Application of ultrasonic and plasma activated water to reduction of sublethal injury rate of food-borne pathogenic bacteria

Abstract

The invention provides application of ultrasonic and plasma activated water to reduction of the sublethal injury rate of food-borne pathogenic bacteria, and belongs to the technical field of biology. The invention provides application of ultrasonic and plasma activated water to reduction of sublethal injury rate of food-borne pathogenic bacteria. According to the method, the food-borne pathogenic bacteria are treated by adopting ultrasonic and plasma activated water, so that the sub-lethal damage rate of the food-borne pathogenic bacteria can be remarkably reduced while the food-borne pathogenic bacteria are greatly reduced, the sterilization effect of the food-borne pathogenic bacteria is improved, and potential safety hazards of foods can be better solved.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and particularly relates to the application of ultrasound-assisted plasma-activated water in reducing the sublethal injury rate of food-borne pathogenic bacteria. Background Art

[0002] Foodborne pathogens are microorganisms that can be transmitted through food and may cause food poisoning, poisoning infections and other related diseases, posing a major public health risk. The most effective strategy to prevent foodborne pathogens is to control food contamination and improve food sterilization technology. Escherichia coli (E. coli) is a common foodborne pathogen belonging to the Enterobacteriaceae family.

[0003] Currently, non-thermal sterilization technology is becoming more and more popular because it can sterilize efficiently and maintain the nutritional and sensory quality of food. Plasma-activated water (PAW) is a functional liquid that is mainly produced by discharging on the water surface or underwater through a low-temperature atmospheric pressure plasma system, which contains various antimicrobial compounds. PAW sterilization has the characteristics of high efficiency and broad spectrum, safety and no residue, and simple operation. Among them, reactive oxygen species (ROS) and reactive nitrogen species (RNS) play an important role in PAW sterilization. However, studies have found that a single PAW treatment cannot completely inactivate microorganisms and will cause sublethal damage (SI) to microorganisms.

[0004] SI refers to a state of damage between the survival and death of microorganisms. SI cells can self-repair and regenerate under favorable conditions, thus posing a potential food safety hazard. At present, the microorganisms that can form SI mainly include Salmonella, Escherichia coli, Staphylococcus aureus, Listeria monocytogenes, etc. A large number of studies have shown that non-thermal processing technologies such as pulsed electric fields, high-pressure carbon dioxide, low-temperature plasma, and ultra-high pressure can induce microorganisms to produce SI. When Escherichia coli cells are exposed to slightly acidic electrolyzed water, the sublethal rate increases with the increase in the processing volume. How to provide a method that can control the number of foodborne pathogens and reduce the SI of foodborne pathogens is of great significance for solving food safety hazards. Summary of the invention

[0005] In view of the problems in the prior art, the object of the present invention is to provide an application of ultrasound-assisted plasma-activated water to reduce the sublethal injury rate of foodborne pathogens.

[0006] The objective of the present invention is achieved through the following technical solutions:

[0007] The invention provides an application of ultrasound-assisted plasma activated water to reduce the sublethal injury rate of foodborne pathogenic bacteria, wherein the power of the ultrasound is 200-600W.

[0008] Preferably, the foodborne pathogenic bacteria include Escherichia coli.

[0009] The present invention provides a method for reducing the sublethal injury rate of foodborne pathogenic bacteria, comprising the following steps:

[0010] The object to be treated is mixed with plasma activated water and then subjected to ultrasonic treatment; the power of the ultrasonic treatment is 200 to 600 W; and the time of the ultrasonic treatment is 3 to 10 minutes.

[0011] Preferably, the power of the ultrasonic treatment is 250 W, and the time of the ultrasonic treatment is 10 min.

[0012] Preferably, the frequency of the ultrasonic treatment is 40 kHz.

[0013] Preferably, the method for preparing the plasma-activated water comprises: subjecting sterile water to plasma ionization treatment to obtain plasma-activated water.

[0014] Preferably, 0.18 MPa compressed air is used as the working gas with a flow rate of 20 to 30 L / min, and the plasma generator probe is placed 0.5 to 1 cm below the sterile water surface for plasma ionization treatment.

[0015] Preferably, the plasma ionization treatment time for each 300 mL of sterile water is 5 to 15 minutes.

[0016] Preferably, the plasma ionization treatment time for each 300 mL of sterile water is 10 minutes.

[0017] The present invention provides application of the method described in the above technical solution in improving the sterilization rate of foodborne pathogenic bacteria.

[0018] Beneficial effects of the present invention:

[0019] The present invention provides an application of ultrasound-assisted plasma activated water to reduce the sublethal injury rate of foodborne pathogens. The present invention uses ultrasound-assisted plasma activated water to treat foodborne pathogens. While greatly reducing foodborne pathogens, it can also significantly reduce the occurrence of sublethal injury rates of foodborne pathogens, improve the sterilization effect of foodborne pathogens, and thus be more conducive to solving food safety hazards. The present invention shows through the results of the examples that the use of ultrasound-assisted plasma activated water to treat Escherichia coli can cause a higher degree of rupture of the Escherichia coli cell membrane, increase the potential difference between the inside and outside of the bacterial cell membrane, increase the fluidity of the cell membrane, change the structure of the cell membrane protein, cause severe oxidative stress in the pathogenic bacteria cells, and lead to a decrease in the activity of antioxidant enzymes in the cells, resulting in damage to the antioxidant enzyme system, inducing DNA oxidation in the cells, and ultimately causing irreversible damage and cell death to foodborne pathogens, thereby improving the sterilization rate while reducing the sublethal injury rate of the bacteria. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative labor.

