Method for effectively inactivating spores in bacillus using chelating resin

Abstract

The present application is a method for effectively inactivating spores in Bacillus by using chelating resin in the field of sterilization. The method uses chelating resin to inactivate spores in Bacillus. Specifically, chelating resin is used to quickly and effectively inactivate spores in Bacillus cereus, Bacillus safensis, Bacillus albus, and Bacillus subtilis. The chelating resin used has low production cost and can be regenerated and reused, achieving effective inactivation of spores in a simple, low-cost, and environmentally friendly manner.

Description

Technical Field

[0001] This invention relates to the field of sterilization technology, and in particular to a method for effectively inactivating spores in Bacillus subtilis using a novel environmentally friendly chelating resin. Background Technology

[0002] Spores are dormant bacterial forms produced by Bacillus bacteria undergoing severe dehydration under nutrient-deficient conditions. They are widely distributed in the environment. Their unique multi-layered structure (exowall, spore coat, outer membrane, cortex, inner membrane, spore nucleus, etc.) maintains internal water content and restricts the entry of harmful substances, making them highly resistant to various adverse conditions. Furthermore, although spore metabolism is not significant in the dormant state, they can still continuously monitor the nutritional status of their surrounding environment. Therefore, under suitable environmental conditions, spores can germinate and resume growth, and can even revive after dormancy for tens of thousands of years.

[0003] As an essential material prerequisite for human survival and development, food quality and safety directly affect the health and safety of the people. Microbial contamination of food is one of the factors that threaten food quality and safety and cause huge economic losses in the food industry. Among them, spores have become the main source of microbial contamination due to their high resistance and resusceptibility. That is, spores are very likely to exist in the final product during processing and can germinate and grow under certain conditions, eventually leading to food spoilage and even causing foodborne diseases, bringing various problems to food safety and preservation. Therefore, sterilization has become a necessary process in food processing.

[0004] Traditional sterilization methods used in the food industry, such as heat sterilization (pasteurization, high-temperature instantaneous sterilization, etc.), while having advantages such as good sterilization effect and convenient operation, may have adverse consequences due to the potential impact of high temperatures on food quality, leading to changes in food color, off-flavors, and nutrient loss. Furthermore, high-temperature sterilization has disadvantages such as environmental pollution and high energy consumption. Most importantly, while heat sterilization can kill pathogenic bacteria and other harmful microorganisms, it is insufficient to kill highly resistant bacterial spores. Emerging non-heat sterilization technologies, such as ultraviolet radiation and ultra-high pressure technology, can maintain relatively low temperatures during processing, minimizing the impact on the color, aroma, taste, and nutritional components of food. However, they are complex to operate, costly, and highly susceptible to various factors, and their spore inactivation effect is not ideal. In addition, there are chemical sterilization technologies, such as those using nitrite and glutaraldehyde. While their spore inactivation effect is more significant than other technologies, they pose problems such as chemical pollution and toxic chemical residues, making them difficult to apply in the food industry. Therefore, finding a method that is simple to operate, low in processing cost, green and environmentally friendly, and can quickly and effectively inactivate spores has become the focus of research and development in the field of sterilization technology.

[0005] Chelating resins are a class of functional polymer materials with cross-linked polymers as the backbone and special chelating groups attached. The atoms constituting these chelating groups have isolated electron pairs that can connect to the empty d orbitals of metal ions through coordinate bonds, thereby chelating metal ions to form multi-ring coordination complexes. Under appropriate conditions, the chelated metal ions can be released. Based on the polymer used in the backbone, chelating resins can be broadly classified into cross-linked polystyrene, polyacrylic acid, polyvinyl alcohol, and natural polymer materials such as chitin, starch, and cellulose. Because the backbone of chelating resins is a three-dimensional structure, insoluble in acids, alkalis, water, and other organic solvents, they have advantages such as low production cost, continuous regeneration, and environmental friendliness. They also exhibit good separation and enrichment effects on heavy metal ions and are simple to operate. Therefore, in recent years, they have been widely used in heavy metal wastewater treatment, chemical production, and the metallurgical industry. However, their application in the field of sterilization technology for spore inactivation has not yet been reported. Summary of the Invention

[0006] To address the aforementioned problems, the primary objective of this invention is to provide a method for effectively inactivating spores in Bacillus using chelating resin, thereby achieving a novel application of chelating resin in effectively inactivating spores in Bacillus.

