CN112655708A - Ultraviolet sterilization synergist (S) -2-hydroxy-4-methyl valeric acid and application thereof - Google Patents
Ultraviolet sterilization synergist (S) -2-hydroxy-4-methyl valeric acid and application thereof Download PDFInfo
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Abstract
The invention belongs to the technical field of ultraviolet sterilization, and discloses an ultraviolet sterilization synergist and a method for sterilizing by combining ultraviolet sterilization synergist with ultraviolet. The research is based on that the A wave band Ultraviolet (UVA) and (S) -2-hydroxy-4-methyl valeric acid have weak sterilization effect (the sterilization amount is 0.5log-1.5log when the single sterilization effect is used), and the combined sterilization effect has obvious synergistic sterilization effect. 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, and particularly relates to an ultraviolet sterilization synergist (S) -2-hydroxy-4-methyl valeric acid and application thereof.
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.
(S) -2-hydroxy-4-methylpentanoic acid has the molecular formula C6H12O3And the molecular mass is 132.158. In order to enhance the ultraviolet sterilization effect, the research of the ultraviolet sterilization effect synergist is an essential step. However, the currently reported organic acids still have little synergistic effect on ultraviolet sterilization, and a new ultraviolet sterilization synergist needs to be supplemented 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 (S) -2-hydroxy-4-methyl valeric acid as an ultraviolet sterilization synergist.
The second purpose of the invention is to provide a method for sterilizing by using (S) -2-hydroxy-4-methyl valeric acid and ultraviolet.
The purpose of the invention is realized by the following technical scheme:
the application of (S) -2-hydroxy-4-methyl valeric acid as an ultraviolet sterilization synergist.
The research of the invention finds that the combination of (S) -2-hydroxy-4-methyl valeric acid and ultraviolet can obviously enhance the ultraviolet sterilization capability, so the compound can be used as a synergist for ultraviolet sterilization.
The invention also provides an ultraviolet sterilization synergist which comprises (S) -2-hydroxy-4-methyl valeric acid.
The invention also provides a method for sterilizing by combining ultraviolet with (S) -2-hydroxy-4-methyl valeric acid, wherein the irradiation time of the ultraviolet is 30min, and the concentration of the (S) -2-hydroxy-4-methyl valeric acid is 1 mM/L.
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 with 1mM/L (S) -2-hydroxy-4-methyl valeric acid, 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) of the sterilization method 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 sterilization effect of Ultraviolet (UVA) in the A wave band and organic acid (S) -2-hydroxy-4-methyl valeric acid is weak (the sterilization amount is 0.5log to 1.5log when the ultraviolet and the organic acid act independently), and the synergistic sterilization effect is obvious after the ultraviolet and the organic acid are combined. 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 (S) -2-hydroxy-4-methyl pentanoic acid is of a source leaf biological brand with the purity of 99%.
The judgment principle is as follows: for example, the bacterial quantity reduced by the experimental group is reduced by 2 log values on the basis of the bacterial quantity reduction of the ultraviolet treatment alone and the organic acid treatment alone, namely the organic acid 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 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;
(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 (S) -2-hydroxy-4-methylpentanoic acid against ATCC25922 strain
1. Experimental materials:
(1) and (3) testing: autoclaved MH broth was cooled for use.
Coli standard strain ATCC25922 (laboratory collection).
2. Preparation work before the test:
(1) 0.1335g of (S) -2-hydroxy-4-methyl pentanoic acid powder is weighed, the solvent is 10mL of pure water, the concentration of (S) -2-hydroxy-4-methyl pentanoic acid is 100mM/L, and the powder is filtered by a filter membrane for standby after dissolution;
(2) the E.coli standard strain ATCC25922 was cultured on MacconyKa agar medium to a suitable size.