[0021] Figure 1 The following are photos of the instruments and equipment used in the embodiments of the present invention;

[0022] Figure 2 This is an experimental flow chart of a method for reducing the sublethal injury rate of Escherichia coli in an embodiment of the present invention;

[0023] Figure 3 It is a statistical result diagram of the reduction of the number of Escherichia coli bacterial liquids in Examples 1 to 3;

[0024] Figure 4 This is a statistical result diagram of the sublethal damage rate of the Escherichia coli liquid of Examples 1 to 3;

[0025] Figure 5 The results of the reduction of the number of Escherichia coli in Example 1 and Examples 4 to 6 are shown;

[0026] Figure 6 This is a graph showing the sublethal damage rate of Escherichia coli in Example 1 and Examples 4 to 6;

[0027] Figure 7 The results of reducing the number of Escherichia coli at different ultrasonic powers in Example 1 and Examples 13 to 15 are shown;

[0028] Figure 8 This is a graph showing the sublethal damage rate of Escherichia coli at different ultrasonic powers in Example 1 and Examples 13 to 15;

[0029] Fig. 9 This is a diagram showing the effect of ultrasound-assisted plasma-activated water on cell membrane morphology;

[0030] Fig.10 This is the result diagram of the effect of different treatments on the absorbance value of Escherichia coli nucleic acid;

[0031] Fig.11 This is the result diagram of the effect of different treatments on the absorbance value of Escherichia coli protein;

[0032] Fig.12 This is the result diagram of the effect of ultrasound-assisted plasma-activated water on cell membrane permeability;

[0033] Fig.13 This is the result diagram of the effect of ultrasound-assisted plasma-activated water on cell membrane potential;

[0034] Fig.14 The results of the different treatments on the ratio of SFAs / UFAs in the cell membrane are shown in the figure;

[0035] Fig.15 This is the result diagram of the effect of ultrasound-assisted plasma-activated water on the structure of cell membrane proteins;

[0036] Fig.16 This is the result diagram of the effect of ultrasound-assisted plasma-activated water on intracellular ROS levels;

[0037] Fig.17 This is the result of the effect of ultrasound-assisted plasma-activated water on the intracellular GSH content;

[0038] Fig.18 This is the result diagram of the effect of ultrasound-assisted plasma-activated water on the activity of intracellular SOD;

[0039] Fig.19 This is the result diagram of the effect of ultrasound-assisted plasma-activated water on the activity of CAT in cells;

[0040] Fig. 20 This is a graph showing the effect of ultrasound-assisted plasma-activated water on intracellular DNA oxidation. DETAILED DESCRIPTION

[0041] The present invention provides an application of ultrasound synergistic plasma activated water to reduce the sublethal injury rate of foodborne pathogens. In the present invention, the foodborne pathogens include Escherichia coli. In an embodiment of the present invention, taking the Escherichia coli strain ATCC25922 as an example, the effect of ultrasound synergistic plasma activated water on reducing the sublethal injury rate of foodborne pathogens is verified. In the present invention, the power of the ultrasound is 200 to 600 W. As an optional embodiment of the present invention, the power of the ultrasound can be 200, 250, 300, 350, 400, 450, 500, 550 or 600 W.

[0042] The present invention uses ultrasound to coordinate plasma activated water to treat foodborne pathogens. While greatly reducing foodborne pathogens, it can also significantly reduce the occurrence of sublethal injury rate of foodborne pathogens, improve the sterilization effect of foodborne pathogens, and thus be more conducive to solving food safety risks.

[0043] The present invention provides a method for reducing the sublethal injury rate of foodborne pathogenic bacteria, comprising the following steps:

[0044] The object to be treated is mixed with plasma activated water and then subjected to ultrasonic treatment; the power of the ultrasonic treatment is 200 to 600 W; and the time of the ultrasonic treatment is 3 to 10 minutes.

[0045] In the present invention, the preparation method of the plasma activated water may include the following steps: subjecting sterile water to plasma ionization treatment to obtain plasma activated water. The present invention does not specifically limit the preparation method of the sterile water, and any conventional method in the art may be used for treatment. As an optional embodiment of the present invention, the plasma ionization treatment includes: using 0.18MPa compressed air as the working gas, with a flow rate of 20-30L / min, and placing the plasma generator probe 0.5-1cm below the sterile water surface for plasma ionization treatment. As an optional embodiment of the present invention, the plasma generator probe is placed 1.0, 0.9, 0.8, 0.7, 0.6 or 0.5cm below the sterile water surface. As an optional embodiment of the present invention, the time for plasma ionization treatment of every 300mL of sterile water can be 5-15min, or 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15min. After the plasma ionization treatment is completed, plasma activated water is obtained.

[0046] After obtaining the plasma activated water, the present invention mixes the plasma activated water with the object to be treated to obtain a plasma object to be treated mixed system. As an optional embodiment of the present invention, the object to be treated may be a liquid. In the present invention, the volume ratio of the object to be treated to the plasma water activated water may be 1:1. In the embodiment of the present invention, a bacterial suspension is taken as an example to verify the effect of the method on reducing the sublethal injury rate of foodborne pathogens.

[0047] After obtaining the mixed system of the objects to be treated by plasma, the present invention performs ultrasonic treatment on the mixed system of the objects to be treated by plasma. As an optional embodiment of the present invention, the power of the ultrasonic treatment can be 200-600W, or 200, 250, 300, 350, 400, 450, 500, 550 or 600W; the time of the ultrasonic treatment can be 3-10min, or 3, 4, 5, 6, 7, 8, 9 or 10min. As an optional embodiment of the present invention, the frequency of the ultrasonic treatment can be 40kHz.

[0048] The present invention provides the application of the method described in the above technical solution in improving the sterilization rate of foodborne pathogens. The present invention shows through the results of the embodiment that the use of ultrasound and plasma activated water to treat Escherichia coli can cause the Escherichia coli cell membrane to rupture to a higher degree, increase the potential difference between the inside and outside of the bacterial cell membrane, increase the fluidity of the cell membrane, change the structure of the cell membrane protein, and cause severe oxidative stress in the pathogenic bacteria cells, and cause the reduction of the activity of the antioxidant enzymes in the cells, resulting in damage to the antioxidant enzyme system, inducing DNA oxidation in the cells, and ultimately causing irreversible damage and cell death to the foodborne pathogens, thereby reducing the sublethal damage rate of the pathogens while improving the sterilization rate. In the present invention, plasma activated water plays a major role in controlling the number of microorganisms, and ultrasonic treatment plays a major role in reducing the sublethal damage rate. The synergistic effect of the two can significantly reduce the occurrence of sublethal damage rates of foodborne pathogens while significantly reducing foodborne pathogens, improve the sterilization effect of foodborne pathogens, and thus be more conducive to solving food safety hazards.

