CN115520931A - A kind of microbial inactivation method based on UVA-LED - Google Patents

Abstract

The invention discloses a microorganism inactivation method based on UVA-LED, belonging to the field of microorganism inactivation. The sterilization method of the present invention comprises the steps of: the microorganisms are irradiated by a UVA-LED light source in a single or multiple irradiation mode. The method provided by the invention is based on UVA-LED, is more green and safe, and has no reactivation phenomenon of microorganisms; through fractionated irradiation, the microbial inactivation efficiency is remarkably improved.

Description

A kind of microbial inactivation method based on UVA-LED

technical field

The invention relates to the field of microorganism inactivation, in particular to a method for inactivating microorganisms based on UVA-LED.

Background technique

Water pollution is a serious threat to human health. To ensure safety, water disinfection has become a necessary process of water treatment. Recently, researchers have developed a series of water disinfection methods. Unlike chemical germicides, disinfection by ultraviolet (UV) light treatment is easy to use, requires no added chemicals, produces few by-products, and is highly effective. Commonly used UV disinfection systems are low pressure (LP) mercury lamps with monochromatic UV radiation (254nm) and medium pressure (MP) mercury lamps covering a broad spectrum. However, they are costly, short-lived, and contain toxic mercury.

In recent years, ultraviolet light-emitting diodes (UV-LEDs) have attracted widespread attention as a new type of UV light source due to their unique characteristics. Compared with low- and medium-pressure mercury lamps, UV LEDs have lower energy requirements and longer lifetimes, and are more environmentally friendly and safer. In addition, UV-LEDs can emit light at any peak wavelength with a narrow distribution. Short-wave ultraviolet light (UVC, <280nm) has been widely used in water disinfection, such as hospital wastewater and purified water. However, UVC-LEDs are expensive and harmful to humans. Moreover, some microorganisms can be activated by light restoration/dark restoration after UVC treatment.

Contents of the invention

The invention provides a method for inactivating microorganisms based on UVA-LED, which can realize the inactivation of microorganisms in water, has a high inactivation rate, and has no revival phenomenon of microorganisms.

The present invention firstly provides a method for sterilizing microorganisms, comprising the following steps: using a UVA-LED light source to irradiate microorganisms, and the light source irradiation mode is single or divided irradiation.

In the above-mentioned sterilization method, the maximum absorption wavelength of the UVA-LED light source is 365nm;

The irradiation power of the UVA-LED light source is ≥180mW/cm ² .

In the above sterilization method, the number of fractional irradiations is 2; the dose of the first irradiation is greater than that of the second, and the interval between fractional irradiations is 5-30 minutes.

Preferably, the interval between the fractional irradiations is 15 minutes.

In the above sterilization method, the microorganism is at least one of Staphylococcus aureus, Escherichia coli and Listeria monocytogenes.

The microorganisms are microorganisms in water.

The pH value of the water is 4-10, specifically 5-8.

Compared with the prior art, the present invention has the following advantages:

(1) The UVC-LED light source is expensive and harmful to the human body, and microorganisms can be revived through light repair/dark repair; the method provided by the present invention is based on UVA-LED, which is greener and safer, and there is no revival of microorganisms.

(2) The method provided by the present invention is based on UVA-LED, and through fractional irradiation, the efficiency of microorganism inactivation is significantly improved.

Description of drawings

Fig. 1 is the schematic diagram of the steps of single irradiation and fractional irradiation of the present invention;

Fig. 2 is the wavelength spectrogram of 365nm UVA-LED used in the present invention;

Figure 3 is a histogram of the number of inactivated colonies compared with single irradiation and fractional irradiation using Staphylococcus aureus as indicator bacteria;

Fig. 4 is the impact of the pH change of aqueous solution on the number of inactivated colonies by irradiation;

Figure 5 is the impact of fractional irradiation on different bacterial strains and the impact of different intervals on the inactivation of bacterial strains; wherein, (A) in Figure 5 is the impact of fractional irradiation on the inactivation of Escherichia coli; (B) is fractional irradiation The impact on the inactivation of Listeria monocytogenes; (C) is the impact of different time intervals on the inactivation of bacterial strains; (D) is the impact of different intervals on the inactivation of bacterial strains;

