Method for inactivating amebic protozoa and intracellular bacteria thereof

Abstract

The invention discloses a method for inactivating amoeba protozoa and intracellular bacteria thereof. The method comprises the following step: performing excimer ultraviolet irradiation with the wavelength of 222nm on the amoeba protozoa or the intracellular bacteria of the amoeba protozoa to inactivate the amoeba protozoa or the intracellular bacteria of the amoeba protozoa. The method provided by the invention not only can effectively inactivate amebic protozoa and intracellular bacteria thereof, but also has the advantages of mild reaction conditions, higher inactivation efficiency, no secondary pollution, no medicament addition and large-scale application.

Description

Technical Field

[0001] The invention belongs to the technical field of water supply system disinfection, and in particular relates to a method for inactivating amoeba and intracellular bacteria thereof by irradiating 222nm excimer ultraviolet rays. Background Art

[0002] Amoebas (single-cell eukaryotes) are protozoa that live in water, soil, and other habitats around the world. Amoebas have been detected in tap water, hospital water supply systems, swimming pools, cooling towers, and drinking water systems. Many amoebas are pathogenic, and some are even fatal to humans. In addition, amoebas also serve as a shelter for pathogens. Studies have found that free-living amoebas serve as carriers of some pathogens (such as Legionella pneumophila, Mycobacterium avium, viruses, fungi, etc.), and can protect intracellular pathogens from the effects of disinfectants during disinfection. Therefore, traditional disinfection methods are difficult to effectively inactivate pathogens inside amoeba cells in water supply systems.

[0003] Ultraviolet light is generally considered to be an environmentally friendly water treatment technology. Due to its advantages of high sterilization efficiency, simple operation and high safety, ultraviolet light irradiation technology has many applications in the field of water treatment. As for the ultraviolet light source used in water treatment, the commercial market mainly uses 254nm low-pressure mercury lamps.

[0004] Excimers are molecules that were once formed but are fleeting in a series of physical and chemical reactions caused by the excitation of the laser mixed gas. When different mixtures of rare gases and halogen gases are filled in the resonant cavity, lasers of different wavelengths will be generated. Excimer ultraviolet lamps have many advantages over traditional low-pressure ultraviolet lamps. For example, excimer lamps have lower energy consumption, smaller size, no environmental pollution, and more optional bands, which can meet the absorption requirements of different pollutants. However, the current research on the use of excimer ultraviolet lamps in water treatment technology is still insufficient, and it is difficult to provide sufficient scientific basis for future practical engineering applications. In particular, to date, there has been no research report on the use of 222nm excimer ultraviolet lamps to inactivate amoeba and its intracellular bacteria. Summary of the invention

[0005] The present invention aims to solve at least one of the technical problems existing in the prior art to a certain extent. To this end, the present invention provides a method for inactivating amoeba and its intracellular bacteria, which can effectively inactivate amoeba and its intracellular bacteria.

[0006] Therefore, in a first aspect of the present invention, the present invention provides a method for inactivating amoeba, comprising:

[0007] The amoeba was inactivated by irradiation with 222 nm wavelength excimer ultraviolet light.

[0008] When excimer ultraviolet light with a wavelength of 222nm is applied to amoeba, the nucleic acid, protein and cell structure of amoeba will be destroyed, causing irreversible damage and leading to its death. Therefore, excimer ultraviolet light with a wavelength of 222nm has an excellent inactivation effect on amoeba.

[0009] In some embodiments, the radiation dose of the excimer ultraviolet light is 15 mJ / cm 2 ~40mJ / cm 2 .

[0010] In some embodiments, the temperature of the excimer ultraviolet irradiation inactivation is 22°C to 28°C.

[0011] In some embodiments, the amoeba exists in the form of: amoeba trophozoites, amoeba spores, and amoeba cysts.

[0012] In some embodiments, the amoeba is provided in the form of an amoeba solution; the initial concentration of the amoeba solution is 5×10 4 / mL~5×10 5 / mL; the pH value of the amoeba solution is 6.0-7.0.