[0007] Another object of the present invention is to provide a method for effectively inactivating spores in Bacillus using chelating resin, the method utilizing... MonoPlus TP 208 chelating resin rapidly and effectively inactivates spores of Bacillus cereus, Bacillus saprolegniasis, Bacillus albus, and Bacillus subtilis. The chelating resin used... MonoPlus TP 208 products meet food-grade standards, have low production costs, and can be recycled and reused. This allows for effective inactivation of spores in a green, environmentally friendly, and pollution-free manner with simple operation and low processing costs.

[0008] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0009] A method for effectively inactivating spores in Bacillus using chelating resin, wherein the method uses chelating resin to inactivate spores in Bacillus.

[0010] Specifically, the following steps are included:

[0011] Take a centrifuge tube, add chelating resin, and add the Bacillus suspension using a pipette;

[0012] Place the centrifuge tubes on a shaker and inactivate them at room temperature for 15 minutes at a speed of 290 rpm.

[0013] After inactivation, the chelating resin in the experimental group was filtered out using a sterile filter, and the filtrate was collected into a new centrifuge tube. The filtrate was then transferred into the centrifuge tube using a pipette, centrifuged at 5000 rcf for 5 mins, and the supernatant was removed. The weight of the bacterial sludge in the tube was then measured and analyzed by flow cytometry after staining reaction in the dark for 15 mins.

[0014] The applicant discovered that the functional group of the chelating resin is a sodium-type iminodiacetic acid group (Na-IDA), as shown below:

[0015]

[0016] After processing, a large amount of Na in the functional groups + The release of these substances disrupts the spore structure to some extent, ultimately leading to spore inactivation. Therefore, chelating resins exhibit a high inactivation rate for Bacillus spores.

[0017] The Bacillus species mentioned are any one or any combination of Bacillus cereus, Bacillus safensis, Bacillus albus, and Bacillus subtilis.

[0018] Further, take four 50ml centrifuge tubes, add 10g of chelating resin to each, and use a pipette (2-10ml) to add 20ml (×4) of bacterial suspension of the four strains respectively in a ratio of 1:2 (resin weight: bacterial suspension volume).

[0019] Furthermore, the above-mentioned strains need to be cultured and spores induced, specifically including the following steps:

[0020] Four target bacteria were activated and expanded. Glycerol tubes of the four target bacteria strains were taken from the bacterial bank (storage temperature: -80℃; protective solution formula: 50% glycerol / bacterial solution = 1:1). After being slowly thawed on ice, the bacterial solution was dipped into the inoculation loop and activated by streaking in three zones on prepared BHIS solid medium. The plates were then placed in an incubator at 37℃ for 24 hours for aerobic incubation. After the appearance of single colonies with the corresponding morphology and color for each bacterial strain, this was considered the first generation of activation. Then, several single colonies were picked from each plate and inoculated into prepared BHIS liquid medium. The plates were placed in a shaker at 37℃ and 200 rpm for 24 hours for aerobic incubation. After the bacterial solution became turbid, the expansion was considered complete, and this was considered the second generation of activation.

[0021] To induce spore formation in four target bacterial strains, four bottles of activated second-generation bacterial cultures were anaerobically incubated at 4°C for 24 hours. The cultures were then transferred to centrifuge tubes using a pipette, centrifuged at 7000 rcf for 5 minutes, and the supernatant was discarded to remove the culture medium components. Physiological saline was then added to each tube and vortexed to mix. One to two drops of malachite green dye were added to each centrifuge tube, and the tubes were vortexed again to ensure sufficient dye contact with the bacterial culture. The tubes were then placed in boiling water for 15 minutes to kill most of the vegetative cells and, on the other hand… To ensure the malachite green dye completely penetrates the spores within the bacteria, remove the centrifuge tube and use a pipette to transfer 20 μl of the dye-bacterial complex onto four sterile glass slides. After drying over an alcohol lamp, wash with deionized water to decolorize the malachite green-stained bacteria. Wipe dry with absorbent paper, then add 1-2 drops of safranin dye to each stained area and let stand for 30 seconds. Restain the decolorized bacteria to make the bacteria and spores exhibit different colors, thus more clearly highlighting the spores and facilitating observation.

