Method for killing spores

Abstract

The invention provides a method for killing spores, and belongs to the technical field of disinfection and sterilization. The spore suspension is subjected to pulsed intense light treatment, and the parameters of the pulsed intense light treatment are as follows: the emission energy is 160-380J, the flashing frequency is 0-60, and the distance from a light source is 0-20cm. According to the method disclosed by the invention, a large amount of free radicals with strong oxidizability can be generated in a short time by specifically limiting parameters of the pulse intense light, and the free radicals have an inactivation effect on spores, so that the effect of killing the spores is improved. Data of the embodiment shows that whether free radicals can be generated in the pulsed light treatment or not is judged by measuring the concentrations of two free radicals (hydroxyl free radicals and superoxide anions) with the strongest oxidation activity in the water body subjected to the pulsed light treatment; and indirectly judging the inactivation effect of the free radicals on the spores by comparing the inactivation quantity of the spores in the intensive pulse light treatment group with the inactivation quantity of the spores in the intensive pulse light treatment group after the free radical scavenger (sodium thiosulfate) is added.

Description

Technical Field

[0001] The present invention relates to the technical field of disinfection and sterilization, and in particular to a method for killing spores. Background Art

[0002] Currently, spores have become a major challenge in the fields of food, medicine, and medical devices due to their strong heat resistance and drug resistance. Although traditional sterilization methods (such as high temperature and high pressure, chemical disinfectants, etc.) are effective, they have problems such as high energy consumption, harm to the environment, and corrosiveness to equipment and materials. Therefore, exploring a new, fast, efficient and environmentally friendly method to kill spores has become a research focus.

[0003] Pulsed light technology is a technology that uses high-intensity, short-duration pulsed light to process materials or substances. Pulsed light equipment discharges a xenon lamp under high voltage, and can release high-intensity broad-spectrum pulsed light (200-1100nm) in a short period of time, including ultraviolet light, visible light and part of infrared light, without causing chemical residues. Free radicals are atoms or molecules containing unpaired electrons, which have high reactivity. Their generation process mainly includes two mechanisms: photochemical reaction and redox reaction. Under the irradiation of high-energy light, water molecules or oxygen molecules in the air can be excited to generate strong oxidizing free radicals, among which hydroxyl radicals (·OH), superoxide anions (O2 - ) have the strongest oxidative activity. These free radicals are extremely chemically active and can quickly oxidize and destroy the cell walls, cell membranes and important internal molecules of microorganisms, further accelerating the death of microorganisms.

[0004] In the related art, the use of strong pulse light to inactivate spores has the problem of poor effect. Summary of the invention

[0005] In view of this, the object of the present invention is to provide a method for killing spores. The method of the present invention can improve the effect of killing spores.

[0006] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0007] The present invention provides a method for killing spores, comprising the following steps:

[0008] The spore suspension is subjected to pulsed strong light treatment, wherein the parameters of the pulsed strong light treatment include: emission energy of 160 to 380 J, flash times of 0 to 60 times, and a distance from the light source of 0 to 20 cm.

[0009] Preferably, the frequency of the pulsed intense light treatment is 1 Hz.

[0010] Preferably, the emission energy is 215-325J.

[0011] Preferably, the distance to the light source is 5 to 10 cm.

[0012] Preferably, the number of flashing lights is 50 times.

[0013] Preferably, the parameters of the pulsed strong light treatment include: emission energy of 215 J, number of flashes of 50 times, and distance from the light source of 10 cm.

[0014] Preferably, the concentration of the spore suspension is 10 7 CFU / mL.

[0015] Preferably, the spores in the spore suspension are Bacillus cereus spores.

[0016] The invention provides a method for killing spores, comprising the following steps: subjecting a spore suspension to pulsed strong light treatment, wherein the parameters of the pulsed strong light treatment include: emission energy of 160 to 380 J, flash times of 0 to 60 times, and a distance from the light source of 0 to 20 cm.

