Method for inducing spore germination based on radio frequency treatment and application thereof

Abstract

The invention relates to a method for inducing spore germination based on radio frequency treatment and application of the method in the field of sterilization. According to the method, electrons and ions in spore contents can vibrate to generate friction and heat energy by utilizing electromagnetic waves generated during radio frequency treatment, so that the spores are effectively stimulated to germinate. In addition, on the basis of utilizing radio frequency to induce spore germination, germinated spores can be killed more efficiently under the same sterilization condition. The method can be applied to the field of food processing, and the spore germination and application method which is high in effectiveness, wide in applicability and easy and convenient to operate is provided on the premise that food safety is guaranteed and nutritional ingredients are reserved.

Description

Technical Field

[0001] The present invention belongs to the technical field of food microbiology, and particularly relates to a method for inducing spore germination by radio frequency treatment and its application. Background Art

[0002] Spores are a kind of dormant bodies produced by spore-forming bacteria, and their structures have outstanding heat resistance, drug resistance and radiation resistance in nature. Spores are widely distributed in the environment. When spores sense that the external environment is suitable for growth and reproduction, the spores will transform from the dormant state to vegetative bacteria, and this process is called spore germination. Spore germination involves changes in the internal chemical components of spores and changes in the permeability of the inner membrane. Moreover, this event will also lead to the loss of the stress resistance of spores, making spores easily killed during the sterilization process. CN113201476A discloses a method for improving the spore germination rate of Bacillus amyloliquefaciens and its adaptability to protein culture media. This invention can effectively improve the spore germination rate by optimizing the culture medium. However, it has application limitations and cannot induce the germination of spores in food raw materials. Therefore, studying the process of inducing spore germination is very important for regulating the survival activities of spores, and at the same time has important practical significance in the fields of food processing and biological protection.

[0003] Clostridium perfringens ( Clostridium perfringens ) is a Gram-positive anaerobic spore-forming pathogenic bacterium and is one of the microorganisms that cause the spoilage of vacuum-packed foods. Due to the extremely strong stress resistance of spores, in traditional food processing, some conventional sterilization methods cannot completely eradicate spores. When consumers accidentally eat foods contaminated with Clostridium perfringens, the toxins produced by it will cause human diseases and even endanger life in severe cases. Although spores can be effectively killed under the conditions of ultra-high pressure and high temperature, it will cause the loss of food nutrients and the decline of texture quality. Therefore, "germinate first and then kill" has been developed and designed as a new spore killing strategy. Chinese invention patent CN105962002A discloses a method for gently killing bacterial spores. This invention uses the spore killing strategy of "germinate first and then kill", and quickly induces spore germination to weaken its resistance by adding nutritional germinants and penetrants, and then gently kills the germinated spores. However, the additional germinants and penetrants added may affect the original flavor of the food and may require corresponding adjustments to the food ingredients, which has certain production limitations. In summary, how to efficiently induce the spore germination of Clostridium perfringens is of great significance for implementing the spore killing strategy of "germinate first and then kill" and avoiding the deterioration of food caused by high temperature and high pressure sterilization.

[0004] In addition, the use of mild heat treatment has also been proven to effectively induce spore germination. However, in complex food systems, when heat is transferred from the outside to the material through general heat conduction, a temperature gradient will exist in the material, resulting in uneven heating of the food. Therefore, a single heat treatment cannot efficiently induce spore germination. Radio frequency technology is a technology that causes electrons and ions in a substance to vibrate through the action of an electromagnetic field, generating friction and heat energy. This technology can uniformly heat the material, and it has been applied in fields such as thawing of frozen foods and killing of bacteria. Chinese invention patent CN115804403A discloses a method for killing thermophilic bacteria by high-pressure microfluidic homogenization combined with radio frequency treatment. This patent combines low-temperature storage technology and radio frequency heating technology on the basis of traditional high-pressure homogenization treatment of bacterial liquid, enhancing the bactericidal effect of high-pressure microfluidic homogenization. However, whether radio frequency treatment can induce spore germination has not been reported.