[0022] Furthermore, it is necessary to determine whether the bacterial counts of the four target bacteria are within the same order of magnitude; otherwise, further dilution should be performed. First, centrifuge the four bacterial suspensions at 7000 rcf for 5 minutes, discard the supernatant to remove culture medium components. This operation avoids the influence of culture medium components on the experimental results and prevents inaccurate results due to spore revival and growth during the experiment. Weigh the four precipitated bacterial sludge samples and dilute each 10-fold with physiological saline. Vortex to mix and prepare bacterial suspensions. Prepare four 2ml centrifuge tubes, add physiological saline to each tube using a pipette, and transfer 10μl of each bacterial suspension into the centrifuge tube. Vortex to mix and use this as the 100-fold dilution group. Perform another 100-fold dilution in the same manner, resulting in a total dilution of 10,000 times. Then, use a flow cytometer (BD Accuri C6) to determine the bacterial count per unit volume.

[0023] The chelating resin is MonoPlus TP 208 chelating resin has iminodiacetic acid as its functional group.

[0024] For chelating resins, to prevent the addition of antifungal Na2CO3 and unpolymerized monomers (such as styrene) during the production process from affecting the test results, pretreatment is necessary. First, wash away small particles and broken resin with deionized water. Then, soak in 2 BV (1 BV = 1 unit resin volume) of 10% NaCl solution for 20 hours. After soaking, rinse thoroughly with 6 BV of deionized water, followed by soaking in 4 BV of 75% ethanol solution for 12 hours to remove air bubbles and allow the resin to fully expand and shrink. Finally, rinse again with 6 BV of deionized water.

[0025] Furthermore, the chelating resin needs to undergo two acid-base cross-regeneration processes before it can be used again. The purpose of this process is to adjust the resin's properties, and the specific steps include the following:

[0026] First, rinse the resin with 40 BV of physiological saline, then wash with 2 BV of deionized water. Next, perform acid regeneration by rinsing the resin with 6-7% HCl at a volume of 2 BV, followed by rinsing with 4 BV of deionized water. Then, perform alkali regeneration by rinsing the resin with 4% NaOH at a volume of 2 BV, followed by rinsing with 2 BV of deionized water. After standing for 19.5 hours, reinject saline by rinsing the resin again with 20 BV of physiological saline. Finally, rinse the resin with deionized water to remove all remaining saline. The regeneration process is now complete.

[0027] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0028] (1) A new application of chelating resin to effectively inactivate spores in Bacillus subtilis;

[0029] (2)Use MonoPlus TP 208 chelating resin rapidly and effectively inactivates spores of Bacillus cereus, Bacillus safensis, Bacillus albus, and Bacillus subtilis, with an inactivation time of 15 minutes and an inactivation rate of over 99%.

[0030] (3) Chelating resin MonoPlus TP 208 products meet food-grade standards, have low production costs, and can be recycled and reused.

[0031] (4) This method is simple to operate and has low processing cost. It can effectively inactivate spores in a green, environmentally friendly and pollution-free manner. Detailed Implementation

[0032] The embodiments of the present invention will be described in detail below with reference to examples. However, those skilled in the art will understand that the following examples are for illustrative purposes only and should not be considered as limiting the scope of the invention. Unless otherwise specified in the examples, conventional conditions or conditions recommended by the manufacturer are followed. Reagents or instruments whose manufacturers are not specified are all commercially available conventional products.

[0033] The chelating resins used in this invention are shown in the table below:

[0034]

[0035] Table 1: MonoPlus TP 208 chelating resin model

[0036] Experiment 1: The spore inactivation effect of chelating resin on four strains of Bacillus.

[0037] 1. Experimental materials.

[0038] 1.1 Experimental instruments: -80℃ refrigerator, vortex mixer, centrifuge, pipette, biosafety cabinet, centrifuge tubes, autoclave, sterile culture dishes, conical flasks, glass rods, inoculation loops, shaker, water bath, microscope, glass slides, BD Accuri C6 flow cytometer.