[0017] Compared with the prior art, the present invention has the following beneficial effects:

[0018] The present invention can generate a large number of free radicals with strong oxidizing properties in a short time by specifically limiting the parameters of the pulsed strong light. These free radicals have an inactivating effect on spores, thereby improving the effect of killing spores.

[0019] The data in the examples show that the present invention determines whether the pulsed strong light treatment can generate free radicals by measuring the concentrations of the two free radicals with the strongest oxidative activity (hydroxyl radicals and superoxide anions) in the water body after the pulsed strong light treatment; and indirectly determines the inactivation effect of free radicals on spores by comparing the number of spores inactivated in the pulsed strong light treatment group and the pulsed strong light treatment group after adding a free radical scavenger (sodium thiosulfate); and by adjusting the pulsed strong light parameters, the inactivation effect on spores is further improved. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 It is a schematic diagram of the structure of the pulsed strong light device of the present invention, wherein 1 is a control module, 2 is a light box, 3 is a germicidal lamp tube, 4 is a quartz window, 5 is a sterilization chamber, 6 is a support table that can be moved up and down, 7 is an electric push rod, 8 is a flat plate, and 9 is a bellows. DETAILED DESCRIPTION

[0021] The present invention provides a method for killing spores, comprising the following steps:

[0022] The spore suspension is subjected to pulsed strong light treatment, wherein the parameters of the pulsed strong light treatment include: emission energy of 160 to 380 J, flash times of 0 to 60 times, and a distance from the light source of 0 to 20 cm.

[0023] In the present invention, the frequency of the pulsed strong light treatment is preferably 1 Hz.

[0024] In the present invention, the emission energy is preferably 215-325 J, specifically 160, 215, 270, 325 or 380 J.

[0025] In the present invention, the distance to the light source is preferably 5 to 10 cm, specifically 5, 7.5, 10, 12.5 or 15 cm.

[0026] In the present invention, the number of flashing lights is preferably 50 times, and specifically can be 10, 20, 30, 40, 50 or 60 times.

[0027] In the present invention, the pulsed intense light treatment preferably uses a xenon lamp.

[0028] In a specific embodiment of the present invention, the parameters of the pulsed strong light treatment include: emission energy of 215 J, number of flashes of 50 times, distance from the light source of 10 cm, and frequency of 1 Hz.

[0029] In the present invention, the concentration of the spore suspension is preferably 10 7 CFU / mL.

[0030] In the present invention, the spores in the spore suspension are preferably Bacillus cereus spores. The present invention has no particular limitation on the source of the Bacillus cereus spores and the method for preparing the spore suspension, and the spores can be prepared using sources and methods well known to those skilled in the art.

[0031] Figure 1 The structure diagram of the pulsed strong light device of the present invention is shown in FIG. 1, wherein 1 is a control module, 2 is a light box, 3 is a germicidal lamp, 4 is a quartz window, 5 is a sterilization chamber, 6 is a support table that can be moved up and down, 7 is an electric push rod, 8 is a flat plate, and 9 is a bellows. Figure 1 The spores are killed in the apparatus shown.

[0032] The technical solutions in the present invention will be described clearly and completely below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0033] Example 1

[0034] exist Figure 1 The experiments were carried out in the pulsed light apparatus shown, using a xenon lamp.

[0035] Determination of free radical concentration in sample solution after pulsed light treatment:

[0036] Take 3mL of ultrapure water in a sterile culture dish with a diameter of 6mm. Place the sterile culture dish in the center of the sterilizer. Operate the control module and randomly set the emission energy to 215J, the number of flashes to 50 times, the frequency to 1Hz, and the distance from the light source to 10cm. After the flash ends, measure the concentration of free radicals generated in the ultrapure water.

[0037] Determination of hydroxyl radical concentration in sample solution after pulsed light treatment:

[0038] Hydroxyl radicals can convert terephthalic acid (TA) into 2-hydroxyterephthalic acid (HTA). HTA has fluorescent properties and has an emission wavelength of 425 nm under an excitation wavelength of 310 nm.