[0013] In some embodiments, the amoeba is derived from water or soil.

[0014] In a second aspect of the present invention, the present invention provides a method for inactivating intracellular bacteria of amoeba, comprising:

[0015] The amoeba or the intracellular bacteria of the amoeba are inactivated by irradiation with excimer ultraviolet light of 222 nm wavelength.

[0016] When the amoeba or the intracellular bacteria of the amoeba are irradiated with the excimer ultraviolet light of 222nm wavelength, the nucleic acid, protein and cell structure of the amoeba and the intracellular bacteria thereof will be destroyed, causing irreversible damage and inactivating them. Therefore, the present invention can effectively inactivate the intracellular bacteria of the amoeba.

[0017] In some embodiments, the radiation dose of the excimer ultraviolet light is 15 mJ / cm 2 ~40mJ / cm 2 .

[0018] In some embodiments, the temperature of the excimer ultraviolet irradiation inactivation is 22°C to 28°C.

[0019] In some embodiments, the amoeba contains 5 to 15 intracellular bacteria, and the intracellular bacteria include Burkholderia; the intracellular bacteria are provided in the form of an amoeba solution containing intracellular bacteria; and the pH value of the amoeba solution containing intracellular bacteria is 6.0 to 7.0.

[0020] Additional aspects and advantages of the present invention will be given in part in the following description and in part will be obvious from the following description, or will be learned through practice of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] The above and / or additional aspects and advantages of the present invention will become apparent and easily understood from the description of the embodiments in conjunction with the following drawings, in which:

[0022] Figure 1 This is a comparison chart of the inactivation effects of amoeba spores under ultraviolet radiation at wavelengths of 254 nm and 222 nm in Example 1 of the present invention;

[0023] Figure 2 This is a comparison chart of the inactivation effect of intracellular bacteria of amoeba in Example 2 of the present invention under ultraviolet radiation of 254nm and 222nm wavelengths;

[0024] Figure 3 This is a comparison chart of the inactivation effects of bacteria in Example 3 of the present invention under ultraviolet radiation at 254nm and 222nm wavelengths. DETAILED DESCRIPTION

[0025] The embodiments of the present invention are described in detail below. The embodiments described below are exemplary and are only used to explain the present invention, and should not be understood as limiting the present invention.

[0026] It should be noted that the terms "first" and "second" are used for descriptive purposes only and should not be understood as indicating or implying relative importance or implicitly indicating the number of the indicated technical features. Therefore, the features defined as "first" and "second" may explicitly or implicitly include one or more of the features. Further, in the description of the present invention, unless otherwise specified, the meaning of "plurality" is two or more.

[0027] The endpoints and any values ​​of the ranges disclosed in this article are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of each range, the endpoint values ​​of each range and the individual point values, and the individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed in this article.

[0028] In this document, the terms “include” or “comprising” are open expressions, that is, including the contents specified in the present invention but not excluding other contents.

[0029] In a first aspect of an embodiment of the present invention, the present invention provides a method for inactivating amoeba, comprising:

[0030] The amoeba was inactivated by irradiation with 222 nm wavelength excimer ultraviolet light.

[0031] Amoeba, which is widely present in nature, is the pathogen of many diseases. Eliminating amoeba can effectively cut off the disease transmission chain. The inventor found that the use of 222nm wavelength excimer ultraviolet irradiation amoeba can effectively inactivate it. Excimer ultraviolet radiation with a wavelength of 222nm can induce an increase in the level of reactive oxygen, which not only destroys the DNA of amoeba, but also destroys its DNA repair enzyme, thereby inhibiting the reactivation of amoeba, making it impossible for amoeba to repair damaged DNA, and then lose the ability to reproduce and survive; at the same time, excimer ultraviolet radiation with a wavelength of 222nm can also destroy the nucleic acid and protein of amoeba, causing irreversible damage and leading to its death. Therefore, the use of 222nm wavelength excimer ultraviolet irradiation to inactivate amoeba has the advantages of mild reaction conditions, high inactivation efficiency, no secondary pollution, no drug addition, and can be applied on a large scale. Thus, the excimer ultraviolet irradiation of 222nm wavelength amoeba has excellent inactivation effect.