[0039] 1.2 Test reagents: LIVE / DEAD TM BacLight TM Bacterial Viability Kit: live and dead fluorescent dyes, physiological saline, 75% ethanol solution, BHI pre-mixed powder, yeast extract, L-cysteine, resazurin, hematoxylin, vitamin K1, ultrapure water, deionized water, HCl solution, NaOH solution, 50% glycerol, malachite green dye, safranin dye.

[0040] 1.3 Experimental Materials: MonoPlus TP 208 chelating resin, Bacillus cereus, Bacillus safensis, Bacillus albus, and Bacillus subtilis.

[0041] 2. Experimental methods.

[0042] 2.1 Culture of the strain and induction of spore formation.

[0043] First, prepare 2L of modified BHIS medium. Weigh out 74g of pre-prepared BHI powder, 10g of yeast extract, 1g of L-cysteine, 0.2mg of resazurin, 5μg of hemolysin, and 0.002mg of vitamin K1. Add ultrapure water to bring the volume to 2L. Stir with a glass rod until the solution is clear. Adjust the pH to 6.3-6.8 with HCl or NaOH solution. Autoclave at 121℃ for 20 minutes. After sterilization, let the solution cool to about 55℃ and pour it into four prepared sterile petri dishes to make BHIS solid medium. Divide the remaining solution into four equal portions to make BHIS liquid medium for later use.

[0044] Four target bacterial strains were activated and expanded. Glycerol tubes of the four target bacterial strains were taken from the bacterial bank (storage temperature: -80℃; protective solution formula: 50% glycerol / bacterial solution = 1:1). After slow thawing on ice, the bacterial solution was dipped into the inoculation loop and activated by streaking in three zones on prepared BHIS solid medium. The plates were then placed in an incubator at 37℃ for 24 hours for aerobic incubation. After the appearance of single colonies with the corresponding morphology and color for each bacterial strain, these were considered as the first generation of activation. Then, several single colonies from each plate were picked and inoculated into prepared BHIS liquid medium. The plates were then placed in a shaker at 37℃ and 200 rpm for 24 hours for aerobic incubation. After the bacterial solution became turbid, the expansion was considered complete, and these were considered as the second generation of activation.

[0045] To induce spore formation in four target bacterial strains, four bottles of activated second-generation bacterial cultures were anaerobically incubated at 4°C for 24 hours. Then, 200 μl of each culture was transferred to four 2 ml centrifuge tubes using a pipette (100-1000 μl). The tubes were centrifuged at 7000 rcf for 5 minutes, the supernatant was discarded, and the culture medium components were removed. 200 μl of physiological saline (0.9% NaCl solution) was added to each tube, and the mixture was vortexed. 1-2 drops of malachite green dye were added to each centrifuge tube, and the mixture was vortexed again to ensure sufficient dye contact with the bacterial culture. The tubes were then placed in boiling water for 15 minutes to kill most of the vegetative cells and ensure complete penetration of the malachite green dye into the spores. The centrifuge tubes were then removed, and the contents were pipetteed (2-20 μl) into the spores. l) Transfer 20 μl of the dye-bacterial complex to four sterile glass slides, dry them over an alcohol lamp, wash them with deionized water to decolorize the peacock green-stained cells, wipe them dry with absorbent paper, add 1-2 drops of safranin dye to each stained area, let stand for 30 seconds, and then re-stain the decolorized cells to make the cells and spores show different colors, so that the spores can be more clearly highlighted and easy to observe. After re-staining, wash off the remaining unstained dye with deionized water again, wipe them dry with absorbent paper, and observe the stained areas under a microscope. The results showed that more than 90% of the stained areas of the four strains were peacock green, and most of them were round with a transparent center. This indicates that most of the spores in the activated second-generation bacterial solution have been induced to form.

[0046] 2.2 Pretreatment of chelating resin and bacterial suspension.