3. (S) -2-hydroxy-4-methyl valeric acid bactericidal effect evaluation experiment:
(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) coli after incubation was released 100-fold using MH broth, approximately 106CFU/mL for standby;
(3) taking a sterile 96-well plate, adding 180 mu of LMH broth culture medium into the 1 st well, and adding 100 mu of LMH broth culture medium into the 2 nd to 11 th wells;
(4) add 20. mu.L 100mM/L (S) -2-hydroxy-4-methyl pentanoic acid into column 1, blow and beat uniformly, suck 100. mu.L to well 2, and so on, suck 100. mu.L to well 10 and discard.
(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, and reading the result.
As shown in Table 2, the Mic value of (S) -2-hydroxy-4-methylpentanoic acid is > 1mM/L, the bacteria are not inhibited from growing significantly at the concentration of (S) -2-hydroxy-4-methylpentanoic acid lower than the Mic value, and the concentration of (S) -2-hydroxy-4-methylpentanoic acid of 1mM/L is finally selected as the UV synergist in consideration of the high cost of use of (S) -2-hydroxy-4-methylpentanoic acid at high concentration and the fact that the (S) -2-hydroxy-4-methylpentanoic acid is diluted to be used at low concentration in practical application.
TABLE 2 MIC of (S) -2-hydroxy-4-methylpentanoic acid against ATCC25922 strain and concentrations selected for the experiment
| Experimental strains | (S) -2-hydroxy-4-methylpentanoic acid Mic | Concentration for experiment |
| ATCC 25922 | >1mM/L | 1mM/L |
Example 3 bactericidal effect of (S) -2-hydroxy-4-methylpentanoic acid on ATCC25922 strain after 30 minutes:
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) 0.1335g of (S) -2-hydroxy-4-methyl pentanoic acid powder is weighed, the solvent is 10mL of pure water, the concentration of (S) -2-hydroxy-4-methyl pentanoic acid is 100mM/L, and the powder is filtered by a filter membrane for standby after dissolution;
(2) the E.coli standard strain ATCC25922 was cultured on MacconyKa medium to a suitable size.
3. (S) -2-hydroxy-4-methyl valeric acid bactericidal effect evaluation experiment:
(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) Adding 1mL of bacterial liquid into a six-hole plate, adding 10 mu L of 100mM/L (S) -2-hydroxy-4-methyl valeric acid, and uniformly mixing to obtain a final concentration of 1 mM/L;
(4) setting control, blank control, 1mM/L (S) -2-hydroxy-4-methyl valeric acid treatment 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.
As shown in Table 3, the number of bacteria in the blank control group was not clearly distinguished from the number of bacteria in the group affected with 1mM/L of (S) -2-hydroxy-4-methylpentanoic acid for 30 min; it was demonstrated that 1mM/L of (S) -2-hydroxy-4-methylpentanoic acid did not change the number of bacteria after 30min treatment of ATCC25922 bacteria. It was shown that 1mM/L of (S) -2-hydroxy-4-methylpentanoic acid was weak in bactericidal effect against ATCC25922, so that (S) -2-hydroxy-4-methylpentanoic acid was finally used in combination with UV at a concentration of 1mM/L for 30min as the final condition.
TABLE 3 fungicidal Effect of (S) -2-hydroxy-4-methylpentanoic acid 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) 0.1335g of (S) -2-hydroxy-4-methyl pentanoic acid powder is weighed, the solvent is 10mL of pure water, the concentration of (S) -2-hydroxy-4-methyl pentanoic acid is 100mM/L, and the powder is filtered by a filter membrane for standby after dissolution;
(3) the E.coli standard strain ATCC25922 was cultured on MacconyKa medium to a suitable size.
3. Ultraviolet synergist effect evaluation experiment:
(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 106；
(3) Adding 1mL of bacterial liquid into a six-hole plate, adding 10 mu L of 100mM/L (S) -2-hydroxy-4-methyl pentanoic acid, and uniformly mixing to obtain a final concentration of 1mM/L
(4) Setting a control, a blank control, treating with (S) -1 mM/L) 2-hydroxy-4-methyl valeric acid for shading, placing into an ultraviolet irradiation box, and irradiating ultraviolet for 30min by a combined action group and an ultraviolet irradiation treatment group;
(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 experimental result is subjected to statistical analysis after three biological repetitions.