[0049] In order to further illustrate the present invention, the technical solution provided by the present invention is described in detail below in conjunction with the accompanying drawings and embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0050] The instruments and equipment used in the embodiments of the present invention are as follows: Figure 1 shown.

[0051] The experimental process in the embodiment of the present invention is as follows Figure 2 shown.

[0052] Configuration of the culture medium used in the examples:

[0053] Preparation of tryptic soy broth (TSB) medium: weigh 30 g of tryptic soy broth medium, add 1000 mL of deionized water, dissolve and sterilize at 121° C. for 15 min, and set aside.

[0054] Preparation of non-selective culture medium: Weigh 40 g TSA culture medium, add 1000 mL deionized water, dissolve and sterilize at 121°C for 15 min, and set aside.

[0055] Preparation of selective culture medium: weigh 40 g TSA culture medium, 30 g NaCl, add 1000 mL deionized water, dissolve and sterilize at 121°C for 15 min, and set aside.

[0056] Preparation of E. coli bacterial suspension used in the following examples: E. coli strain (ATCC25922) was inoculated into tryptic soy agar (TSA) and cultured for 24 h. A single isolated colony was selected and inoculated into sterile tryptic soy broth (TSB), cultured at 37°C until the logarithmic growth phase, then centrifuged at 5000×g for 5 min to remove the supernatant, washed the bacteria twice with sterile saline and suspended in sterile saline, and the bacterial density was adjusted to about 10 8 CFU / mL.

[0057] Example 1

[0058] A method for reducing the sublethal injury rate of Escherichia coli, comprising the following steps:

[0059] (1) Preparation of PAW

[0060] PAW was prepared using a non-thermal atmospheric pressure plasma system (PG-1000Z / D, Nanjing Suman Electronics Co., Ltd.). The system consists of a plasma nozzle, a high-voltage generator (800W), and a gas control device. Compressed air of 0.18MPa was used as the working gas with a flow rate of 22.5L / min. The plasma in vivo device probe was placed 1cm below the surface of 300mL sterile water for plasma ionization treatment. The plasma activated water (PAW) was generated by ionization for 10min.

[0061] (2) Sterilization

[0062] Take 5 mL of Escherichia coli suspension and place it in a sterile centrifuge tube. Add 5 mL of PAW and place it in an ultrasonic equipment tank (KQ-100DE). Set the frequency to 40 kHz, the power to 250 W, the temperature to 20 °C, and sterilize for 10 minutes.

[0063] (3) Detection

[0064] Take 1 mL of the treated Escherichia coli suspension and dilute it with sterile saline in a 10-fold gradient to an appropriate concentration. Take 1 mL of the diluted bacterial suspension and mix it with non-selective culture medium TSA and selective culture medium TSA (containing 3% NaCl) respectively, culture at 37°C for 48 hours, and then record the number of colonies.

[0065] Bacterial reduction number = logarithm of untreated bacterial suspension colonies - logarithm of non-selective culture medium colonies;

[0066] Sublethal injury rate = (number of colonies on non-selective medium - number of colonies on selective medium) / number of colonies on non-selective medium × 100%.

[0067] This embodiment is recorded as: US-PAW10, sterilization 10 minutes.

[0068] Example 2

[0069] A method for reducing the sublethal damage rate of Escherichia coli, the steps are the same as those of Example 1, the only difference being that the ionization time in step (1) is 5 minutes. This example is denoted as US-PAW5, sterilization for 10 minutes.

[0070] Example 3

[0071] A method for reducing the sublethal damage rate of Escherichia coli, the steps are the same as those of Example 1, the only difference being that the ionization time in step (1) is 15 minutes. This example is recorded as: US-PAW15, sterilization 10 minutes.

[0072] Example 4

[0073] A method for reducing the sublethal damage rate of Escherichia coli, the steps are the same as those in Example 1, the only difference being that the sterilization treatment time in step (2) is 7 minutes. This example is recorded as: US-PAW10, sterilization 7 minutes.

[0074] Example 5

[0075] A method for reducing the sublethal damage rate of Escherichia coli, the steps are the same as those in Example 1, the only difference being that the sterilization treatment time in step (2) is 5 minutes. This example is recorded as: US-PAW10, sterilization 5 minutes.

[0076] Example 6

[0077] A method for reducing the sublethal damage rate of Escherichia coli, the steps are the same as those in Example 1, the only difference being that the sterilization treatment time in step (2) is 3 minutes. This example is recorded as: US-PAW10, sterilization 3 minutes.

[0078] Example 7

[0079] A method for reducing the sublethal damage rate of Escherichia coli, the steps are the same as those of Example 2, the only difference being that the sterilization treatment time in step (2) is 7 minutes. This example is recorded as: US-PAW5, sterilization 7 minutes.

[0080] Example 8

[0081] A method for reducing the sublethal damage rate of Escherichia coli, the steps are the same as those in Example 2, the only difference being that the sterilization treatment time in step (2) is 5 minutes. This example is denoted as US-PAW5, sterilization 5 minutes.

[0082] Example 9

[0083] A method for reducing the sublethal damage rate of Escherichia coli, the steps are the same as those in Example 2, the only difference being that the sterilization treatment time in step (2) is 3 minutes. This example is recorded as: US-PAW5, sterilization 3 minutes.

[0084] Example 10

[0085] A method for reducing the sublethal damage rate of Escherichia coli, the steps are the same as those of Example 3, the only difference being that the sterilization treatment time in step (2) is 7 minutes. This example is recorded as: US-PAW15, sterilization 7 minutes.

[0086] Embodiment 11

[0087] A method for reducing the sublethal damage rate of Escherichia coli, the steps are the same as those in Example 3, the only difference being that the sterilization treatment time in step (2) is 5 minutes. This example is recorded as: US-PAW15, sterilization 5 minutes.

[0088] Example 12

[0089] A method for reducing the sublethal damage rate of Escherichia coli, the steps are the same as those in Example 3, the only difference being that the sterilization treatment time in step (2) is 3 minutes. This example is recorded as: US-PAW15, sterilization 3 minutes.

