3. Bactericidal experiment:

[0057] (1) Inoculate Escherichia coli BW 25113 single colony in a centrifuge tube containing 20 mL of LB broth, and place it in a 37°C shaking bed at 180 rpm for 3 h. Then take out the centrifuge tube.

[0058] (2) Centrifuge the cultured bacterial solution at 5500 rpm for 3 min, resuspend with an equal volume of PBS buffer and dilute 10 times, and the bacterial amount is 10 7 CFU / mL, ready for use.

[0059] (3) The system used in this experiment is 1 mL, and the bacterial solution is the bacterial solution in (2):

[0060] A) Control group: 0.7 mL PBS + 0.2 mL PBS + 0.1 mL bacterial solution;

[0061] B) Single drug group: 0.7 mL PBS + 0.1 mL A / B drug + 0.1 mL PBS + 0.1 mL bacterial solution;

[0062] C) Combined group: 0.7 mL bacterial solution + 0.1 mL A drug + 0.1 mL B drug + 0.1 mL bacterial solution.

[0063] (4) This test sets up 9 groups:

[0064] E. coli BW25113: ① control group; ② single lemon essential oil group: 1 / 2 MIC (2.5%) lemon essential oil; ③ single lemon essential oil group: 1 / 4 MIC (1.25%) lemon essential oil; ④ single silver nitrate group: 0.25 μg / mL silver nitrate; ⑤ single silver nitrate group: 0.5 μg / mL silver nitrate; ⑥ combination group: 1 / 4 MIC lemon essential oil + 0.25 μg / mL silver nitrate; ⑦ combination group: 1 / 4 MIC lemon essential oil + 0.5 μg / mL silver nitrate; ⑧ combination group: 1 / 2 MIC lemon essential oil + 0.25 μg / mL silver nitrate; ⑨ combination group: 1 / 2 MIC lemon essential oil + 0.5 μg / mL silver nitrate;

[0065] (5) After the system is prepared, it is cultured at 37°C for 1 h on a 180 rpm shaker.

[0066] (6) At 1 h, 100 μL of bacterial solution is taken from each tube and added to a 2 ml centrifuge tube containing 900 μL of PBS buffer for ten-fold gradient dilution. After dilution, 25 μL is taken and dropped on LB agar medium, which is incubated in a 37°C incubator for 16-18 h for colony counting. The experimental results are statistically analyzed after three biological repetitions.

[0067] The results are shown in Table 2 and Figure 1 As shown in Table 2 and

[0068] Table 2 Killing effect of lemon essential oil and silver nitrate combination on E. coli wild strain BW25113

[0069]

[0070] Example 3 Evaluation of the combined killing effect of lemon essential oil and silver nitrate on Staphylococcus aureus ATCC 29213

[0071] 1. Experimental materials:

[0072] (1) Test: LB broth, PBS buffer and ultrapure water sterilized by high pressure are cooled for standby.

[0073] (2) Lemon essential oil: Guangzhou Wenyuan Essential Oil Co., Ltd.; Silver nitrate: brand of Macklin, MW 169.87

[0074] Test strain: Staphylococcus aureus ATCC 29213 (laboratory preservation).

[0075] 2. Preparation before test:

[0076] (1) Staphylococcus aureus ATCC 29213 was subcultured on agar medium and cultured to an appropriate size.

[0077] (2) 0.1 g of silver nitrate powder was weighed on a precision balance in a centrifuge tube, 10 mL of sterile water was added for dissolution, the silver nitrate concentration was 10 mg / mL, vortex mixing was performed, and filtration was performed. The filtrate was physically sterilized through a filter membrane with a diameter of 0.22 μm, and was divided into sterilized centrifuge tubes and stored in a -20°C refrigerator.

[0078] 3. Bactericidal test:

[0079] (1) Staphylococcus aureus ATCC 29213 single colony was inoculated in a centrifuge tube containing 20 mL of LB broth, and was placed in a 37°C shaker at 180 rpm for 3 h. The centrifuge tube was removed.

[0080] (2) The cultured bacterial solution was centrifuged at 5500 rpm for 3 min, and was resuspended with PBS buffer at the same volume and diluted 10 times. The bacterial amount was 10 7 CFU / mL, ready for use.

[0081] (3) The system used in this experiment was 1 mL, and the bacterial solution was the bacterial solution in (2):

[0082] A) Control group: 0.7 mL PBS + 0.2 mL PBS + 0.1 mL bacterial solution;

[0083] B) Single drug group: 0.7 mL PBS + 0.1 mL A / B drug + 0.1 mL PBS + 0.1 mL bacterial solution;

[0084] C) Combined group: 0.7 mL bacterial solution + 0.1 mL A drug + 0.1 mL B drug + 0.1 mL bacterial solution.