[0047] To prevent the addition of antifungal Na2CO3 and unpolymerized monomers (such as styrene) during the production of the chelating resin from affecting the test results, pretreatment is necessary before the test. First, wash away small particles and broken resin with deionized water. Then, soak the resin in 2 BV (1 BV = 1 unit resin volume) of 10% NaCl solution for 20 hours. After soaking, rinse thoroughly with 6 BV of deionized water, followed by soaking in 4 BV of 75% ethanol solution for 12 hours to remove air bubbles and allow the resin to fully expand and shrink. Finally, rinse again with 6 BV of deionized water and set aside for use.

[0048] Since the initial bacterial counts of the four strains were unknown, it was necessary to determine whether the bacterial counts were on the same order of magnitude before the experiment; otherwise, further dilution was required. First, the four bacterial suspensions were centrifuged at 7000 rcf for 5 minutes, the supernatant was discarded, and the culture medium components were removed. This operation avoids the influence of culture medium components on the experimental results and prevents inaccurate results due to spore revival and growth during the experiment. The four precipitated bacterial sludge samples were collected, weighed, and diluted 10-fold with physiological saline. After vortexing, bacterial suspensions were prepared. Four 2ml centrifuge tubes were prepared, and 990μl of physiological saline was added to each tube using a pipette (100-1000μl). 10μl of each bacterial suspension was then transferred to the centrifuge tube and vortexed. This was used as a 100-fold dilution group. This group was then diluted 100-fold again in the same manner, resulting in a total dilution of 10,000 times. The bacterial count was then determined using a flow cytometer (BDAccuri C6). The results showed that the bacterial count in the suspension of Bacillus cereus was 9830 cells / μL; the bacterial count in the suspension of Bacillus saforticus was 9765 cells / μL; the bacterial count in the suspension of Bacillus albus was 9658 cells / μL; and the bacterial count in the suspension of Bacillus subtilis was 9850 cells / μL. This indicates that the initial bacterial counts in the suspensions of the four strains were all on the same order of magnitude, and therefore no further treatment was required.

[0049] 2.3 Inactivation test for spores.

[0050] Take four 50ml centrifuge tubes, add 10g of chelating resin to each, and use a pipette (2-10ml) to add 20ml (×4) of bacterial suspension of the four strains in a 1:2 ratio (resin weight: bacterial suspension volume). This serves as the experimental group. Take another four 50ml centrifuge tubes, add only 20ml (×4) of bacterial suspension of the four strains, without adding chelating resin. This serves as the control group.

[0051] Both the experimental and control groups were placed on a shaker and inactivated at room temperature for 15 minutes at a speed of 290 r / min.

[0052] After inactivation, the chelating resin in the experimental group was filtered out using a 200-mesh sterile filter. The filtrate was collected into new 50ml centrifuge tubes (×4). Using a pipette (100-1000μl), 1ml of the filtrate from both the control and experimental groups was transferred into 2ml centrifuge tubes (×24, 3 replicates). The tubes were centrifuged at 5000rcf for 5 minutes, the supernatant was discarded, and the bacterial sludge in each tube was weighed. Each tube was diluted 10-fold with physiological saline and vortexed to mix. This was used as the 10-fold dilution group. Another 24 2ml centrifuge tubes were prepared. 900μl of physiological saline was added to each tube using a pipette (100-1000μl). Then, 100μl of the bacterial suspension from each of the 10-fold dilution groups was transferred to the corresponding 900μl of physiological saline to create the 100-fold dilution group.

[0053] Take 24 centrifuge tubes (2 ml each), and add 987 μl of physiological saline to each tube using a pipette (100-1000 μl). (LIVE / DEAD) TM BacLight TM Add 1.5 μl each of the two dyes Syto-9 and Pi from the Bacterial Viability Kit to each centrifuge tube. Finally, add 2-10 μl of the bacterial suspension diluted 100 times with a pipette and vortex to mix. After staining in the dark for 15 mins, analyze the results using a flow cytometer.

[0054] 2.4 Data Analysis.

[0055] All experimental data are expressed as (mean ± standard deviation).

[0056] 2.5 Regeneration treatment of chelating resin.