[0005] Based on the above analysis, the present invention provides a new technology for inducing spore germination by radio frequency treatment and using the strategy of "germinate first and then kill" for spore killing, which has great practical application significance and value. Summary of the Invention

[0006] The main object of the present invention is to provide a method for inducing spore germination based on radio frequency treatment and its application, so as to overcome the deficiencies in the prior art.

[0007] To achieve the above invention object, the present invention adopts the following technical solutions: One aspect of the present invention provides a method for inducing spore germination based on radio frequency treatment, including two stages of radio frequency activation treatment and germination treatment of spores to induce spore germination.

[0008] Preferably, the spores are Clostridium perfringens ( Clostridium perfringens ) spores.

[0009] Preferably, the radio frequency activation treatment is a single radio frequency treatment.

[0010] Further, the single radio frequency treatment includes placing the spore suspension at the central position of the treatment chamber of the radio frequency instrument, in direct contact with the lower electrode plate, at a distance of 5-10 cm from the upper electrode plate, and treating for 15-35 min at a radio frequency of 39.7-41.7 MHz and a radio frequency power of 290-300 W.

[0011] Preferably, after radio frequency treatment, it is treated in an ice-water bath for 15-30 min.

[0012] Specifically, the present invention provides a method for inducing spore germination based on radio frequency treatment, including the following steps: S1. Radio frequency activation: After treating the spore suspension with a radio frequency instrument for 15 - 35 min, maintain it in an ice - water bath for 15 - 30 min to obtain activated spores. S2. Spore germination: Incubate the activated spores obtained in step S1 at 30 - 40 °C for 30 - 50 min to obtain germinated spores.

[0013] Preferably, the number of spores in the spore suspension described in step S1 is 10 3 ~10 8 CFU / mL.

[0014] More preferably, the solvent of the spore suspension is a Tris - HCl buffer solution with a pH of 6.9 - 7.1.

[0015] "Germinate first and then kill" has been developed and designed as a new spore killing strategy. Briefly, the core of such a strategy is to weaken the resistance of spores by inducing germination and then kill them under milder sterilization conditions, thereby improving the sterilization efficiency.

[0016] On the other hand, the present invention provides a sterilization method based on radio - frequency - induced spore germination, including the application of the method of inducing spore germination by radio - frequency treatment in the field of sterilization.

[0017] Specifically, in the sterilization method based on radio - frequency - induced spore germination, the obtained germinated spores are further heated at 85 - 95 °C for 10 - 20 min to kill the spores.

[0018] The present invention uses a method based on a single radio frequency to induce spore germination, utilizes the spore killing strategy of "germinate first and then kill", realizes the application of this method in the field of spore killing. On the basis of inducing spore germination by radio - frequency treatment, heat activation is used to kill some of the spores germinated after radio - frequency treatment and further activate the ungerminated spores, thereby further improving the application effect of radio - frequency - induced spore germination in the field of spore killing.

[0019] As another preferred embodiment, the radio - frequency activation method is a combined treatment of radio frequency and heat.

[0020] Further, the combined treatment of radio frequency and heat is specifically to treat with a radio frequency instrument for 15 - 35 min, maintain it in an ice - water bath for 15 - 30 min, and then treat at 75 - 80 °C for 10 - 20 min.

[0021] As one of the purposes of the invention, the present invention also provides an application of a sterilization method based on radio - frequency - induced spore germination, including inducing spore germination by the combined treatment of radio frequency and heat and then killing the spores.

[0022] Specifically, the sterilization method based on radio frequency-induced spore germination includes subjecting the germinated spores treated by the method of inducing spore germination by combining radio frequency and heat treatment to heating at 85-95 °C for 10-20 min, and the spores can be killed.

[0023] As one of the objects of the invention, the invention also provides an application of the sterilization method based on radio frequency-induced spore germination in the field of food processing.

[0024] Compared with the prior art, the invention has at least the following beneficial effects: (1) The invention mainly uses the electromagnetic waves generated during radio frequency treatment to cause the electrons and ions in the spore contents to vibrate, generating friction and heat energy, with outstanding germination efficiency. Compared with the traditional heat shock method, the invention can significantly induce spore germination by radio frequency treatment under lower temperature conditions.