Figure 6 is the effect of light restoration and dark restoration on fractionated irradiation inactivated bacterial strains;

Fig. 7 is the comparison of the effect of fractional irradiation on bacterial strain damage; wherein (A) in Fig. 7 is the effect of single irradiation on bacterial strain overflow protein; (B) is the influence of fractional irradiation on bacterial strain overflow protein; (C) is single irradiation The effect of sub-irradiation and fractional irradiation on the MDA content of the strain; (D)-(F) is the effect of fractional irradiation on the cell morphology of the strain observed by scanning electron microscope; (G)-(I) is the effect of fractional irradiation on the strain observed by transmission electron microscope Microscopic influence;

Figure 8 is the inactivation mechanism data of fractionated irradiation in Example 4; wherein, (A) in Figure 8 is a diagram of the interval irradiation sterilization mechanism; (B) is an active oxygen scavenger incubated for 30 minutes in advance to inactivate the interval irradiation The effect on the number of colonies; (C) is the effect of adding active oxygen scavenger without incubation, and direct irradiation on the number of inactivated colonies by interval irradiation; (D) is the change of relative content of active oxygen at different times after the first irradiation; (E) is Changes in the intracellular glutathione (GSH) content of the strains at different times after the first irradiation.

detailed description

The present invention will be further described in detail below in conjunction with specific embodiments, and the given examples are only for clarifying the present invention, not for limiting the scope of the present invention.

The experimental methods in the following examples are conventional methods unless otherwise specified.

Quantitative experiments in the following examples were all set up to repeat the experiments three times, and the results were averaged.

The materials and reagents used in the following examples can be obtained from commercial sources unless otherwise specified.

The preparation method of the LB broth medium in the following examples is as follows: Tryptone (OXOID, U.S.) 10g, Yeast Extract (OXOID, U.S.) 5g, Sodium Chloride (Beijing Solaibao Technology Co., Ltd., China) 10g, Add 15-20 g of agar powder (Beijing Suo Lai Bao Technology Co., Ltd., China) to the solid medium; add double distilled water to 1000 mL.

Staphylococcus aureus ATCC6538, Escherichia coli O157:H7 ATCC35150 and Listeria monocytogenes ATCC10403s used in the following examples were all purchased.

The used experimental material and experimental method of following embodiment are as follows:

(1) The strain cultivation and counting methods are as follows: Staphylococcus aureus ATCC6538, Escherichia coli O157:H7ATCC35150 and Listeria monocytogenes ATCC10403s were resuscitated twice in LB broth medium at 37°C and 220 rpm before use. Count the number of colonies in water using the agar plate method. Briefly, serially diluted with 0.9% saline, and spread on LB agar medium. Plates were incubated at 37°C until colonies were sufficiently distinct to be counted (approximately 24 hours).

(2) Preparation of microbial suspension: the bacterial classification recovered twice in the above (1) was inoculated and serially diluted in 0.9% normal saline, so that the initial concentration of Staphylococcus aureus, Escherichia coli or Listeria monocytogenes was about to 10 ⁸ CFU/mL to obtain a microbial suspension.

(3) Fig. 1 is a schematic diagram of the steps of single irradiation and fractional irradiation of the present invention, single irradiation is continuous light treatment of water samples; fractional irradiation has the same irradiation energy as single irradiation, but the light source is turned off for a period of time in the middle, and then Then perform a second irradiation.

(4) Properties of UVA-LED light source: The UVA-LED light source used in the examples was purchased from Zhongshan Bingyi Electronic Technology Co., Ltd., China.

The wavelength scanning diagram is shown in Figure 2, and the light intensity is the strongest at 365nm, and the detailed properties of the light source are shown in Table 1.

Table 1 Specific properties of UVA light source

Embodiment 1, disinfection and sterilization

The light intensity of the UVA-LED light source is 183mW/cm ² . Microbial suspensions were prepared by dilution with 0.9% saline. 2 mL of microbial suspension was added to a 6-well plate at an initial concentration of 10 ⁸ CFU/mL, and then exposed to a UVA-LED light source. The dose of UVA radiation varies according to the species of bacteria. The time interval of irradiating the water sample in batches is 5min-30min, and the logarithmic value of the number of inactivated colonies indicates the inactivation efficiency.