[0057] The chelating resin needs to undergo two acid-base cross-regeneration processes before it can be used again, in order to adjust the resin's properties. First, rinse the resin with 40 BV of physiological saline, then wash it with 2 BV of deionized water. Next, perform acid regeneration by rinsing the resin with 6-7% HCl at a volume of 2 BV, followed by rinsing with 4 BV of deionized water. Then, perform alkali regeneration by rinsing the resin with 4% NaOH at a volume of 2 BV, followed by rinsing with 2 BV of deionized water. After standing for 19.5 hours, reinject saline by rinsing the resin again with 20 BV of physiological saline. Finally, rinse the resin with deionized water to remove any remaining saline. The regeneration process is now complete.

[0058] 3. Results and Discussion.

[0059]

[0060] Table 2: Average bacterial count, activity, and inactivation rate of the control and experimental groups

[0061] The average bacterial count, activity, and inactivation rate of the control and experimental groups are shown in Table 2. As can be seen from Table 2, the initial bacterial counts of the four strains in the control groups were all on the same order of magnitude: Bacillus cereus: 9987±313 cells / μl; Bacillus saforticus: 9883±62 cells / μl; Bacillus albus: 9691±84 cells / μl; and Bacillus subtilis: 9921±64 cells / μl. This result is consistent with the initial bacterial count results of the four strains verified before the experiment.

[0062] For *Bacillus cereus*, the initial average activity of the control group without chelating resin inactivation treatment was 97.61±0.97%, while the average activity of the experimental group after chelating resin inactivation treatment decreased to 0.13±0.01%, with an average inactivation rate of 99.87%. For *Bacillus saforticus*, the initial average activity of the control group without chelating resin inactivation treatment was 97.33±0.79%, while the average activity of the experimental group after chelating resin inactivation treatment decreased to 0.15±0.01%, with an average inactivation rate of 99.85%. For Bacillus albus, the initial average activity of the control group without chelating resin inactivation treatment was 96.88±1.9%, while the average activity of the experimental group after chelating resin inactivation treatment decreased to 0.13±0.02%, with an average inactivation rate of 99.87%. For Bacillus subtilis, the initial average activity of the control group without chelating resin inactivation treatment was 98.19±0.37%, while the average activity of the experimental group after chelating resin inactivation treatment decreased to 0.14±0.01%, with an average inactivation rate of 99.86%.

[0063] In summary, the chelating resin achieved a spore inactivation rate of over 99% for all four bacterial strains. The mechanism of spore inactivation is due to the resin's functional group being a sodium-type iminodiacetic acid group (Na-IDA). After treatment, the large amount of Na in the functional group... + The released chelating resin disrupts the spore structure to some extent, ultimately leading to spore inactivation. This result demonstrates that the chelating resin exhibits a high inactivation rate for spores from the four tested Bacillus strains. Based on its inactivation mechanism, it can be further applied to inactivate spores from most Bacillus strains, particularly pathogenic bacteria. This provides a better solution for food contamination caused by microorganisms in the food industry. Therefore, this method, which can rapidly and effectively inactivate pathogenic Bacillus while ensuring simple operation, low processing costs, and environmental friendliness, is crucial and important for the food industry.

[0064] Furthermore, since chelating resins are currently widely used only in the treatment of heavy metal wastewater, chemical production, and metallurgical industries, there are no reports on their use in the inactivation of spores in the field of sterilization technology. Therefore, this method has opened up a new field of application for chelating resins and has certain application potential and commercial value.

[0065] The above are merely preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for effectively inactivating spores in Bacillus using chelating resin, characterized in that... The method uses chelating resin to inactivate spores in Bacillus; the chelating resin is Lewatit® MonoPlus TP208 chelating resin, and its functional group is iminodiacetic acid; The Bacillus species mentioned are any one or any combination of Bacillus cereus, Bacillus safortus, Bacillus albus, and Bacillus subtilis.

2. The method for effectively inactivating spores in Bacillus using chelating resin according to claim 1, characterized in that... Includes the following steps: Take a centrifuge tube, add chelating resin, and add the Bacillus suspension using a pipette; Place the centrifuge tubes on a shaker and inactivate them at room temperature for 15 min at a speed of 290 r / min.

3. The method for effectively inactivating spores in Bacillus using chelating resin according to claim 1, characterized in that... Take four 50ml centrifuge tubes, add 10g of chelating resin to each, and use a 2-10ml pipette to add 20ml of bacterial suspension of each of the four strains in a ratio of resin weight to bacterial suspension volume of 1:

2.