[0090] Comparative Example 1

[0091] A method for reducing the sublethal damage rate of Escherichia coli, the steps are the same as those of Example 1, except that step (1) is omitted, the plasma activated water in step (2) of Example 1 is adjusted to deionized water, and the sterilization treatment time is 10 minutes. This comparative example is recorded as US, sterilization for 10 minutes.

[0092] Comparative Example 2

[0093] A method for reducing the sublethal damage rate of Escherichia coli, the steps are the same as those of Example 1, except that step (1) is omitted, the plasma activated water in step (2) of Example 1 is adjusted to deionized water, and the sterilization treatment time is 7 minutes. This comparative example is recorded as US, sterilization 7 minutes.

[0094] Comparative Example 3

[0095] A method for reducing the sublethal damage rate of Escherichia coli, the steps are the same as those of Example 1, except that step (1) is omitted, the plasma activated water in step (2) of Example 1 is adjusted to deionized water, and the sterilization treatment time is 5 minutes. This comparative example is recorded as US, sterilization 5 minutes.

[0096] Comparative Example 4

[0097] A method for reducing the sublethal damage rate of Escherichia coli, the steps are the same as those of Example 1, except that step (1) is omitted, the plasma activated water in step (2) of Example 1 is adjusted to deionized water, and the sterilization treatment time is 3 minutes. This comparative example is recorded as US, sterilization 3 minutes.

[0098] Comparative Example 5

[0099] A method for reducing the sublethal damage rate of Escherichia coli, the steps are the same as those of Example 1, the only difference is that the ultrasonic treatment in step (2) is omitted, and the other conditions are the same as those of Example 1. This comparative example is recorded as: PAW10, sterilization 10 minutes.

[0100] Comparative Example 6

[0101] A method for reducing the sublethal damage rate of Escherichia coli, the steps are the same as those of Example 2, the only difference is that the ultrasonic treatment in step (2) is omitted, and the other conditions are the same as those of Example 2. This comparative example is recorded as: PAW5, sterilization for 10 minutes.

[0102] Comparative Example 7

[0103] A method for reducing the sublethal damage rate of Escherichia coli, the steps are the same as those of Example 3, the only difference is that the ultrasonic treatment in step (2) is omitted, and the other conditions are the same as those of Example 3. This comparative example is recorded as: PAW15, sterilization for 10 minutes.

[0104] Comparative Example 8

[0105] A method for reducing the sublethal damage rate of Escherichia coli, the steps are the same as those of Example 4, the only difference is that the ultrasonic treatment in step (2) is omitted, and the other conditions are the same as those of Example 4. This comparative example is recorded as: PAW10, sterilization 7min.

[0106] Comparative Example 9

[0107] A method for reducing the sublethal damage rate of Escherichia coli, the steps are the same as those of Example 5, the only difference is that the ultrasonic treatment in step (2) is omitted, and the other conditions are the same as those of Example 5. This comparative example is recorded as: PAW10, sterilization 5 minutes.

[0108] Comparative Example 10

[0109] A method for reducing the sublethal damage rate of Escherichia coli, the steps are the same as those of Example 6, the only difference is that the ultrasonic treatment in step (2) is omitted, and the other conditions are the same as those of Example 6. This comparative example is recorded as: PAW10, sterilization for 3 minutes.

[0110] Comparative Example 11

[0111] A method for reducing the sublethal damage rate of Escherichia coli, the steps are the same as those of Example 7, the only difference is that the ultrasonic treatment in step (2) is omitted, and the other conditions are the same as those of Example 7. This comparative example is recorded as: PAW5, sterilization 7min.

[0112] Comparative Example 12

[0113] A method for reducing the sublethal damage rate of Escherichia coli, the steps are the same as those of Example 8, the only difference is that the ultrasonic treatment in step (2) is omitted, and the other conditions are the same as those of Example 8. This comparative example is recorded as: PAW5, sterilization for 5 minutes.

[0114] Comparative Example 13

[0115] A method for reducing the sublethal damage rate of Escherichia coli, the steps are the same as those of Example 9, the only difference is that the ultrasonic treatment in step (2) is omitted, and the other conditions are the same as those of Example 9. This comparative example is recorded as: PAW5, sterilization for 3 minutes.

[0116] Comparative Example 14

[0117] A method for reducing the sublethal damage rate of Escherichia coli, the steps are the same as those of Example 10, the only difference is that the ultrasonic treatment in step (2) is omitted, and the other conditions are the same as those of Example 10. This comparative example is recorded as: PAW15, sterilization 7min.

[0118] Comparative Example 15

[0119] A method for reducing the sublethal damage rate of Escherichia coli, the steps are the same as those of Example 11, the only difference is that the ultrasonic treatment in step (2) is omitted, and the other conditions are the same as those of Example 11. This comparative example is recorded as: PAW15, sterilization 5min.

[0120] Comparative Example 16

[0121] A method for reducing the sublethal damage rate of Escherichia coli, the steps are the same as those of Example 12, the only difference is that the ultrasonic treatment in step (2) is omitted, and the other conditions are the same as those of Example 12. This comparative example is recorded as: PAW15, sterilization for 3 minutes.

[0122] Embodiment 13

[0123] A method for reducing the sublethal damage rate of Escherichia coli, the steps are the same as those in Example 1, the only difference is that the power of ultrasound is 200W.

[0124] Embodiment 14

[0125] A method for reducing the sublethal damage rate of Escherichia coli, the steps are the same as those in Example 1, the only difference is that the power of ultrasound is 400W.

[0126] Embodiment 15

[0127] A method for reducing the sublethal damage rate of Escherichia coli, the steps are the same as those in Example 1, the only difference is that the power of ultrasound is 600W.

[0128] Application Example 1

[0129] 1. The effects of different treatments of Examples 1 to 12 and Comparative Examples 1 to 16 on the reduction of Escherichia coli counts are shown in Table 1. The effects of different treatments of Examples 1 to 12 and Comparative Examples 1 to 16 on the sublethal injury rate of Escherichia coli are shown in Table 2.

[0130] Table 1 Effects of different treatments on the reduction of Escherichia coli counts

[0131]

[0132] Note: US: ultrasound; PAW: plasma activated water, without ultrasound; US-PAW: ultrasound-assisted plasma activated water, 5, 10, 15 represents activated water ionization for 5, 10, 15 min. Capital letters indicate significant differences between the same line (p<0.05), and lowercase letters indicate significant differences between the same column (p<0.05). Same below.