(1) Comparing the inactivation effect of single irradiation and fractional irradiation on Staphylococcus aureus

Different UVA energies were used to treat the microbial suspension containing Staphylococcus aureus, and the single irradiation was irradiated with a light intensity of 183mW/cm ^{2 ;} time, and then at an interval of 15 minutes, and then irradiated for a certain period of time. The sum of the single irradiation energy and fractional irradiation energy is the same. In the 37.8J/cm ² irradiation group, the fractional irradiation energy was divided into two parts: 27.0J/cm ² and 10.8J/cm ² . In the 43.2J/cm ² irradiation group, the fractional irradiation energy was divided into two parts: 32.4J/cm ² and 10.8J/cm ² . In the 54.0J/cm ² irradiation group, the fractional irradiation energy was divided into two parts: 37.8J/cm ² and 16.2J/cm ² .

The results obtained are shown in AC in FIG. 3 . From AC in FIG. 3 , it can be known that the number of inactivated colonies of Staphylococcus aureus increases gradually with the increase of UVA irradiation dose. And compared with single irradiation, fractional irradiation significantly improves the inactivation efficiency of UVA. For example, under the condition of 43.2J/cm ² single irradiation, the number of inactivated colonies is 1.3log, while under the condition of fractional irradiation, The number of inactivated colonies was 2.4log. The kinetic relationship between the irradiation dose and the number of inactivated colonies is shown in D in Figure 3. Whether it is a single irradiation or fractional irradiation, there is a good linear relationship between the irradiation dose and the number of inactivated colonies.

(2) Using UVA-LED light source with light intensity of 183mW/cm ² to irradiate microbial suspension in different energies, the irradiation energy is 39.6J/cm ² +19.8J/cm ² , 32.4J/cm ² + 16.2J/cm ² and 27.0J/cm ² +10.8J/cm ² , the interval time is 15min; adjust the pH of the Staphylococcus aureus microbial suspension to 5, 6, 7, 8, and explore the inactivation of the strain by changing the pH of the aqueous solution Impact. The results are shown in Figure 4. Within the pH range of 5.0 to 8.0, pH changes did not affect the inactivation of strains by fractional irradiation, indicating that fractional irradiation can be applied to aqueous solutions of different pHs.

(3) Taking Escherichia coli (E.coli) and Listeria monocytogenes (L.monocytogenes) as the research objects, under different irradiation energy conditions, explore the effect of fractional irradiation on the inactivation of different strains. The microbial suspension was irradiated with a UVA-LED light source at a light intensity of 183mW/cm ² in a single and multiple times, and the interval between multiple irradiations was 15min. The total irradiation energy of Escherichia coli was 21.6J/cm ² and 14.4J/cm ² respectively. In the 21.6J/cm ² fractional irradiation group, the irradiation energy was divided into two parts: 14.4J/cm ² and 7.2J/cm ² . In the 14.4J/cm ² fractionated irradiation group, the irradiation energy was divided into two parts: 10.8J/cm ² and 3.6J/cm ² . The total irradiation energy of Listeria monocytogenes was 54.0J/cm ² and 37.8J/cm ² respectively. In the fractionated irradiation group of 54.0J/cm ² , the irradiation energy was divided into two parts: 37.8J/cm ² and 16.2J/cm ² . In the fractionated irradiation group of 37.8J/cm ² , the irradiation energy was divided into two parts: 27.0J/cm ² and 10.8J/cm ² . The results are shown in A and B in Figure 5, whether it is Escherichia coli or Listeria monocytogenes, the fractional irradiation significantly improved the inactivation efficiency of the UVA-LED light source for the strains.