[0025] (2) During the process of inducing germination of the spore suspension by radio frequency treatment, the temperature conditions of all treatment steps are maintained below 95 °C, thereby ensuring that when the method is applied to the food field, the loss of food nutrition and the decline in quality caused by high temperature and high pressure can be avoided as much as possible.

[0026] (3) The method for spore germination provided by the invention does not need to be used in combination with a spore germination agent, nor does it need to change the main formula of the food, and can significantly induce spore germination, avoiding an increase in production costs. Description of the Drawings

[0027] Figure 1 Absorbance (OD 600 ) of the Clostridium perfringens spore suspension in Examples 1-2 and Comparative Examples 1-2 of the present invention.

[0028] Figure 2 Fluorescence intensity of dipicolinic acid (DPA) of the Clostridium perfringens spore suspension in Examples 1-2 and Comparative Examples 1-2 of the present invention.

[0029] Figure 3 Germination amount of the Clostridium perfringens spore suspension in Examples 1-2 and Comparative Examples 1-2 of the present invention.

[0030] Figure 4A Observation result of the Clostridium perfringens spores in Comparative Example 1 of the present invention under a phase contrast microscope.

[0031] Figure 4B Observation result of the Clostridium perfringens spores in Comparative Example 2 of the present invention under a phase contrast microscope.

[0032] Figure 4C Observation result of the Clostridium perfringens spores in Example 1 of the present invention under a phase contrast microscope.

[0033] Figure 4D This is the observation result of Clostridium perfringens spores under a phase contrast microscope in Example 2 of the present invention.

[0034] Figure 5 This is the inactivation amount of Clostridium perfringens spores in the spore suspensions of Examples 4 - 5 and Comparative Examples 3 - 4 of the present invention. Detailed implementation manners

[0035] The technical solutions of the present invention will be described in more detail below in conjunction with several examples and drawings, so that the present invention can be more easily understood by those skilled in the art, and thus the protection scope of the present invention can be more clearly and definitely defined. However, the present invention is not limited to the following examples.

[0036] The raw materials, reagents and experimental methods used in the following examples and comparative examples are as follows: (1) Clostridium perfringens ( Clostridium perfringens ), preservation number NCTC 8238, British Type Culture Collection.

[0037] (2) Thioglycollate Medium (FTG) medium was purchased from Guangdong Huankai Microbial Science and Technology Co., Ltd.

[0038] (3) Duncan Strong (DS) medium was prepared by mixing 15.0 g of peptone, 4.0 g of yeast extract, 10.0 g of disodium hydrogen phosphate, 4.0 g of soluble starch, 1.0 g of sodium thioglycollate and 1000 mL of distilled water evenly, and adjusting the pH value to 7.8 with 1 mol / L sodium hydroxide solution. Peptone was purchased from Qingdao Hi-Tech Industrial Park Haibo Biotechnology Co., Ltd.; yeast extract was purchased from Thermo Fisher Scientific; disodium hydrogen phosphate was purchased from Sangon Biotech (Shanghai) Co., Ltd.; soluble starch was purchased from Sinopharm Chemical Reagent Co., Ltd.; sodium thioglycollate was purchased from Shanghai Yuanye Bio-Technology Co., Ltd.

[0039] (4) Brain Heart Infusion Agar (BHI) medium was purchased from Qingdao Hi-Tech Industrial Park Haibo Biotechnology Co., Ltd.

[0040] (5) Cultivation of Clostridium perfringens: The cryopreserved Clostridium perfringens was inoculated into DS medium with an inoculation amount of 1% (V / V), and anaerobically cultured at 37°C for 24 h to fully activate the bacteria. The activated Clostridium perfringens was inoculated into FTG medium with an inoculation amount of 1% (V / V), and anaerobically cultured at 37°C for 12 h, and subcultured continuously for 3 times to obtain the third-generation bacterial liquid.

[0041] Preparation and purification of Clostridium perfringens spores: Inoculate the third-generation bacterial solution prepared in (5) into DS medium at an inoculation amount of 2% (V / V), and anaerobically culture at 37 °C for 24 h to obtain a spore suspension. Centrifuge the spore suspension at 12,000 g for 6 min at 4 °C to obtain a spore precipitate. Then, wash the precipitate six times repeatedly with sterile water at 4 °C, resuspend the precipitate in sterile water to obtain an initial spore suspension, and use a phase contrast microscope to check the purity of the prepared spore suspension.