[0133] Table 2 Effects of different treatments on sublethal damage rate of Escherichia coli

[0134]

[0135] Among them, the bacterial liquid counting results of Example 1, Comparative Example 1 and Comparative Example 5 are shown in Table 3.

[0136] Table 3 Bacterial liquid counting results of Example 1, Comparative Example 1 and Comparative Example 5

[0137] Example 1 Comparative Example 1 Comparative Example 5 Reduced bacterial count (logCFU / mL) 4.89±0.07 0.19±0.04 3.59±0.08 Sublethal injury rate (%) 13.30±2.15 16.13±3.25 28.14±1.45

[0138] It can be seen from Table 3 that the sterilization effect of Escherichia coli under the conditions of Example 1 is better, the number of Escherichia coli bacteria is higher, and the sublethal loss rate is lower. Compared with the treatment with plasma activated water alone, the sterilization effect of Escherichia coli in Example 1 is significant; the number of bacteria reduced by plasma activated water alone is 3.59±0.08logCFU / mL, and the sublethal damage rate is significantly higher than that in Example 1; compared with the ultrasonic treatment alone, the number of Escherichia coli bacteria in Example 1 is significantly increased, the sublethal damage rate is also significantly reduced, and the sterilization effect is significant.

[0139] Among them, the statistical results of the bacterial liquid of Examples 1 to 3 are as follows Figure 3 and Figure 4 shown. Figure 3 It is a statistical result diagram of the reduction of the number of Escherichia coli bacterial liquids in Examples 1 to 3; Figure 4This is a statistical result diagram of the sublethal damage rate of the Escherichia coli liquid in Examples 1 to 3.

[0140] Depend on Figure 3 and Figure 4 It can be seen that with the extension of ionization time, the number of Escherichia coli in the bacterial solution continued to decrease, and the number of reduced bacteria continued to increase. When the ionization time was 10min and 15min, there was no significant difference in the number of reduced bacteria of Escherichia coli, but the sublethal damage rate of Escherichia coli was significantly different. When the ionization time was 10min, the sublethal damage rate of Escherichia coli was lower.

[0141] The number of Escherichia coli reduced in Example 1 and Examples 4 to 6 is as follows Figure 5 The sublethal injury rates of Escherichia coli in Examples 1 and 4 to 6 are shown in Figure 6 As shown. Figures 5-6 It can be seen that with the extension of treatment time, the number of Escherichia coli in the bacterial solution continued to increase, showing a significant difference. The sublethal damage rate of Escherichia coli showed a decreasing trend. When the sterilization treatment time was 10 minutes, the sublethal damage rate of Escherichia coli was low.

[0142] 2. The statistical results of bacterial liquid under different ultrasonic powers in Examples 1 and 13 to 15 are shown in Tables 4 and Figures 7-8 shown.

[0143] Table 4 Effects of different ultrasonic powers on the reduction of Escherichia coli counts and sublethal damage rates

[0144] Embodiment 13 Example 1 Embodiment 14 Embodiment 15 Reduced bacterial count (logCFU / mL) 3.10±0.56 4.89±0.07 5.01±1.24 5.34±0.54 Sublethal injury rate (%) 35.14±1.51 13.30±2.15 25.14±4.51 30.24±1.46

[0145] From Table 4 and Figures 7-8 It can be seen that with the increase of ultrasonic power, the number of Escherichia coli in the bacterial solution showed an upward trend, and the number of bacteria reduced when the ultrasonic power was 600 W was higher, but the difference was not significant when the ultrasonic power was 400 W. The sublethal damage rate of Escherichia coli was lower when the ultrasonic power was 250 W, and the sublethal damage rate of Escherichia coli also increased with the increase of ultrasonic power.

[0146] 3. Effects of ultrasound-assisted plasma-activated water on cell membrane structure and oxidative damage

[0147] Determination of cell membrane structure indicators: After being treated with different groups, Escherichia coli was centrifuged at 4°C, 12000×g for 2 min, and the supernatant was collected.

[0148] Reference "Bactericidal effect and mechanism of plasma activated water on Shewanella putrefaciens[J]. Food Science, 2023, 44(09): 25-31." Methods Cell membrane permeability, extracellular nucleic acid and protein content, and cell membrane potential were determined.

[0149] Reference: "Study on the antibacterial mechanism of cinnamaldehyde on Escherichia coli and its synergistic sterilization effect with pulsed electric field [D]. South China University of Technology, 2019." The fatty acid composition of the cell membrane was determined by GC-MS method.

[0150] Reference: "Research on the mechanism of naringenin synergistically killing Escherichia coli and Staphylococcus aureus with pulsed electric field [D]. South China University of Technology, 2019." Potassium iodide (KI) labeling was used for fluorescence spectroscopy analysis of the influence on cell membrane protein structure.

[0151] Determination of oxidative loss index: The treated bacterial solution was centrifuged at 3000×g for 3 min, and the bacterial sludge was collected and washed with PBS buffer for later use. The Escherichia coli sample was obtained by ultrasonic disruption (300W, 3min) under ice bath conditions. Reference "Study on the inactivation effect and mechanism of plasma activated water on Salmonella [J]. Food Industry Science and Technology, 2021, 42(8): 138-143." Determination of intracellular ROS content.

[0152] After the protein concentration of each group of samples was determined using the BCA kit, the SOD activity, CAT content and GSH content of the samples were detected using the corresponding kits.

[0153] 8-OHdG ELISA kit was used to detect DNA oxidative damage in each group of samples.

[0154] The cell membrane structure and oxidative damage treated in Example 1, Comparative Example 1 and Comparative Example 5 were observed, and untreated bacterial solution was used as a negative control.