[0085] (4) This test sets up 9 groups:

[0086] Staphylococcus aureus ATCC 29213: ① control group; ② single lemon essential oil group: 1 / 2 MIC (2.5%) lemon essential oil; ③ single lemon essential oil group: 1 / 4 MIC (1.25%) lemon essential oil; ④ single silver nitrate group: 0.25 μg / mL silver nitrate; ⑤ single silver nitrate group: 0.5 μg / mL silver nitrate; ⑥ combination group: 1 / 4 MIC lemon essential oil + 0.25 μg / mL silver nitrate; ⑦ combination group: 1 / 4 MIC lemon essential oil + 0.5 μg / mL silver nitrate; ⑧ combination group: 1 / 2 MIC lemon essential oil + 0.25 μg / mL silver nitrate; ⑨ combination group: 1 / 2 MIC lemon essential oil + 0.5 μg / mL silver nitrate;

[0087] (5) After the system is prepared, it is cultured at 37°C for 1 h on a 180 rpm shaker.

[0088] (6) At 1 h, 100 μL of bacterial solution is taken from each tube and added to a 2 ml centrifuge tube containing 900 μL of PBS buffer for ten-fold gradient dilution. After dilution, 25 μL is taken and dropped on LB agar medium, which is incubated in a 37°C incubator for 16-18 h for colony counting. The experimental results are statistically analyzed after three biological repeats.

[0089] The results are shown in Table 3 and Figure 2 As shown in Table 3 and

[0090] Table 3 Killing effect of lemon essential oil and silver nitrate combination on Staphylococcus aureus quality control strain ATCC 29213

[0091]

[0092]

[0093] Example 4 Evaluation of the combined killing effect of lemon essential oil and silver nitrate on multi-drug resistant Escherichia coli NP109

[0094] 1. Experimental materials:

[0095] (1) Test: LB broth, PBS buffer and ultrapure water sterilized by high pressure were cooled for standby use.

[0096] (2) Lime essential oil: Guangzhou Yuyuan Essential Oil Co., Ltd.

[0097] Silver nitrate: MacLaren brand, MW 169.87

[0098] Test strain: Escherichia coli multi-drug resistant bacteria NP109 (laboratory preservation).

[0099] 2. Preparation before the test:

[0100] (1) Subculture Escherichia coli multi-drug resistant bacteria NP109 on agar medium and culture to the appropriate size.

[0101] (2) Weigh 0.1 g of silver nitrate powder in a centrifuge tube, add 10 mL of sterile water for dissolution, the concentration of silver nitrate is 10 mg / mL, vortex to mix and filter. The filtrate is physically sterilized through a filter membrane with a diameter of 0.22 μm, and is divided into sterile centrifuge tubes and stored in a -20°C freezer.

[0102] 3. Bactericidal experiment:

[0103] (1) Inoculate Escherichia coli multi-drug resistant bacteria NP109 single colony in a centrifuge tube containing 20 mL of LB broth, and place it in a 37°C shaker at 180 rpm for 3 h. Then take out the centrifuge tube.

[0104] (2) Centrifuge the cultured bacterial solution at 5500 rpm for 3 min, resuspend with an equal volume of PBS buffer and dilute 10 times. The bacterial amount is 10 7 CFU / mL, ready for use.

[0105] (3) The system used in this experiment is 1 mL, and the bacterial solution is the bacterial solution in (2):

[0106] A) Control group: 0.7 mL PBS + 0.2 mL PBS + 0.1 mL bacterial solution;

[0107] B) Single drug group: 0.7 mL PBS + 0.1 mL A / B drug + 0.1 mL PBS + 0.1 mL bacterial solution;

[0108] C) Combined group: 0.7 mL bacterial solution + 0.1 mL A drug + 0.1 mL B drug + 0.1 mL bacterial solution.

[0109] (4) This test sets up 9 groups:

[0110] Escherichia coli resistant bacteria NP109: ① control group; ② lime essential oil alone group: 1 / 2MIC (2.5%) lime essential oil; ③ lime essential oil alone group: 1 / 4MIC (1.25%) lime essential oil; ④ silver nitrate alone group: 0.25μg / mL silver nitrate; ⑤ silver nitrate alone group: 0.5μg / mL silver nitrate; ⑥ combined drug group: 1 / 4MIC lime essential oil + 0.25μg / mL silver nitrate; ⑦ combined drug group: 1 / 4MIC lime essential oil + 0.5μg / mL silver nitrate; ⑧ combined drug group: 1 / 2MIC lime essential oil + 0.25μg / mL silver nitrate; ⑨ combined drug group: 1 / 2MIC lime essential oil + 0.5μg / mL silver nitrate;

[0111] (5) After the system is prepared, shake and incubate at 37°C and 180 rpm for 1 h.

[0112] (6) At 1 hour, 100 μL of bacterial solution was aspirated from each tube and added to a 2 ml centrifuge tube containing 900 μL of PBS buffer for ten-fold gradient dilution. After dilution, 25 μL was aspirated and dropped onto LB agar medium. The culture was incubated in a 37°C incubator for 16 to 18 hours, and the colonies were counted. The experimental results were statistically analyzed after three biological replicates.