[0042] (7)Homogenization treatment of the spore suspension: Then, take the initial spore suspension prepared in (6), centrifuge at 12,000 g for 6 min at 4 °C to obtain a spore precipitate. Resuspend the precipitate in Tris-HCl buffer with a pH of 7, and adjust the initial spore number of the spore suspension to 1×10 8 CFU / mL for subsequent experiments.

[0043] The technical solution of the present invention will be described in detail below through specific examples.

[0044] Example 1 This example provides a method for inducing spore germination based on radio frequency treatment, and the specific steps are as follows: S1. Spore activation: Adjust the initial spore concentration of the Clostridium perfringens spore suspension to 1×10 8 CFU / mL with Tris-HCl buffer with a pH of 7 to prepare an initial spore suspension. Place the initial spore suspension at the center of the treatment chamber of the radio frequency instrument, in direct contact with the lower electrode plate, 8 cm away from the upper electrode plate, and treat the spore suspension at a radio frequency of 40.68 MHz and a radio frequency power of 300 W for 25 min, and then maintain it in an ice-water bath for 16 min to obtain activated spores; S2. Spore germination: Incubate the activated spores obtained in step S1 at 37 °C for 30 min to obtain germinated spores.

[0045] Example 2 This example provides a method for inducing spore germination based on radio frequency treatment, and the specific steps are as follows: S1. Spore activation: Adjust the initial spore concentration of the Clostridium perfringens spore suspension to 1×10 8 CFU / mL with Tris-HCl buffer with a pH of 7 to prepare an initial spore suspension. Place the initial spore suspension at the center of the treatment chamber of the radio frequency instrument, in direct contact with the lower electrode plate, 8 cm away from the upper electrode plate, and treat the spore suspension at a radio frequency of 40.68 MHz and a radio frequency power of 300 W for 25 min, and then maintain it in an ice-water bath for 16 min; then, heat it to 80 °C and treat it for 10 min to obtain activated spores; S2, Spore germination: Incubate the activated spores obtained in step S1 at 37 °C for 30 min to obtain germinated spores.

[0046] Example 3 This example provides a method for inducing spore germination based on radio frequency treatment. The specific steps are as follows: S1, Spore activation: Adjust the initial spore concentration of the Clostridium perfringens spore suspension to 1×10 8 CFU / mL with Tris-HCl buffer at pH 7 to prepare the initial spore suspension. Place the initial spore suspension at the center of the treatment chamber of the radio frequency instrument, in direct contact with the lower electrode plate, 8 cm away from the upper electrode plate, and treat the spore suspension at a radio frequency of 40.68 MHz and a radio frequency power of 300 W for 35 min, then maintain it in an ice-water bath for 16 min to obtain activated spores; S2, Spore germination: Incubate the activated spores obtained in step S1 at 37 °C for 30 min to obtain germinated spores.

[0047] Example 4 This example provides an application of inducing spore germination based on radio frequency treatment. The specific steps are as follows: Heat the germinated spores obtained in Example 1 at 90 °C for 10 min to complete the killing of the germinated spores.

[0048] Example 5 This example provides an application of inducing spore germination based on radio frequency treatment. The specific steps are as follows: Heat the germinated spores obtained in Example 2 at 90 °C for 10 min to complete the killing of the germinated spores.

[0049] Example 6 This example provides an application of inducing spore germination based on radio frequency treatment. The specific steps are as follows: Heat the germinated spores obtained in Example 3 at 90 °C for 10 min to complete the killing of the germinated spores.

[0050] Comparative Example 1 This comparative example provides a method for spore germination. The specific steps are as follows: Adjust the initial spore concentration of the Clostridium perfringens spore suspension to 1×10 8 CFU / mL with Tris-HCl buffer at pH 7 to prepare the initial spore suspension. Incubate the initial spore suspension at 37 °C for 30 min as the germination state of spores in the natural environment.