In this experiment, a growth control, a UV control, a 1mM/L (S) -2-hydroxy-4-methylpentanoic acid control, and a purple plus 1mM/L (S) -2-hydroxy-4-methylpentanoic acid test were set, and the assay was carried out according to the method of example 1.
The results are shown in Table 4, 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 1mM/L (S) -2-hydroxy-4-methyl valeric acid has no significant inhibitory effect on Escherichia coli ATCC 25922; the combined action result of 30min shows that the synergistic effect of ultraviolet rays and 1mM/L (S) -2-hydroxy-4-methyl valeric acid has obvious bactericidal effect on Escherichia coli ATCC 25922.
TABLE 4 Bactericidal Effect of the combination of UV and (S) -2-hydroxy-4-methylpentanoic acid 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 (7)
- The application of (S) -2-hydroxy-4-methyl valeric acid as ultraviolet sterilizing synergist.
- 2. An ultraviolet sterilization synergist is characterized in that the sterilization synergist comprises (S) -2-hydroxy-4-methyl valeric acid.
- 3. A method for sterilizing by combining ultraviolet with (S) -2-hydroxy-4-methyl valeric acid is characterized in that the irradiation time of the ultraviolet is 30min, and the concentration of the (S) -2-hydroxy-4-methyl valeric acid is 1 mM/L.
- 4. The method for sterilization by ultraviolet combination of (S) -2-hydroxy-4-methylpentanoic acid as claimed in claim 3, wherein the ultraviolet intensity is 2.4-3.0mW/cm2。
- 5. The method for sterilization by ultraviolet combination of (S) -2-hydroxy-4-methylpentanoic acid as claimed in claim 4, wherein the ultraviolet irradiation is performed by preheating for 30 min.
- 6. The method of UV-sterilization in combination with (S) -2-hydroxy-4-methylpentanoic acid as claimed in claim 5, comprising the steps of:(1) mixing the bacterial liquid with 1mM/L (S) -2-hydroxy-4-methyl valeric acid, 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.
- 7. The method for sterilizing by ultraviolet combination of (S) -2-hydroxy-4-methyl pentanoic acid organic acid as claimed in claim 6, wherein the final concentration of the bacterial liquid in the step (1) is 106CFU/mL。
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| CN202011429772.9A CN112655708A (en) | 2020-12-09 | 2020-12-09 | Ultraviolet sterilization synergist (S) -2-hydroxy-4-methyl valeric acid and application thereof |
| PCT/CN2021/118335 WO2022121420A1 (en) | 2020-12-09 | 2021-09-14 | Ultraviolet sterilization synergist and sterilization method using same in combination with ultraviolet |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| WO2022121420A1 (en) * | 2020-12-09 | 2022-06-16 | 华南农业大学 | Ultraviolet sterilization synergist and sterilization method using same in combination with ultraviolet |
Citations (1)
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| CN1128621A (en) * | 1994-07-06 | 1996-08-14 | 吴羽化学工业株式会社 | Process for sterilization of food |
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Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1128621A (en) * | 1994-07-06 | 1996-08-14 | 吴羽化学工业株式会社 | Process for sterilization of food |
Non-Patent Citations (5)
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| ACS: "13748-90-8", 《STN REGISTRY》 * |
| M. SAKKO ET AL.: "2-hydroxyisocaproic acid is fungicidal for Candida and Aspergillus species", 《MYCOSES》 * |
| MARJUT ET AL: "Antimicrobial activity and suitability of 2-hydroxyisocaproic acid for the treatment of root canal infections", 《ACTA UNIV. OUL》 * |
| 王罗春等: "《农村饮用水安全保障》", 31 January 2018, 冶金工业出版社 * |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2022121420A1 (en) * | 2020-12-09 | 2022-06-16 | 华南农业大学 | Ultraviolet sterilization synergist and sterilization method using same in combination with ultraviolet |
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Application publication date: 20210416 |