[0032] In some embodiments of the present invention, the radiation dose of the excimer ultraviolet light is 15 mJ / c 2 ~40mJ / cm 2 . Thus, the amoeba can be effectively inactivated.

[0033] As an example, the radiation dose is 15 mJ / cm 2 、17mJ / cm 2 、19mJ / cm 2 、21mJ / cm 2 、23mJ / cm 2 、25mJ / cm 2 、27mJ / cm 2 、29mJ / cm 2 、31mJ / cm 2 、33mJ / cm 2 、35mJ / cm 2 、37mJ / cm 2 、39mJ / cm 2 、40mJ / cm 2 wait.

[0034] In some embodiments of the present invention, the temperature of the excimer ultraviolet irradiation inactivation is 22° C. to 28° C. Thus, the amoeba can be effectively killed.

[0035] As an example, the inactivation temperature is 22°C, 23°C, 24°C, 25°C, 26°C, 27°C, 28°C, etc.

[0036] In some embodiments of the present invention, the existence forms of the amoeba include: amoeba trophozoites, amoeba spores, and amoeba cysts.

[0037] Among the forms of existence of amoeba, amoeba trophozoites are the active and parasitic forms of amoeba, with irregular shapes and sizes that vary according to species; amoeba spores and amoeba cysts are the dormant and infective forms of amoeba, with protective outer walls for resisting adverse environmental conditions, and have strong resistance to the external environment. Spores and cysts can be hatched under suitable conditions to release trophozoites. According to an embodiment of the present invention, amoeba spores can be effectively inactivated after being irradiated with excimer ultraviolet rays of a wavelength of 222nm, and are therefore also applicable to inactivating amoeba trophozoites. Thus, amoeba can be effectively killed.

[0038] In some embodiments of the present invention, the amoeba is provided in the form of an amoeba solution; the initial concentration of the amoeba solution is 5×10 4 / mL~5×10 5 / mL; the pH value of the amoeba solution is 6.0-7.0.

[0039] In some embodiments of the present invention, the amoeba originates from water or soil.

[0040] In a second aspect of the embodiments of the present invention, the present invention provides a method for inactivating intracellular bacteria of amoeba, comprising:

[0041] The amoeba or the intracellular bacteria of the amoeba are inactivated by irradiation with excimer ultraviolet light of 222 nm wavelength.

[0042] The inventors have found that the intracellular bacteria of amoeba can be effectively inactivated by irradiating them with excimer ultraviolet rays of 222nm wavelength. During the irradiation process, amoeba is preferentially irradiated, and the amoeba is first inactivated or killed, and the protective effect on the intracellular bacteria is lost. Then the intracellular bacteria of amoeba continue to be irradiated with ultraviolet rays, and the cell structure of the intracellular bacteria is destroyed by the excimer ultraviolet rays of 222nm wavelength, resulting in cell inactivation or death. Thus, the present invention can effectively kill the intracellular bacteria of amoeba.

[0043] In some embodiments of the present invention, the radiation dose of the excimer ultraviolet light is 15 mJ / cm2 ~40mJ / cm 2 Thus, the intracellular bacteria of amoeba can be effectively inactivated.

[0044] As an example, the radiation dose is 15 mJ / cm 2 、17mJ / cm 2 、19mJ / cm 2 、21mJ / cm 2 、23mJ / cm 2 、25mJ / cm 2 、27mJ / cm 2 、29mJ / cm 2 、31mJ / cm 2 、33mJ / cm 2 、35mJ / cm 2 、37mJ / cm 2 、39mJ / cm 2 、40mJ / cm 2 wait.

[0045] In some embodiments of the present invention, the temperature of the excimer ultraviolet irradiation inactivation is 22° C. to 28° C. Thus, the intracellular bacteria of amoeba can be effectively killed.

[0046] As an example, the inactivation temperature is 22°C, 23°C, 24°C, 25°C, 26°C, 27°C, 28°C, etc.