(4) Optimizing the conditions for fractional irradiation:

Taking Staphylococcus aureus as the indicator bacteria, under the condition of total irradiation energy of 43.2J/cm ² , the intervals between two irradiations were 0min, 5min, 15min and 30min respectively. Investigate the effect of interval time on the inactivation of strains. Results As shown in C in Fig. 5, the inactivation efficiency does not increase as the interval time increases, and the number of inactivated colonies is the largest when the interval is 15 minutes.

Secondly, the interval method also has a great influence on the inactivation of the strain. Taking Staphylococcus aureus as the indicator bacteria, under the conditions of irradiation energy of 43.2J/cm ² and interval time of 15min, a single irradiation of 43.2J/cm ² The dose is divided into two different radiation doses, 10.8+32.4 means that the first radiation dose is 10.8J/cm ² , and the second radiation dose is 32.4J/cm ² . 21.6+21.6 means that the first and second irradiation doses are both 21.6J/cm ² . 32.4+10.8 means that the first irradiation dose is 32.4J/cm ² . The second irradiation dose is 10.8J/cm ² .

The effect of different segmentation methods on the inactivation of strains is shown in D in Figure 5. Under the condition that the first irradiation dose is greater than the second irradiation dose, the inactivation effect on the strain is the best.

Embodiment 2, the impact of fractional irradiation on bacterial strain restoration

Treat the microbial suspension containing Staphylococcus aureus with different UVA energies, add 2mL of the microbial suspension to a 6-well plate with an initial concentration of 10 ⁸ CFU/mL, and then expose to UVA-LED light source, the light source intensity is still 183mw /cm ² . The samples were treated by single irradiation and fractional irradiation, the interval time of fractional irradiation was 15min, 54J/cm ² and 72J/cm ² were used to treat the microbial suspension. In the fractionated irradiation group of 54.0J/cm ² , the irradiation energy was divided into two parts: 37.8J/cm ² and 16.2J/cm ² . In the 72.0J/cm ² fractionated irradiation group, the irradiation energy was divided into two parts: 43.2J/cm ² and 28.8J/cm ² . After the irradiation was stopped, 0.1 mL of the irradiated microbial suspension was taken to count (Nt), and 1.5 mL of the irradiated suspension was transferred to a sterile transparent test tube for the photoreactivation experiment of Staphylococcus aureus. For Staphylococcus aureus dark repair experiments, transfer 1.5 mL of the irradiated microbial suspension into sterile aluminum foil-wrapped transparent test tubes. Since the irradiated bacteria are affected by the temperature, these aluminum foil-wrapped and unwrapped transparent test tubes containing 1.5mL of the irradiated bacteria solution were placed in a constant temperature shaker (25°C, 60rpm, Shanghai Zhichu desktop full-temperature shaking The incubator shaker ZQTY-90V) was used for the resurrection experiment. There are 2 fluorescent tubes inside the constant temperature shaker, after incubation for 0.5h, 1h, 2h, 3h, and 5h, samples were taken and plated for counting (N1). The photoreactivation efficiency of the bacterial solution is evaluated by the percentage of resurrection, and the calculation method is as follows, N0 represents the number of colonies (CFU/mL) in the stock solution before inactivation, Nt represents the number of colonies in the sample after inactivation, and N1 represents the number of colonies in the sample after inactivation. The number of colonies after treatment for a certain period of time under light or dark conditions.

Percent resurrection (%)=(Log ₁₀ N ₁ -Log ₁₀ N _t )/(Log ₁₀ N ₀ -Log ₁₀ N _t )

Light repair/dark repair is one of the main factors affecting the application of UVC, so we explored the effect of fractional irradiation on the light repair/dark repair of strains, the results are shown in Figure 6, the effect of fractional irradiation on the light repair and Dark repair has no effect, and shows a sustained inactivation effect on the strain in the later stage; among them, in Figure 6 (A) is 54J/cm ² light repair; (B) is 72J/cm ² light repair; (C) is 54J /cm ² dark repair; (D) is 72J/cm ² dark repair.