[0155] (1) The effect of ultrasound-assisted plasma-activated water on cell membrane morphology is shown in the figure Fig. 9 As shown (×10000). Fig. 9 As shown, the untreated bacterial sample Escherichia coli is regular rod-shaped, the cells are plump and uniform, and the surface is complete and smooth. After being treated with Comparative Example 1, the morphology of Escherichia coli remains basically unchanged. After being treated with Comparative Example 5, the Escherichia coli cells are slightly wrinkled and the surface becomes rough. After being treated with Example 1, obvious wrinkles and depressions appear on the surface of Escherichia coli cells. The above results show that the synergistic treatment of Example 1 damages the cell morphology of Escherichia coli, which may further affect its normal physiological metabolism and ultimately lead to its inactivation.

[0156] (2) The effects of ultrasound-assisted plasma-activated water on cell membrane integrity are shown in Table 5 and Figures 10-11 As shown, Fig.10 The results of the effects of different treatments on the absorbance of E. coli nucleic acid; Fig.11The results of the effects of different treatments on the absorbance of E. coli proteins. Nucleic acids and proteins have characteristic peaks at 260nm and 280nm in the ultraviolet region, respectively, and their concentrations are proportional to the absorbance. Therefore, the integrity of the cell membrane can be inferred from the leakage content of extracellular nucleic acids and proteins.

[0157] Table 5 Effects of ultrasound-assisted plasma-activated water on cell membrane integrity

[0158] Group A260 A280 Untreated bacterial samples 0.034±0.005c 0.010±0.000c Comparative Example 1 0.034±0.003c 0.029±0.008b Comparative Example 5 0.048±0.001b 0.050±0.000a Example 1 0.055±0.001a 0.049±0.001a

[0159] Note: Different lowercase letters in the same column indicate significant differences (p<0.05), the same below.

[0160] From Table 5 and Figures 10-11 The results show that there is a small amount of leakage in the untreated bacterial sample, which may be caused by the death of some cells in the normal life cycle. Compared with the untreated bacterial sample, the amount of Escherichia coli nucleic acid leakage treated with Comparative Example 1 was not significant (p>0.05). After treatment with Comparative Example 5 and Example 1, the absorbance value at 260nm was significantly (p<0.05) higher than that of the untreated bacterial sample group, indicating that the treatment with Comparative Example 5 and Example 1 promoted the leakage of nucleic acid. Compared with the treatment with Comparative Example 5, the absorbance at 260nm treated with Example 1 increased significantly (p<0.05), reaching 0.05. The trend of protein leakage is consistent with that of nucleic acid.

[0161] (3) The effects of ultrasound-assisted plasma-activated water on cell membrane permeability are shown in Table 6 and Fig.12 shown.

[0162] The effects of different treatments on the permeability of the E. coli cell membrane were evaluated by measuring the relative fluorescence intensity of PI. Fig.12 shown.

[0163] Table 6 Effects of different treatments on the permeability of Escherichia coli cell membrane

[0164] Group PI fluorescence intensity (%) Untreated bacterial samples 100.00±0.00c Comparative Example 1 141.96±13.87c Comparative Example 5 9,761.29±460.15b Example 1 13,086.86±542.12a

[0165] From Table 6 and Fig.12 It can be analyzed that, compared with the untreated bacterial sample, the treatment of comparative example 1 did not significantly change the relative fluorescence intensity of Escherichia coli (p>0.05). After the treatment of comparative example 5 and example 1, the fluorescence intensity increased significantly (p<0.05). The relative fluorescence intensity of PI in the treatment of example 1 was 1.34 times higher than that in the treatment of comparative example 5 (p<0.05).

[0166] The antibacterial effect of PAW is closely related to its physicochemical properties, including pH, conductivity and ORP. The latest research shows that ultrasonic treatment increases ORP, O3, NO 2-, OH· content. These highly oxidizing substances change the permeability of bacterial cell membranes to a certain extent. From the results, compared with the individuals in Comparative Example 5, the treatment in Example 1 significantly improved membrane permeability and promoted the leakage of nucleic acids and proteins. It is speculated that this is because the shock wave generated by the bursting of bubbles formed by US cavitation creates instantaneous holes in the cell membrane, making it easier for PAW to penetrate into the cells and accelerate the outflow of nucleic acids and proteins from the cells. This may be the main reason for the significant increase in leakage due to the synergistic treatment.

[0167] (4) The effects of ultrasound-assisted plasma-activated water on cell membrane potential are shown in Table 7 and Fig.13 shown.

[0168] Table 7 Effects of ultrasound-assisted plasma-activated water on cell membrane potential

[0169] Group DiBAC4(3) relative fluorescence intensity (%) Untreated bacterial samples 100.00±0.00c Comparative Example 1 100.40±2.20c Comparative Example 5 107.69±1.15b Example 1 113.32±0.55a

[0170] Cell membrane potential plays a crucial role in regulating bacterial physiology and behavior. Fig.13 As shown, there is no significant difference in the relative fluorescence intensity after treatment in Comparative Example 1 compared with the untreated bacterial sample. After treatment in Comparative Example 5 and Example 1, the relative fluorescence intensity increased significantly (p>0.05), increasing by 7.69% and 13.32%, respectively. The increase in cell membrane potential indicates that the cell is in a depolarized state. Studies have shown that the depolarization of the cell membrane can affect the K + 、Na + , Ca 2+ Plasma channels increase the potential difference between the inside and outside of the bacterial cell membrane, causing irreversible damage to the cells and ultimately leading to cell death. This result is consistent with the trend of cell membrane permeability and microbial bacterial count reduction.

[0171] (5) The effect of ultrasound-assisted plasma activated water on the fatty acid composition of the membrane is shown in Figures 8 and Fig.14 shown. Fig.14 This is a graph showing the effects of different treatments on the ratio of SFAs / UFAs in the cell membrane.