[0051] Comparative Example 2 This comparative example provides a method for germinating spores, and the specific steps are as follows: S1. Spore activation: Adjust the initial spore concentration of the Clostridium perfringens spore suspension to 1×10 8 CFU / mL with Tris-HCl buffer at pH 7 to prepare the initial spore suspension. After treating the initial spore suspension at 80 °C for 10 min, maintain it in an ice-water bath for 16 min to obtain activated spores; S2. Spore germination: Incubate the spore suspension treated in step S1 at 37 °C for 30 min to obtain germinated spores.

[0052] Comparative Example 3 This comparative example provides a method for killing spores, and the specific steps are as follows: Heat the germinated spores obtained based on Comparative Example 1 at 90 °C for 10 min to complete the killing of the germinated spores.

[0053] Comparative Example 4 This comparative example provides a method for killing spores, and the specific steps are as follows: Heat the germinated spores obtained based on Comparative Example 2 at 90 °C for 10 min to complete the killing of the germinated spores.

[0054] Test Example Test the OD 600 value, DPA fluorescence intensity value, germination amount, and microscopic morphology under a phase contrast microscope of the spores after being treated in Examples 1-2 and Comparative Examples 1-2. At the same time, test the inactivation amount of the spores after being treated in Examples 3-4 and Comparative Examples 3-4. The specific test methods are as follows: (1) Determination of the OD 600 value of spores Take 200 μL of the spore suspension samples after being treated in Examples 1-2 and Comparative Examples 1-2 onto a 96-well plate, and then use a microplate reader to measure the absorbance value at a wavelength of 600 nm. The results are as Figure 1 shown.

[0055] (2) Determination of DPA fluorescence intensity Take 1 mL of the spore suspension samples after being treated in Examples 1-2 and Comparative Examples 1-2, centrifuge at 12000 g for 6 min at 4 °C, and collect the supernatant. Then mix the collected supernatant with an equal volume of terbium chloride hexahydrate reagent with a concentration of 20 μmol / L and a pH of 5.6 evenly, and take 200 μL of the mixed solution onto a 96-well plate. Use a microplate reader to measure the DPA fluorescence intensity at an excitation wavelength of 270 nm and an emission wavelength of 545 nm. The results are as Figure 2 shown.

[0056] (3) Determination of the amount of germinated spores Take 1 mL of the spore suspension sample treated according to Examples 1-2 and Comparative Examples 1-2, place it in 9 mL of 0.9% sodium chloride solution, and dilute it stepwise. Select an appropriate dilution gradient and spread it on a BHI plate. After anaerobic incubation at 37 °C for 18 h, count the colonies to determine the amount of germinated spores. The results are as Figure 3 shown.

[0057] (4) Microscopic morphology observation of spores Take 20 μL of the spore suspension samples of Examples 1-2 and Comparative Examples 1-2 that have not been subjected to final sterilization treatment, spread them evenly on a glass slide, and dry and fix the spores with an alcohol lamp. Then wash the dried glass slide with deionized water and dry the water on the alcohol lamp again. Finally, observe the microscopic morphology of the spores after treatment according to Examples 1-2 and Comparative Examples 1-2 using a phase contrast microscope. The results are as Figure 4A (Comparative Example 1), Figure 4B (Comparative Example 2), Figure 4C (Example 1) and Figure 4D (Example 2) shown.

[0058] (5) Determination of the inactivated amount of spores Take 1 mL of the spore suspension with an initial spore count of 1×10 8 CFU / mL after homogenization treatment. Heat it at 75 °C for 20 min and then cool it to room temperature. Place it in 9 mL of 0.9% sodium chloride solution and dilute it stepwise. Select an appropriate dilution gradient and spread it on a BHI plate. After anaerobic incubation at 37 °C for 18 h, count the colonies to determine the initial spore count. Take another 1 mL of the spore suspension sample treated according to Examples 4-5 and Comparative Examples 3-4, place it in 9 mL of 0.9% sodium chloride solution, and dilute it stepwise. Select an appropriate dilution gradient and spread it on a BHI plate. After anaerobic incubation at 37 °C for 18 h, count the colonies to determine the remaining spore count after the killing treatment. The inactivated amount of spores is expressed as the difference between the initial spore count and the remaining spore count. The results are as Figure 5 shown.