[0047] In some embodiments of the present invention, the amoeba contains 5 to 15 intracellular bacteria, and the intracellular bacteria include Burkholderia; the intracellular bacteria are provided in the form of an amoeba solution containing intracellular bacteria; and the pH value of the amoeba solution containing intracellular bacteria is 6.0 to 7.0.

[0048] The scheme of the present invention will be explained below in conjunction with the embodiments. It will be appreciated by those skilled in the art that the following embodiments are only used to illustrate the present invention and should not be considered as limiting the scope of the present invention. Where specific techniques or conditions are not indicated in the embodiments, the techniques or conditions described in the literature in this area or the product specifications are used. The reagents or instruments used are not indicated by the manufacturer and are all conventional products that can be obtained commercially.

[0049] Example 1: Inactivation of amoeba spores

[0050] (1) Cultivation of amoeba spores:

[0051] The frozen amoeba spores were mixed with Klebsiella pneumoniae suspension and inoculated on SM / 5 agar medium. The medium components included: 2g D-glucose, 2g yeast extract, 2g Peptone, 0.2 g MgCl 2 , 1.9gKH 2 PO 4 , 1g K 2 HPO 4 After culturing for 5 days at 21°C under light, the amoeba spores on the SM / 5 agar medium were picked up with an inoculating loop into KK2 buffer (containing 2.25 g KH 2 PO 4 and 0.67 g K 2 HPO 4 ) in a solution with a pH of 6.5 and counted using a hemocytometer under a microscope. The initial concentration of amoeba spores in KK2 buffer was 5×10 5 Pieces / mL.

[0052] (2) Inactivation by ultraviolet irradiation:

[0053] The amoeba spore solution was stirred evenly using a magnetic stirrer, and then the amoeba spore solution was placed in a 254nm low-pressure ultraviolet parallel beam instrument (control group) and a 222nm wavelength excimer ultraviolet parallel beam instrument (experimental group) for inactivation test, and the inactivation temperature was 25°C. Samples were taken at different ultraviolet irradiation times (different irradiation times correspond to different ultraviolet radiation doses). Three independent experiments were performed for each ultraviolet radiation dose.

[0054] (3) Statistical analysis

[0055] Inactivation of amoeba spores is defined as the log reduction (N 0 / N), where N 0 and N are the number of surviving amoeba spores before and after irradiation, respectively. The number of amoeba spores was determined using the most probable number (MPN) method, and the statistical results are shown in Tables 1 and Figure 1 At the same time, the inactivation rate (%) of amoeba spores under different ultraviolet radiation doses was statistically analyzed, and the statistical results are shown in Table 2.

[0056] Table 1 Comparison of the inactivation effects of amoeba spores under 254nm and 222nm wavelength ultraviolet radiation

[0057]

[0058]

[0059] Table 2 Inactivation rate of amoeba spores after ultraviolet radiation at 254nm and 222nm wavelengths

[0060]

[0061] The experimental results are as follows Figure 1 As shown by Figure 1 It can be seen that when the radiation dose reaches 20mJ / cm 2 Above and 18mJ / cm 2 When the radiation dose is greater than 20 mJ / cm, the inactivation rate of amoeba spores by ultraviolet rays of 254 nm and 222 nm can reach 4 log, that is, the inactivation rate reaches more than 99.99%. As shown in Table 2, when the 222 nm wavelength ultraviolet rays are used to inactivate amoeba spores, the radiation dose is greater than 20 mJ / cm 2 When the inactivation rate of amoeba spores is close to 100%, it should be noted that the inactivation of amoeba spores requires a higher dose of ultraviolet radiation than the inactivation of amoeba trophozoites and amoeba cysts.

[0062] Therefore, it is shown that the present invention can effectively inactivate amoeba by irradiating amoeba with excimer ultraviolet light of 222nm wavelength.