[0039] Determination of superoxide anion concentration in sample solution after pulsed light treatment:

[0040] Superoxide anions can oxidize hydroxylamine hydrochloride to generate nitrite. Under the action of p-aminobenzenesulfonic acid and α-naphthylamine, nitrite generates a pink azo compound. The product has a characteristic absorption peak at 530nm. The content of superoxide anions can be quantitatively detected based on the change in absorbance.

[0041] Spore culture

[0042] B.cereusATCC 14579 was purchased from Qingdao Haibo Biotechnology Co., Ltd., Shandong Province. The strain was inoculated into nutrient broth medium (Hope Bio-Technology Co., Ltd., Qingdao, Shandong, China) and cultured for 14 hours. 100L of the cultured bacteria were evenly spread on manganese sulfate nutrient agar medium (Hope Bio-Technology Co., Ltd., Qingdao, Shandong, China) and placed in a 37°C incubator for 5 days. After that, the plate was taken out and placed at room temperature for 3 days. The spore purity was observed by spore staining. When the purity was >95%, the spores were scraped with sterile water, centrifuged 5 times, and resuspended with sterile water (10 8 CFU / mL) and stored at 4°C for future use.

[0043] Inactivation effect of pulsed intense light on Bacillus cereus spores:

[0044] The original spore suspension was diluted to a concentration of 10 with sterile water. 7 CFU / mL was used for the experiment. Take 3mL with a concentration of 10 7CFU / mL of spore suspension in a sterile petri dish with a diameter of 6mm. Place the sterile petri dish in the center of the sterilizer. Operate the control module and randomly set the emission energy to 215J, the number of flashes to 50 times, the frequency to 1Hz, and the distance from the light source to 10cm. After the flash ends, dilute the spore suspension to a suitable gradient, take 100mL of the dilution, evenly spread it on the plate counting agar medium, and incubate at 37℃ for 12h before counting.

[0045] After adding free radical scavengers and removing free radicals, the inactivation effect of pulsed light on Bacillus cereus spores:

[0046] The original spore suspension was diluted to a concentration of 10 with a 0.5% sodium thiosulfate solution. 7 CFU / mL was used for the experiment. The operation method was the same as above.

[0047] Experimental Results

[0048] Table 1 Test results of hydroxyl radical and superoxide anion concentration in the solution after pulsed strong light treatment. The blank control in Table 1 is not treated in any way. 3 mL of ultrapure water is added to the sterile culture dish, and then the concentration of free radicals is measured. It can be seen that after short-term irradiation with pulsed strong light, the oxygen molecules in the water are stimulated to produce trace hydroxyl radicals and superoxide anions. These free radicals may damage the spore structure, thereby improving the inactivation effect of pulsed strong light on spores.

[0049] Table 1 Hydroxyl radical and superoxide anion concentrations in the solution after pulsed light treatment

[0050] Different treatment Hydroxyl radical concentration (μM) Superoxide anion concentration (μM) Blank control 0.04±0.01 0.00±0.00 Pulse light treatment 1.12±0.08 4.21±0.04

[0051] Table 2 shows the effect data of pulsed strong light killing spores after adding free radical scavengers. It can be seen that after pulsed strong light treatment, the spore inactivation amount is 2.97±0.06log(CFU / mL), while after adding free radical scavengers, the inactivation amount is significantly reduced to 1.96±0.13log(CFU / mL). In summary, pulsed strong light can quickly induce the generation of free radicals, and the generated free radicals have the ability to kill spores.