4. The method for effectively inactivating spores in Bacillus using chelating resin according to claim 3, characterized in that... The above-mentioned strains need to be cultured and spores induced, specifically including the following steps: Four target bacteria were activated and expanded. Glycerol tubes of the four target bacteria were taken from the bacterial bank. The storage temperature of the glycerol tubes was -80℃. The protective solution formula was 50% glycerol / bacterial solution = 1:

1. After slow thawing on ice, the bacterial solution was dipped with an inoculation loop and activated by streaking in three zones on prepared BHIS solid medium. The plates were then placed in an incubator at 37℃ and aerobically cultured for 24 hours. After the appearance of single colonies with the corresponding morphology and color of each bacterial cell, these were considered as the first generation of activation. Then, several single colonies were picked from each plate and inoculated into prepared BHIS liquid medium. The plates were placed in a shaker at 37℃ and 200 rpm and aerobically cultured for 24 hours. After the bacterial solutions became turbid, these were considered as the second generation of activation. To induce spore formation in four target bacterial strains, four bottles of activated second-generation bacterial solutions were anaerobically placed in a refrigerator at 4°C for 24 hours. The solutions were then transferred to centrifuge tubes using a pipette, centrifuged at 7000 rcf for 5 minutes, the supernatant was discarded to remove the culture medium components, and physiological saline was added to each tube and vortexed. One to two drops of malachite green dye were added to each centrifuge tube, and the tubes were vortexed again to ensure sufficient contact between the dye and the bacterial solution. The tubes were then placed in boiling water for 15 minutes. After removing the centrifuge tubes, 20 μl of the dye-bacterial solution complex was transferred to four sterile glass slides using a pipette. The slides were dried over an alcohol lamp and washed with deionized water to decolorize the malachite green-stained bacterial cells. One to two drops of safranin dye were added to each stained area, and the slides were allowed to stand for 30 seconds. The decolorized bacterial cells were then counterstained to produce different colors for the bacterial cells and spores.

5. The method for effectively inactivating spores in Bacillus using chelating resin according to claim 4, characterized in that... To determine whether the bacterial counts of the four target bacteria are on the same order of magnitude, the four bacterial suspensions were first centrifuged at 7000 rcf for 5 minutes. The supernatant was poured off, the culture medium components were removed, and the four precipitated bacterial sludge samples were collected, weighed, and diluted 10-fold with physiological saline. After vortexing and mixing, bacterial suspensions were prepared. Four 2 ml centrifuge tubes were prepared, and physiological saline was added to each tube using a pipette. 10 μl of each bacterial suspension was then transferred to the centrifuge tube and vortexed and mixed. This was used as a 100-fold dilution group. The same method was used to perform another 100-fold dilution, resulting in a total dilution of 10,000 times. The bacterial count was then determined by flow cytometry.

6. The method for effectively inactivating spores in Bacillus using chelating resin according to claim 1, characterized in that... The chelating resin requires pretreatment. First, wash away small particles and broken resin with deionized water, then soak in 2 BV of 10% NaCl solution for 20 hours, where 1 BV = 1 unit resin volume. After soaking, rinse thoroughly with 6 BV of deionized water, then soak in 4 BV of 75% ethanol solution for 12 hours to remove air bubbles and allow the resin to fully expand and contract. Finally, rinse thoroughly again with 6 BV of deionized water.

7. The method for effectively inactivating spores in Bacillus using chelating resin according to claim 6, characterized in that... The chelating resin needs to undergo two acid-base cross-regeneration processes before it can be used again. The specific steps are as follows: First, rinse the resin with 40 BV of physiological saline, then wash with 2 BV of deionized water. Next, perform acid regeneration by rinsing the resin with 6-7% HCl at a volume of 2 BV, followed by rinsing with 4 BV of deionized water. Then, perform alkali regeneration by rinsing the resin with 4% NaOH at a volume of 2 BV, followed by rinsing with 2 BV of deionized water. After standing for 19.5 hours, reinject saline by rinsing the resin again with 20 BV of physiological saline. Finally, rinse the resin with deionized water to remove all remaining saline. The regeneration process is now complete.

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