[0063] Example 2: Inactivation of intracellular bacteria of amoeba

[0064] (1) Cultivation of intracellular bacteria of amoeba:

[0065] Burkholderia agricolaris B1qs70 was inoculated on SM / 5 agar medium at 30°C and cultured for 3 days, and then colonies were collected and suspended in KK2 buffer.

[0066] Take the amoeba spores obtained in Example 1 and culture them in 1×10 5 The amoeba spores were mixed with 200 μL Klebsiella pneumoniae suspension (OD600 = 1.5) and 10 μL Burkholderia suspension (OD600 = 1.5), inoculated on SM / 5 agar medium, and incubated at 21°C for 5 days. The initial concentration of Burkholderia was 5×10 6 CFU / mL, the concentration of amoeba spores is 5×10 5 / mL (equivalent to 10 intracellular bacteria in each amoeba spore).

[0067] Amoeba spores containing bacteria were collected, suspended in a centrifuge tube with KK2 buffer, pH 6.5, and counted on a hemocytometer using a light microscope.

[0068] (2) Inactivation by ultraviolet irradiation:

[0069] The intracellular bacteria solution of amoeba was stirred evenly using a magnetic stirrer, and then the intracellular bacteria solution of amoeba was placed in a 254nm low-pressure ultraviolet parallel beam instrument (control group) and a 222nm wavelength excimer ultraviolet parallel beam instrument (experimental group) for inactivation test, and the inactivation temperature was 25°C. Samples were taken at different ultraviolet irradiation times (different irradiation times correspond to different ultraviolet radiation doses). Three independent experiments were performed for each ultraviolet radiation dose.

[0070] (3) Release of intracellular bacteria:

[0071] In a 2.0 mL centrifuge tube, the intracellular bacteria solution of amoeba was mixed with 0.3 mL of lysis beads (ZR BashinaBead) with diameters of 0.5 mm and 0.1 mm, respectively, and broken on a lysis device (FastPrep-24 5G) at an oscillation speed of 4.5 M / s and continuous intermittent oscillation (oscillation 40 s, rest 3 min) four times. Microscopic observation confirmed that all amoeba spores had been broken and the intracellular bacteria were released.

[0072] (4) Statistical analysis:

[0073] Inactivation of intracellular bacteria was defined as log reduction (N 0 / N), where N 0 and N are the number of surviving intracellular bacteria before and after irradiation, respectively. The released intracellular bacteria were counted using the CFU method, and the statistical results are shown in Table 3 and Figure 2 At the same time, the inactivation rate (%) of intracellular bacteria of amoeba under different ultraviolet radiation doses was statistically analyzed, and the statistical results are shown in Table 4.

[0074] Table 3 Comparison of inactivation effects of intracellular bacteria of amoeba under ultraviolet radiation at 254nm and 222nm wavelengths

[0075]

[0076]

[0077] Table 4 Inactivation rate of intracellular bacteria of amoeba after ultraviolet radiation at 254nm and 222nm wavelengths

[0078]

[0079] The experimental results are as follows Figure 2 As shown by Figure 2It can be seen that the inactivation of intracellular bacteria by ultraviolet light of 254nm wavelength has a tailing phenomenon, indicating that the increase in dose cannot effectively inactivate intracellular bacteria. This is because after the amoeba spores are inactivated, they can still absorb some ultraviolet rays, so only a part of the ultraviolet rays can reach the bacteria in the cells. When ultraviolet light of 222nm wavelength is used for irradiation and inactivation, when the radiation dose is greater than 18mJ / cm 2 When the ultraviolet radiation dose of 222nm wavelength is greater than 20mJ / cm 2 When the concentration of 5% iodine was 0.1447 g / cm2, the inactivation rate of intracellular bacteria of amoeba was close to 100%.

[0080] Therefore, it is shown that the present invention can effectively inactivate the intracellular bacteria of amoeba by irradiating the intracellular bacteria of amoeba with ultraviolet rays of 222nm wavelength.