[0059] (6) Data statistical analysis All experiments were repeated three times, and the results were expressed as mean ± standard deviation. The experimental data were analyzed for significant differences using the Tukey procedure in SPSS Statistics software. Different lowercase letters a to d in the figure indicate significant differences in the results between different test groups ( P < 0.05).

[0060] Result analysis The ability of the present invention to induce the germination of Clostridium perfringens spores by radio frequency treatment and the application effects will be specifically described below in conjunction with the accompanying drawings.

[0061] Ungerminated spores have a relatively high optical density because the water content in their cores is reduced and the light transmittance is low. When the spores begin to germinate, the internal water content increases, resulting in a change in their optical properties. This change can be represented by measuring the OD 600 value, because the light scattering by the germinated spores decreases, causing the OD 600 value to decrease. As Figure 1 shown, Comparative Example 1 can be used to represent the initial OD 600 value of the spores under natural conditions. For Comparative Example 2 after conventional heat shock treatment at 80 °C for 10 min, compared with Comparative Example 1, the OD 600 value showed a significant decrease ( P < 0.05). In addition, the results of Example 1 showed that under the condition of only using radio frequency heating, the decrease in OD 600 was significantly lower than that of Comparative Example 1 and Comparative Example 2. This also proves that radio frequency heating can effectively induce the germination of spores, which may be because the heating mechanism of radio frequency is not traditional heat transfer, but frictional heat generated by the vibration of electrons and ions, which can improve the heating efficiency and induce more spores to germinate. The OD 600 value of Example 2 after combined treatment of radio frequency treatment and heat activation showed the greatest degree of decrease, indicating that the method of combining radio frequency and heat treatment achieved the best spore germination effect, and there may be a synergistic effect.

[0062] When the spores are ungerminated, DPA is tightly surrounded inside the core of the spores, and only a relatively low fluorescence intensity can be detected. When the spores germinate, the DPA inside the spore core is released, and the higher the detected DPA fluorescence intensity value, the more spores have germinated. As Figure 2 shown, compared with Comparative Example 1, the DPA fluorescence intensity values of Comparative Example 2, Example 1 and Example 2 all increased significantly ( P < 0.05). This further proves that radio frequency heating can significantly induce the germination of spores. It is worth mentioning that, numerically, the method of combining radio frequency and heat treatment has the highest DPA fluorescence intensity. This may be because during radio frequency treatment, the endogenous heating effect of the electromagnetic field can effectively wake up the dormant spores, causing some spores to release DPA. When further heat activation treatment is carried out, more spores can be further induced to germinate.

[0063] As Figure 3As shown, Comparative Example 1 showed the lowest amount of spore germination. The results of Example 1 and Example 2 indicated that both radio frequency treatment and the combined treatment of radio frequency and heat activation could significantly increase the amount of spore germination, activating more spores. It is worth noting that there was no significant difference in the amount of spore germination between Example 2 and Comparative Example 2. This might be because after the radio frequency treatment in Example 2, some spores had germinated and lost their heat resistance, so inactivation of some spores occurred during the further heat activation treatment. Therefore, further analysis is needed in combination with Figure 5 the results of the inactivation amount.

[0064] As Figure 4A shown, the center of the spores in Comparative Example 1 was bright and transparent, basically in the bright state, indicating that no obvious spore germination occurred. However, as Figure 4B , Figure 4C and Figure 4D shown, the transparency of the centers of the spores in Comparative Example 2, Example 1 and Example 2 decreased, showing a change from the bright state to the dark state. This very intuitively shows that both radio frequency treatment and the combined treatment of radio frequency and heat activation played a role in promoting spore germination.