[0172] Table 8 Effects of different treatments on cell membrane fatty acid composition

[0173] fatty acid(%) Untreated bacterial samples Comparative Example 1 Comparative Example 5 Example 1 C4:0 18.83±1.95a 13.12±1.36ab 7.75±5.15b 8.04±0.01b C12:0 0.53±0.09c 1.17±0.01c 2.87±0.64b 4.17±0.45a C14:0 3.15±0.84a 3.26±0.51a 3.27±0.14a 3.21±0.39a C15:0 3.42±0.96a 3.54±0.59a 4.17±0.29a 4.47±0.56a C16:0 46.71±0.37b 53.09±1.76a 49.46±2.86b 38.26±0.02c C17:0 1.91±0.50a 1.95±0.34a 2.20±0.17a 2.14±0.23a C18:0 3.19±0.09b 3.14±0.41b 4.71±0.02a 5.31±0.43a C16:1 13.56±0.65a 9.03±0.73b 12.69±0.79a 14.07±1.13a C18:1 1.02±0.31b 1.14±0.05ab 0.85±0.27b 1.69±0.31a C18:2 3.56±0.44b 4.21±0.36ab 3.74±0.22ab 4.46±0.21a SFAs 79.67±0.50b 81.60±1.01a 78.27±0.32b 74.24±0.19c UFAs 20.49±0.33b 18.40±1.01c 21.73±0.32b 25.76±0.19a

[0174] Bacteria can reduce damage by regulating the structure of fatty acids, regulating the fluidity of cell membranes, maintaining membrane stability and normal physiological functions. This experiment detected the composition of fatty acids in Escherichia coli cell membranes after different treatments by GC-MS. As shown in Table 8, compared with the untreated bacterial samples, the proportion of SFAs increased and the proportion of UFAs decreased after treatment in Comparative Example 1. After treatment in Example 1, the proportion of SFAs (saturated fatty acids) was significantly reduced from 79.67±0.50% to 74.24±0.19%, and the proportion of UFAs (unsaturated fatty acids) was significantly increased from 20.49±0.33% to 25.76±0.19%. In particular, the reduction in the proportion of SFAs was mainly due to the change in butyric acid (C4:0). After treatment in Example 1, the butyric acid level decreased by 57.30%. The increase in the proportion of UFAs was mainly attributed to the change in the content of linoleic acid (C18:2), which was significantly increased from 3.56±0.44% to 4.46±0.21%. In addition, compared with Comparative Example 5, the proportion of SFAs in Example 1 group was significantly reduced by 5.15%, and the proportion of UFAs was significantly increased by 18.55%.

[0175] Unsaturated branched-chain fatty acids increased cell membrane fluidity, while saturated long-chain fatty acids decreased cell membrane fluidity. The ratio of SFA / UFAs after different treatments was calculated, as shown in Table 9 and Fig.14 shown.

[0176] Table 9 Ratio of SFA / UFAs after different treatments

[0177] Group Saturated fatty acid / unsaturated fatty acid ratio Untreated bacterial samples 3.90±0.09b Comparative Example 1 4.45±0.30a Comparative Example 5 3.60±0.07b Example 1 2.88±0.03c

[0178] From Table 9 and Fig.14 It can be analyzed that the treatment of comparative example 1 changed the ratio of membrane fatty acid composition and increased the ratio of SFA / UFAs. This shows that after the treatment of comparative example 1, the fluidity of the E. coli cell membrane decreased, causing the cells to be in a defensive state when resisting environmental pressure. After the treatment of Example 1, the ratio of SFA / UFA dropped to 2.88±0.03%, which was reduced by 20.00% compared with comparative example 5. This shows that after the treatment of Example 1, the membrane fluidity of E. coli was enhanced and the ability to resist external conditions was reduced. Therefore, the material exchange inside and outside the cell was enhanced, affecting the physiological metabolism of the cell.

[0179] (6) Effects of ultrasound-assisted plasma-activated water on the structure of cell membrane proteins Fig.15 shown. Fig.15Effects of different KI concentrations (a, c and e) and different treatments (b, d and f) on the fluorescence intensity of amino acid residues Phe (a and b, λex = 258 nm), Trp (c and d, λex = 280 nm) and Tyr (e and f, λex = 296 nm), Note: KI concentrations (from top to bottom) are 0, 5, 10, 15, 20, 25, 30 mM, respectively.

[0180] This experiment uses endogenous fluorescence intensity to explore the effects of treatment on the structure of Escherichia coli membrane proteins. Amino acid residues including tryptophan (Trp), tyrosine (Tyr) and phenylalanine (Phe) are the main fluorophores in membrane proteins. KI can quench the fluorescence of surface residues of membrane proteins, but does not affect the luminescence spectrum of residues inside membrane proteins. Therefore, based on the effect of KI on the fluorescence of these amino acid residues, the effects of different treatments on the positions of Trp, Tyr and Phe were explored.

[0181] KI affects the fluorescence emission spectra of Phe, Trp and Tyr residues in Escherichia coli membrane proteins ( Fig.15 a, c and e in Figure 1). As the concentration of KI increases, the fluorescence intensity of Phe decreases significantly, indicating that Phe residues are mainly located outside. There is no obvious quenching effect on Trp and Tyr residues, indicating that Trp and Tyr residues are mainly located in the membrane. Fig.15 As shown in b, d, and f. Compared with Comparative Examples 1 and 5, the maximum emission peak of the amino acid residues in Example 1 group was significantly reduced, indicating that the combination of PAW and US treatment showed a complementary effect. For Trp and Tyr residues, the fluorescence peak of Example 1 group was lower than the maximum KI concentration, indicating that compared with KI, the treatment of Example 1 can more significantly change the cell membrane structure. At the same time, after treatment with US-PAW, the fluorescence spectrum showed a slight blue shift, indicating that Example 1 acts on cell membrane proteins, causing changes in protein conformation and changing the microenvironment of Phe, Trp and Tyr. The stability of membrane protein conformation is crucial to the growth and vitality of bacteria. The results of this study are consistent with the results of ultrasound combined with sodium hypochlorite. By combining ultrasound treatment, the fluorescence peak of amino acid residues is reduced, thereby changing the structure of cell membrane proteins, ultimately leading to cell confusion, decomposition and death.

[0182] (7) The effects of ultrasound-assisted plasma-activated water on intracellular ROS levels are shown in Table 10 and Fig.16 shown.