[0081] Example 3: Inactivation of bacteria

[0082] (1) Inactivation by ultraviolet irradiation:

[0083] The Burkholderia solution of Example 2 was taken, and the bacterial concentration in the bacterial solution was 5×10 6 CFU / mL. Use a magnetic stirrer to stir the bacterial solution evenly, and then place the bacterial solution under a 254nm low-pressure ultraviolet parallel beam instrument (control group) and a 222nm wavelength excimer ultraviolet parallel beam instrument (experimental group) for inactivation test, and the inactivation temperature is 25°C. Samples are taken at different ultraviolet irradiation times (different irradiation times correspond to different ultraviolet radiation doses). Three independent experiments were performed for each ultraviolet radiation dose.

[0084] (2) Statistical analysis:

[0085] Bacterial inactivation was defined as log reduction (N 0 / N), where N 0 and N are the number of surviving bacteria before and after irradiation, respectively. The bacteria were counted using the CFU method. The statistical results are shown in Table 5 and Figure 3 shown.

[0086] Table 5 Comparison of the inactivation effect of bacteria under 254nm and 222nm wavelength ultraviolet radiation

[0087]

[0088] The experimental results are as follows Figure 3 As shown by Figure 3 It can be seen that in the process of inactivating bacteria, because the bacteria exist directly in the environment rather than in the form of intracellular bacteria in the amoeba cells, there is no protection from the amoeba cells. Therefore, when the radiation dose reaches 15mJ / cm2 At this time, both 254nm and 222nm wavelength ultraviolet rays can inactivate bacteria by 4log, that is, the inactivation rate is 99.99%, but the sterilization effect of 254nm wavelength ultraviolet rays is better.

[0089] This shows that irradiating bacteria with 222nm wavelength ultraviolet rays can effectively inactivate bacteria.

[0090] In the description of this specification, the description with reference to the terms "one embodiment", "some embodiments", "example", "specific example", or "some examples" etc. means that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic representations of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described may be combined in any one or more embodiments or examples in a suitable manner. In addition, those skilled in the art may combine and combine the different embodiments or examples described in this specification and the features of the different embodiments or examples, without contradiction.

[0091] Although the embodiments of the present invention have been shown and described above, it is to be understood that the above embodiments are exemplary and are not to be construed as limitations of the present invention. A person skilled in the art may change, modify, replace and vary the above embodiments within the scope of the present invention.

Claims

1. A method for inactivating amoeba, characterized in that: include: The amoeba was inactivated by irradiation with 222 nm wavelength excimer ultraviolet light.

2. The method according to claim 1, characterized in that The radiation dose of the excimer ultraviolet light is 15 mJ / cm 2 ~40mJ / cm 2 .

3. The method according to claim 1 or 2, characterized in that: The temperature for inactivation by ultraviolet irradiation of the excimer is 22°C to 28°C.

4. The method according to claim 1 or 2, characterized in that: The existence forms of the amoeba include: amoeba trophozoites, amoeba spores, and amoeba cysts.

5. The method according to any one of claims 1 to 4, characterized in that: The amoeba is provided in the form of an amoeba solution; The initial concentration of the amoeba solution was 5×10 4 / mL~5×10 5 Pieces / mL; The pH value of the amoeba solution is 6.0-7.

0.

6. The method according to any one of claims 1 to 5, characterized in that: The amoeba originates from water or soil.

7. A method for inactivating intracellular bacteria of amoeba, characterized in that: include: The amoeba or the intracellular bacteria of the amoeba are inactivated by irradiation with excimer ultraviolet light of 222 nm wavelength.

8. The method according to claim 7, characterized in that The radiation dose of the excimer ultraviolet light is 15 mJ / cm 2 ~40mJ / cm 2 .

9. The method according to claim 7 or 8, characterized in that: The temperature for inactivation by ultraviolet irradiation of the excimer is 22°C to 28°C.

10. The method according to any one of claims 7 to 9, characterized in that: The amoeba contains 5 to 15 intracellular bacteria, and the intracellular bacteria include Burkholderia; The intracellular bacteria are provided in the form of an amoeba solution containing the intracellular bacteria; The pH value of the amoeba solution containing intracellular bacteria is 6.0-7.0.

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