[0113] The results are shown in Table 4 and Figure 3 As shown in the figure, bacteria grew normally in the blank control group, indicating that the multidrug-resistant Escherichia coli NP109 could grow normally under the experimental conditions; in the single-drug group, the multidrug-resistant Escherichia coli NP109 did not decrease significantly compared with the essential oil group alone, indicating that the essential oil group alone had no obvious inhibitory effect on the multidrug-resistant Escherichia coli NP109; the same results also showed that the use of AgNO3 alone had a significant inhibitory effect on the multidrug-resistant Escherichia coli NP109; the results of the combined group showed that the synergistic effect of lime essential oil and silver nitrate had a significant bactericidal effect on the multidrug-resistant Escherichia coli NP109.

[0114] Table 4 Bactericidal effect of lime essential oil and silver nitrate combined with multidrug-resistant Escherichia coli NP109

[0115]

[0116]

[0117] Example 5 Evaluation of the deodorizing effect of lime essential oil combined with silver nitrate

[0118] 1. Experimental materials:

[0119] (1) Test: Prepare a portable gas detector and a sealed aluminum box for use.

[0120] (2) Lime essential oil: Guangzhou Wuyuan Essential Oil Co., Ltd.; Silver nitrate: McLean brand, MW169.87

[0121] Test simulated farm odor: ammonia water.

[0122] 2. Preparation before the test:

[0123] (1) The portable gas detector was debugged and self-checked.

[0124] 3. Deodorization test:

[0125] (1) The gas detector was turned on and placed in the detection box after debugging, and waited for 3 min to ensure the stability of the instrument.

[0126] (2) The dose of ammonia water used in this experiment was 10 μL, which was dropped on the fixed site of the detection box: control group: 10 μL of ammonia water.

[0127] (3) This test set 9 groups:

[0128] Ammonia water: ① control group; ② single lime essential oil group: 1 / 2 MIC (2.5%) lime essential oil; ③ single lime essential oil group: 1 / 4 MIC (1.25%) lime essential oil; ④ single silver nitrate group: 0.25 μg / mL silver nitrate; ⑤ single silver nitrate group: 0.5 μg / mL silver nitrate; ⑥ combined drug group: 1 / 2 MIC lime essential oil + 0.25 μg / mL silver nitrate; ⑦ combined drug group: 1 / 2 MIC lime essential oil + 0.5 μg / mL silver nitrate; ⑧ combined drug group: 1 / 4 MIC lime essential oil + 0.25 μg / mL silver nitrate; ⑨ combined drug group: 1 / 4 MIC lime essential oil + 0.5 μg / mL silver nitrate;

[0129] (4) After the liquid was dropped, the detection was started, and the detection value was recorded every 3 min;

[0130] (5) Result determination: 0-19 is below the warning line, no alarm; 20-49 is low-level alarm; 50-100 is high-level alarm. (This value directly represents the concentration data value of ammonia gas degradation)

[0131] Table 5 Deodorization effect of lime essential oil and silver nitrate combination on ammonia water

[0132]

[0133] The results are shown in Table 5. The detection value in the blank control group was 100, indicating that the odor detection was stable under the experimental conditions. The results of the single drug group and the combined group showed that the single and combined effects of lime essential oil and silver nitrate on the deodorization of ammonia water were significant, but the combined effect on the deodorization of ammonia water was better.

[0134] Obviously, the above embodiments of the present application are merely for the purpose of clearly illustrating the technical solutions of the present application, and are not intended to limit the specific implementation manners of the present application. Any modification, equivalent replacement and improvement made within the spirit and principle of the present application shall be included in the protection scope of the present application.

Claims

1. An antibacterial deodorizing composition, characterized in that: The antibacterial and deodorizing composition is composed of lime essential oil and silver nitrate; the concentration of lime essential oil in the antibacterial and deodorizing composition is 1%-5%, and the concentration of silver nitrate is 0.25-0.5 μg / mL.

2. Use of the antibacterial and deodorizing composition according to claim 1 in the preparation of antibacterial and deodorizing products.

3. The use according to claim 2, characterized in that The antibacterial targets of the product are Escherichia coli, multidrug-resistant Escherichia coli and / or Staphylococcus aureus.

4. The use according to claim 2, characterized in that The deodorizing object of the product is ammonia gas or ammonia water.

5. The use according to claim 2, characterized in that The product is in the form of a solid, liquid, pump spray or semisolid gel.

6. An antibacterial and deodorizing product, characterized in that: The invention comprises the antibacterial and deodorizing composition according to claim 1.

7. The antibacterial and deodorizing product according to claim 6, characterized in that: The solvent of the antibacterial and deodorizing product is water.

8. The antibacterial and deodorizing product according to claim 7, characterized in that: The concentration of the lime essential oil is 1-5%.

9. A bacteriostatic deodorization method, characterized in that: The antibacterial and deodorizing product according to claim 8 is directly sprayed in the form of a diluted solution.

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