[0183] Table 10 Effects of ultrasound-assisted plasma-activated water on intracellular ROS levels

[0184] Group DCFH-DA relative fluorescence intensity (%) Untreated bacterial samples 100.00±0.00c Comparative Example 1 132.01±0.23c Comparative Example 5 714.78±48.73b Example 1 776.94±35.34a

[0185] The content of ROS is an important signal of normal physiological function. Fig.16 As shown, the relative fluorescence intensity of E. coli in Comparative Example 1 did not increase significantly compared with the untreated bacterial sample (p>0.05). However, after treatment with Comparative Example 5 and Example 1, the ROS level increased significantly (p<0.05). The relative fluorescence intensity of the Example 1 group was 8.70% higher than that of the Comparative Example 5, showing a significant difference (p<0.05). This indicates that the combined treatment caused a severe oxidative stress response in the cells.

[0186] (8) The effects of ultrasound-assisted plasma-activated water on the activity levels of intracellular antioxidant oxidases are shown in Tables 11 and Figures 17 to 19 shown.

[0187] Table 11 Effects of ultrasound-assisted plasma-activated water on the activity levels of intracellular antioxidant oxidases

[0188]

[0189]

[0190] GSH content, SOD activity and CAT content reflect the level of intracellular antioxidants. Figures 17 to 19 As shown, compared with the untreated bacterial samples, the GSH content in the three treated Escherichia coli was significantly reduced (p<0.05). The treatment of Example 1 caused the intracellular GSH content to drop to 44.71 μmol / L, which was significantly lower than that of the comparative example 5 group (p<0.05). SOD is an antioxidant metalloenzyme, which is considered to be the first line of defense against ROS, and its activity largely determines the resistance of bacteria to oxidative stress. SOD catalyzes the dismutation of superoxide anion radicals to produce O2 and H2O2, which is the core regulatory factor of microbial ROS levels. After treatment with comparative example 1, the intracellular antioxidant enzymes were activated and the SOD activity increased significantly (p<0.05). The treatment of comparative example 5 and example 1 significantly reduced the SOD activity (p<0.05). Compared with the untreated bacterial samples, the treatment of example 1 reduced the SOD activity of Escherichia coli by 58.40%. When treated with comparative example 1, the CAT activity increased significantly (p<0.05), and the body mobilized the CAT enzyme to decompose hydrogen peroxide to cope with the oxidative damage caused by free radicals. However, after being treated with Example 1, the CAT activity was significantly decreased by 19.50% (p<0.05).

[0191] The treatment of comparative example 1 had no significant effect on the ROS content. It may be due to the low frequency of ultrasound treated with E. coli, which cannot directly damage the cells and induce the accumulation of ROS in the cells. When external factors invade the cells, the intracellular antioxidant enzymes of E. coli are activated, thereby playing a defensive role in the cells. The SOD activity was enhanced after the treatment of comparative example 1, indicating that the cells are in a defensive state and defense is achieved by increasing energy metabolism. The treatment of Example 1 significantly reduced the SOD activity, CAT activity, and GSH content, indicating that the active substances produced by the synergistic treatment can lead to a decrease in the activity of intracellular antioxidant enzymes, resulting in damage to the antioxidant enzyme system. ROS content is negatively correlated with SOD, CAT, and GSH activity. In addition, since the plasma membrane and cell wall of Gram-negative bacteria are thin, ROS causes the loss of permeability of the damaged cell membrane, and intracellular substances leak out from both, causing irreversible damage and cell death.

[0192] (9) The effects of ultrasound-assisted plasma-activated water on intracellular DNA oxidation are shown in Table 12 and Fig. 20 shown.

[0193] Table 12 Effects of ultrasound-assisted plasma-activated water on intracellular DNA oxidation

[0194] Group 8-OHdG content (ng / mL) Untreated bacterial samples 4.74±0.12c Comparative Example 1 4.72±0.67c Comparative Example 5 5.46±0.10b Example 1 6.28±0.16a

[0195] 8-OHdG is the most commonly used marker for evaluating DNA oxidation, and its production often leads to strand misreading, mutation, and cell death. The effects of different treatments on E. coli DNA oxidation products are shown in Tables 12 and Fig. 20 As shown. When E. coli was treated with the three sterilization methods, the 8-OHdG content of E. coli increased significantly. Among them, the 8-OHdG content of E. coli treated with Example 1 was as high as 6.28±0.16 (ng / mL), which was significantly increased by 33.05% and 15.02% compared with the treatments of Comparative Example 1 and Comparative Example 5, respectively. This shows that ultrasound synergizes with plasma activated water to induce DNA oxidation in E. coli. The experimental results are consistent with the results of the influence of ROS.

[0196] In summary, the present invention uses ultrasound synergistic plasma activated water to treat foodborne pathogens. While greatly reducing foodborne pathogens, it can also significantly reduce the occurrence of sublethal injury rate of foodborne pathogens and improve the sterilization effect of foodborne pathogens.

[0197] Although the above embodiment describes the present invention in detail, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the protection scope of the present invention.

Claims

1. Application of ultrasound synergistic plasma activated water to reduce sublethal injury rate of foodborne pathogens, wherein the power of the ultrasound is 200-600W.

2. The use according to claim 1, characterized in that: The foodborne pathogens include Escherichia coli.

3. A method for reducing the sublethal injury rate of foodborne pathogens, characterized in that: The following steps are involved: The object to be treated is mixed with plasma activated water and then subjected to ultrasonic treatment; the power of the ultrasonic treatment is 200 to 600 W; and the time of the ultrasonic treatment is 3 to 10 minutes.

4. The method according to claim 3, characterized in that: The power of the ultrasonic treatment is 250W, and the time of the ultrasonic treatment is 10 minutes.

5. The method according to claim 3 or 4, characterized in that: The frequency of the ultrasonic treatment was 40 kHz.

6. The method according to claim 3, characterized in that: The method for preparing plasma activated water comprises: subjecting sterile water to plasma ionization treatment to obtain plasma activated water.

7. The method according to claim 6, characterized in that: Use 0.18MPa compressed air as the working gas with a flow rate of 20-30L / min, and place the plasma generator probe 0.5-1cm below the sterile water surface for plasma ionization treatment.

8. The method according to claim 6 or 7, characterized in that: The plasma ionization treatment time for each 300 mL of sterile water is 5 to 15 minutes.

9. The method according to claim 8, characterized in that: The plasma ionization treatment time for each 300 mL of sterile water is 10 minutes.

10. Use of the method according to any one of claims 3 to 9 in improving the sterilization rate of foodborne pathogenic bacteria.

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