Embodiment 3, fractional irradiation damages the bacterial strain

2 mL of microbial suspension was added to a 6-well plate at an initial concentration of 10 ⁸ CFU/mL, and then exposed to a UVA-LED light source with an intensity of 183 mw/cm ² . The microbial suspension containing Staphylococcus aureus was treated with 43.2J/cm ² and 72.0J/cm ² in single irradiation and fractional irradiation respectively, and the interval of fractional irradiation was 15min. In the 43.2J/cm ² fractionated irradiation group, the irradiation energy was divided into two parts: 32.4J/cm and 10.8J/cm ² . In the 72.0J/cm ² fractionated irradiation group, the irradiation energy was divided into two parts: 43.2J/cm ² and 28.8J/cm ² . After the irradiation, the supernatant was collected by centrifugation, and the protein content was determined. Exploring the damage effect of fractional irradiation on bacterial strains from the microstructure. Proteins are an important building block of bacteria, and when bacteria are damaged, intracellular proteins are released. As shown in A and B in Figure 7, after fractional irradiation, the amount of intracellular protein release was greater than that of single irradiation. It shows that the fractional irradiation has a greater damage effect on the strain and is more conducive to the inactivation of the strain.

2 mL of microbial suspension was added to a 6-well plate at an initial concentration of 10 ⁸ CFU/mL, and then exposed to a UVA-LED light source with an intensity of 183 mw/cm ² . The microbial suspension containing Staphylococcus aureus was treated with 72.0J/cm ² single irradiation and fractional irradiation respectively, and the interval time between fractional irradiation was 15min. In the 72.0J/cm ² fractionated irradiation group, the irradiation energy was divided into two parts: 43.2J/cm ² and 28.8J/cm ² . After the irradiation, the supernatant was collected by centrifugation, and the content of malondialdehyde was determined. Malondialdehyde (MDA), as a cytotoxic substance, represents the degree of lipid peroxidation of cell membrane. MDA content was determined by TBA (thiobarbituric acid) method. As shown in C in Figure 7, compared with single irradiation, the MDA content of the strain increased more after fractional irradiation, indicating that the strain suffered more serious oxidative damage.

2 mL of microbial suspension was added to a 6-well plate at an initial concentration of 10 ⁸ CFU/mL, and then exposed to a UVA-LED light source with an intensity of 183 mw/cm ² . The microbial suspension containing Staphylococcus aureus was treated with 43.2J/cm ² single irradiation and fractional irradiation respectively, and the interval time between fractional irradiation was 15min. In the 43.2J/cm ² fractionated irradiation group, the irradiation energy was divided into two parts: 32.4J/cm ² and 10.8J/cm ² . After the irradiation, the precipitate was centrifuged and used for scanning electron microscope and transmission electron microscope observation. The samples of the control group were not subjected to irradiation treatment (that is, the 10 ⁸ CFU/mL microbial suspension was directly fixed with electron microscope fixative, and then used for electron microscope observation treatment) . Scanning electron microscope observes the impact of fractional irradiation on bacterial strain morphology, as shown in the scanning electron micrograph in Figure 7 (see DF in Figure 7, wherein D is the control group, E is the sample after single irradiation, and F is the sample after fractional irradiation). It was shown that the Staphylococcus aureus cells in the control group were intact and spherical, while after UVA treatment, the surface of the Staphylococcus aureus cells began to wrinkle. Cells in the fractionated irradiation group had more debris on their surface. Transmission electron microscope has observed identical result, as shown in Fig. 7 transmission electron microscope figure (seeing GI among Fig. 7, and wherein G is control group, and H is the sample after single irradiation, and I is the sample after fractional irradiation), the control group's Staphylococcus aureus cells are in good condition, and the intracellular environment, cell membrane and cell wall are also good. In contrast, the cytoplasm of S. aureus in the single irradiation group was lost. In addition, the S. aureus cells in the fractionated irradiation group were incomplete and lysed.

Embodiment 4, the inactivation mechanism of fractionated irradiation

We hypothesized that it was due to the interaction of some active substances with S. aureus cells during the interval, increasing the sensitivity of S. aureus cells to UVA, which eventually led to the strain being more easily inactivated (A in Fig. 8).