[0052] Table 2 Effect of pulsed light killing spores after adding free radical scavengers Unit: log (CFU / mL)

[0053] Different treatment Number of spores killed Pulse light treatment 3.39±0.03 After adding the scavenger, pulsed light treatment 1.96±0.13

[0054] Example 2

[0055] The same as Example 1, the only difference is that the parameters of the pulsed strong light are different. The effects of different pulsed strong light parameters on killing spores are shown in Tables 3 to 5. It can be seen that the present invention uses a single factor experiment to select the optimal parameters. First, the emission energy intensity is determined. When the energy is 215J, the inactivation quantity is significantly increased compared to when the energy is 160J, and then with the increase of energy, the inactivation effect is not significantly improved, and considering the energy consumption problem in actual use, 215J is selected as the optimal emission energy; then the optimal irradiation distance is determined. When the distance increases from 15cm to 10cm, the inactivation effect is significantly improved, and when the distance increases from 10cm to 5cm, the inactivation effect is not significantly improved, and considering the actual application, 10cm is determined as the optimal irradiation distance; finally, the number of flashes is determined. When the number of flashes increases from 10 times to 50 times in sequence, the inactivation effect is significantly improved. When the number of flashes is 60 times, the inactivation effect is not significant compared with 50 times, therefore, 50 times is selected as the best number of flashes. The optimal experimental parameters were finally determined to be an emission energy of 215 J, an irradiation distance of 10 cm, and 50 flashes.

[0056] Table 3 Killing effect of different emission energies on spores Unit: log (CFU / mL)

[0057] Emission energy / J Flashing times / times Frequency / Hz Distance from light source / cm Number of spores killed 160 40 1 10 <![CDATA[2.43±0.11 c ]]> 215 40 1 10 <![CDATA[3.46±0.15 b ]]> 270 40 1 10 <![CDATA[3.64±0.08 ab ]]> 325 40 1 10 <![CDATA[3.74±0.06 ab ]]> 380 40 1 10 <![CDATA[3.97±0.20 a ]]>

[0058] Note: Different lowercase letters indicate significant differences among groups (p<0.05).

[0059] Table 4 Killing effect of different irradiation distances on spores Unit: log (CFU / mL)

[0060] Emission energy / J Flashing times / times Frequency / Hz Distance from light source / cm Number of spores killed 215 40 1 5 <![CDATA[3.84±0.11 a ]]> 215 40 1 7.5 <![CDATA[3.81±0.07 a ]]> 215 40 1 10 <![CDATA[3.66±0.03 a ]]> 215 40 1 12.5 <![CDATA[3.11±0.09 b ]]> 215 40 1 15 <![CDATA[2.71±0.08 c ]]>

[0061] Note: Different lowercase letters indicate significant differences among groups (p<0.05).

[0062] Table 5 The killing effect of different flash times on spores Unit: log (CFU / mL)

[0063]

[0064]

[0065] Note: Different lowercase letters indicate significant differences among groups (p<0.05).

[0066] In summary, the present invention explores a method for killing spores, which has the advantages of high efficiency and green environmental protection, and achieves the purpose of sterilization while avoiding concerns about chemical residues.

[0067] The above description is only a preferred embodiment of the present invention and does not limit the present invention in any form. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be regarded as the protection scope of the present invention.

Claims

1. A method for killing spores, characterized in that: The following steps are involved: The spore suspension is subjected to pulsed strong light treatment, wherein the parameters of the pulsed strong light treatment include: emission energy of 160 to 380 J, flash times of 0 to 60 times, and a distance from the light source of 0 to 20 cm.

2. The method according to claim 1, characterized in that The frequency of the pulsed intense light treatment is 1 Hz.

3. The method according to claim 1 or 2, characterized in that: The emission energy is 215-325J.

4. The method according to claim 1 or 2, characterized in that: The distance from the light source is 5 to 10 cm.

5. The method according to claim 1 or 2, characterized in that: The number of flashing lights is 50 times.

6. The method according to claim 1, characterized in that The parameters of the pulsed strong light treatment include: emission energy of 215 J, number of flashes of 50 times, and distance from the light source of 10 cm.

7. The method according to claim 1, characterized in that The concentration of the spore suspension is 10 7 CFU / mL.

8. The method according to claim 1 or 7, characterized in that: The spores in the spore suspension are Bacillus cereus spores.

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