[0065] As Figure 5 shown, without radio frequency and heat treatment, the spore inactivation amount of Comparative Example 3 was only 0.30 lgCFU / mL. Under the conditions of single heat shock and single radio frequency treatment, Comparative Example 4 and Example 4 showed similar spore inactivation amounts, which were 0.56 lg CFU / mL and 0.49 lg CFU / mL respectively, and there was no significant difference between them ( P >0.05). This indicates that the single radio frequency treatment has achieved the effect of heat shock-induced spore germination and can effectively implement the spore killing strategy of "germinate first and then kill". In addition, after the combined treatment of radio frequency and heat activation, the spore inactivation amount of Example 5 reached 1.33 lg CFU / mL, and the killing efficiency of spores reached more than 95%. This might be because during the radio frequency heating process, the high-frequency electromagnetic field acts on the electrons and ions inside the spores, causing them to vibrate turbulently, generating friction and heat energy loss, and efficiently converting electrical energy into heat energy to uniformly heat the spore suspension. This caused certain germination of the spores. During the further heat activation, some spores germinated after the radio frequency treatment could be killed, and more spores were induced to germinate. Therefore, more DPA was released and OD 600 also decreased more significantly. At the same time, the germination of the spores made the spores lose their heat resistance, resulting in more spores losing their activity during the sterilization process at 90°C. In addition, this method is also applicable to Clostridium perfringens ( Clostridium perfringensIn addition to its effectiveness against Clostridium perfringens spores, it also has the effect of inducing the germination of other spores. Since the effect is similar to that of Clostridium perfringens spores, similar results will not be repeated. In summary, radio frequency treatment can effectively induce the germination of spores and has achieved remarkable results in the application of spore killing that "germinates first and then kills". This provides a new and highly practical spore control method for food storage and the protection of its quality and nutritional components.

[0066] It should be noted that the above are only illustrative embodiments of the present invention, and the purpose is to explain the present invention in detail and is not used to limit the present invention. Although those skilled in the art can still modify the technical solutions described in the foregoing embodiments or perform equivalent replacements on some of the technical features. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principles of the present invention to adapt specific situations or materials to the teachings of the present invention shall be included in the protection scope of the present invention.

Claims

1. A method for inducing spore germination based on radio frequency treatment, characterized in that: The method comprises two stages: radio frequency activation treatment of spores and germination treatment to induce spore germination.

2. The method for inducing spore germination based on radio frequency treatment according to claim 1, characterized in that: The RF activation process is a single RF process.

3. The method for inducing spore germination based on radio frequency treatment according to claim 2, characterized in that: The radio frequency activation treatment includes placing the spore suspension at the center of the radio frequency instrument treatment chamber, in direct contact with the lower electrode plate, 5 to 10 cm away from the upper electrode plate, and treating it for 15 to 35 minutes at a radio frequency of 39.7 to 41.7 MHz and a radio frequency power of 290 to 300 W.

4. The method for inducing spore germination based on radio frequency treatment according to claim 1, characterized in that: The germination treatment includes keeping the temperature at 30-40° C. for 30-50 min.

5. The method for inducing spore germination based on radio frequency treatment according to any one of claims 1 to 4, characterized in that: The spores are Clostridium perfringens ( Clostridium perfringens ) spores.

6. The method for inducing spore germination based on radio frequency treatment according to any one of claims 1 to 4, characterized in that: The following steps are involved: S1. Radiofrequency activation treatment: Use a single radiofrequency to treat the spore suspension for 15 to 35 minutes, and maintain it in an ice water bath for 15 to 30 minutes to obtain activated spores; S2. Germination treatment: The activated spores obtained in step S1 are kept at 30-40° C. for 30-50 min to obtain germinated spores.

7. The method for inducing spore germination based on radio frequency treatment according to claim 6, characterized in that: In S1, the number of spores in the spore suspension is 10 3 ~10 8 CFU / mL, pH is 6.9-7.

1.

8. The method for inducing spore germination based on radio frequency treatment according to claim 6, characterized in that: In S1, the radio frequency activation treatment also includes a combination of radio frequency and heating; The radio frequency and heating combined method comprises maintaining the spore suspension after S1 radio frequency treatment in an ice water bath, heating it to 75-80° C. for 10-20 min, and then subjecting it to S2 treatment to obtain activated spores.

9. A sterilization method based on radio frequency induced spore germination, characterized in that: The germinated spores obtained according to any one of claims 1 to 8 are heated at 85 to 95° C. for 10 to 20 minutes.

10. Use of the sterilization method based on radio frequency induced spore germination according to claim 9 in the field of food processing.

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