2 mL of microbial suspension was added to a 6-well plate at an initial concentration of 10 ⁸ CFU/mL, and then exposed to a UVA-LED light source with an intensity of 183 mw/cm ² . The microbial suspension containing Staphylococcus aureus was treated with 43.2J/cm ² single irradiation and fractional irradiation respectively, and the interval time between fractional irradiation was 15min. In the 43.2J/cm ² fractional irradiation group, the irradiation energy was divided into two parts, 32.4J/cm ² and 10.8J/cm ² , and the inactivation mechanism was explored under this condition.

Detecting the role of reactive oxygen species in fractionated irradiation using free radical scavengers, mannitol scavenging OH, mannitol concentration 0.5M (Beijing Solaibao Technology Co., Ltd., China, CAS: 69-65-8), peroxidation Catalase was used to remove H ₂ O ₂ , and the concentration of catalase was 1 mg/mL (Beijing Suolaibao Technology Co., Ltd., China, CAS: 9001-05-2). In order to completely dissolve the scavenger in water and penetrate into the cells, the scavenger and Staphylococcus aureus cells were incubated with the Staphylococcus aureus cells in the dark for 30 min in a constant temperature shaker (25°C, 80rpm) before light treatment. After the incubation, perform single irradiation or fractional irradiation, the irradiation energy is ^43.2J / ^{cm 2} . After the end of the irradiation, the plates were counted to determine the influence of free radicals on the fractionated irradiation. As shown in B in Figure 8, compared with the control group, when mannitol scavenged OH, the survival rate of Staphylococcus aureus increased significantly, and its value was even higher than that of the single-irradiation group, indicating that OH was exposed to important role in. Furthermore, no significant difference was observed for strain inactivation in the presence of catalase, suggesting that hydrogen peroxide plays little role in the strain inactivation process. Interestingly, when the strain was not pre-incubated with the scavenger, direct single irradiation or fractional irradiation (irradiation energy was 43.2J/cm ² , fractional irradiation group, irradiation energy was divided into 32.4J/cm ² and 10.8J/cm 2 cm ² with an interval of 15 min.), there was no difference between the mannitol group and the control group (C in Figure 8). This result suggests that intracellular ·OH is the key to water disinfection, rather than aqueous ·OH.

Taking Staphylococcus aureus as indicator bacteria, the irradiation energy was 43.2J/cm ² , and the fractional irradiation group was divided into two parts: 32.4J/cm ² and 10.8J/cm ² , with an interval of 15 minutes. Under fractional irradiation conditions, the changes of ROS and GSH with time were measured after the first irradiation. The fluorescent probe DCFH-DA method was used to measure ROS, and the colorimetric method was used to measure GSH (purchased from: Nanjing Jiancheng Bioengineering Institute). As shown in D and E in Fig. 8, the fluorescence intensity decreased with increasing time interval, indicating that the ROS in the water samples decreased. In contrast, GSH content increased with time. Glutathione is a free radical scavenger and cell protector. Therefore, increased glutathione levels can reduce cell damage. The reason why the long time interval is unfavorable to the inactivated strains by fractional irradiation may be due to the gradual decrease of intracellular ROS and the gradual increase of GSH content.

Claims (7)

1. A method for sterilizing microorganisms, comprising the steps of: using a UVA-LED light source to irradiate microorganisms, and the light source irradiation mode is a single or fractional irradiation. 2. The sterilization method according to claim 1, characterized in that: the maximum absorption wavelength of the UVA-LED light source is 365nm; The irradiation power of the UVA-LED light source is ≥180mW/cm ² . 3. The sterilization method according to claim 1 or 2, characterized in that: the number of fractional irradiations is 2 times; the first irradiation dose is greater than the second, and the interval between fractional irradiations is 5-5. 30min. 4. The sterilization method according to claim 3, characterized in that: the interval time between the fractional irradiations is 15 minutes. 5. The sterilization method according to any one of claims 1-4, characterized in that: the microorganism is at least one of Staphylococcus aureus, Escherichia coli and Listeria monocytogenes. 6. The sterilization method according to any one of claims 1-5, characterized in that: the microorganisms are microorganisms in water. 7. The sterilization method according to claim 6, characterized in that: the pH value of the water is 4-10.

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