Preparation method of nano ozone water and application of nano ozone water in killing food-borne pathogenic bacteria

Abstract

The invention discloses a preparation method of nano ozone water and application of the nano ozone water in killing food-borne pathogenic bacteria, and belongs to the technical field of bactericides. The nano ozone water is prepared by utilizing the ultra-fine bubble generator and the ozone generator, and the prepared nano ozone water is high in stability, not easy to decompose and high in practicability. The nano ozone water can achieve an effective killing effect on escherichia coli, salmonella, enterococcus, campylobacter and clinical multi-drug-resistant bacteria thereof, and a new resource is provided for preparing a safe and reliable antibacterial product.

Description

Technical Field

[0001] The present invention relates to the technical field of fungicides, and particularly to a preparation method of nano-ozone water and its application in killing foodborne pathogenic bacteria. Background Art

[0002] In the food industry, chlorine and hydrogen peroxide are the most commonly used disinfectants, but the residues they release may have potential toxic effects. Ozone is a natural substance in the atmosphere, and its characteristics of quick bactericidal effect and no pollution have attracted the attention of food scientists. As early as June 26, 2001, the U.S. Food and Drug Administration has officially approved the use of ozone as an antibacterial agent for treatment, storage and processing in the food production process, and defined it as a generally recognized safe element, that is, a food additive considered safe for human health.

[0003] Ozone gas is toxic, and the olfactory perception threshold of humans for ozone is very low. Long-term inhalation of high-concentration ozone will cause permanent damage to organs such as the heart and lungs of the human body. The liquid form of ozone can effectively solve the disadvantages of the gas form. Ozone can be directly dissolved in water to prepare ozone water with a certain concentration, but the ozone water prepared by this method is extremely prone to decomposition. Now, the nano-bubble generation technology is combined with the ozone generation technology. By using the characteristics of small nano-bubble particle size and strong gas dissolution ability, the dissolution of ozone in water can be promoted to overcome the instability of ozone water prepared by ordinary methods. At the same time, nano-bubble ozone water with a certain concentration can be quickly prepared by adjusting the intake air volume and water intake.

[0004] At the same time, the traditional plate counting method can characterize the disinfection effect of disinfectants, but the accurate and comprehensive evaluation of disinfectants cannot be achieved in actual applications. In addition, there is currently a lack of systematic evaluation research on the disinfection effects of nano-ozone water on various foodborne pathogenic bacteria. In particular, there is no literature report on the disinfection effect of nano-ozone water on Escherichia coli. Summary of the Invention

[0005] The purpose of the present invention is to provide a preparation method of nano-ozone water and its application in killing foodborne pathogenic bacteria to solve the problems existing in the above-mentioned prior art. The nano-ozone water prepared by the present invention has high stability, is not easily decomposed, and has strong practicability. The nano-ozone water can achieve effective disinfection effects on Escherichia coli, Salmonella, Enterococcus and Campylobacter and their multi-drug resistant bacteria, providing a new resource for the preparation of safe and reliable antibacterial products.

[0006] To achieve the above purpose, the present invention provides the following solutions:

[0007] The present invention provides a method for preparing nano-ozone water. By using an ultra-fine bubble generator and an ozone generator, the inlet pressure of oxygen is set at 0.4 - 0.6 MPa, the gas pressure gauge of the ozone generator is controlled within the range of 0.15 - 0.2 MPa, the flow regulating valve of the gas flow meter is set at 80 - 120 mL / min, and the gas is discharged at a flow rate of 30 s. While introducing the gas, ultrasonic treatment is carried out to prepare the nano-ozone water.

[0008] The present invention provides the application of the nano-ozone water prepared by the above preparation method in the preparation of a bacterial disinfection product, and the bacterial disinfection product has the function of inhibiting and / or killing foodborne pathogenic bacteria; the foodborne pathogenic bacteria include Escherichia coli.

[0009] Optionally, the foodborne pathogenic bacteria further include Campylobacter, Salmonella, and Enterococcus.

[0010] Optionally, the concentration of the nano-ozone water is not less than 2 mg / L.

[0011] The present invention also provides a bacterial disinfection product, and the active ingredient in the bacterial disinfection product is the nano-ozone water prepared by the above preparation method.

[0012] The present invention also provides the application of the nano-ozone water prepared by the above preparation method in the preparation of a bacterial lysate, and the bacterial lysate has the function of destroying the bacterial cell membrane; the bacteria include Escherichia coli.

[0013] Optionally, the bacteria further include Campylobacter, Salmonella, and Enterococcus.

[0014] Optionally, the concentration of the nano-ozone water is not less than 8 mg / L.

[0015] The present invention also provides a bacterial lysate, and the active ingredient in the bacterial lysate is the nano-ozone water prepared by the above preparation method.

[0016] The present invention discloses the following technical effects:

[0017] The present invention discloses a method for preparing nano-ozone water, which is prepared by combining an ozone generator and an ultra-fine bubble generator. Its characterization data is detected by a nano-particle size analyzer. The initial concentration of the nano-ozone water prepared with a gas pressure of 0.5 MPa and the nano-bubble generator for 10 min is 8.0 mg / L, meeting the nano-level standard. After standing for 1, 2, and 3 h, its nano-particle size remains below 10 μm. It is feasible to prepare nano-ozone water by this method, and the prepared nano-ozone water has high stability and strong practicability. At the same time, the present invention proves that nano-ozone water can achieve effective disinfection effects on Escherichia coli, Salmonella, Enterococcus, Campylobacter, and their multi-drug resistant bacteria, providing a new resource for the preparation of safe and reliable antibacterial products. Brief Description of the Drawings

[0018] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for use in the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can also be obtained based on these drawings.

[0019] Figure 1 Effect of temperature on the stability of nano-ozone water;

[0020] Figure 2 Effect of initial concentration on the stability of nano-ozone water;

[0021] Figure 3 Bactericidal effect of nano-ozone water treatment on Escherichia coli; where A is the logarithm of the killing of Escherichia coli ATCC25922 by nano-ozone water at different concentrations; B is the logarithm of the killing of Escherichia coli ATCC25922 by nano-ozone water at different treatment times; C is the logarithm of the killing of Escherichia coli with different drug resistance phenotypes by nano-ozone water;

[0022] Figure 4 Effect of nano-ozone water treatment on the biofilm formation ability of Escherichia coli; where A is the standard strain of Escherichia coli ATCC25922, B is the multi-drug resistant strain GK-15, C is the multi-drug resistant strain HZ-130, D is the multi-drug resistant strain SW2-1; "*": p < 0.05; "**": p < 0.01; "***": p < 0.001; "****": p < 0.0001;

[0023] Figure 5 Field emission scanning electron microscopy (SEM), transmission electron microscopy (TEM) and room temperature ultrathin section TEM images of Escherichia coli before and after nano-ozone water treatment; where A is the standard strain of Escherichia coli ATCC25922, B is the multi-drug resistant strain GK-15, C is the multi-drug resistant strain HZ-130, D is the multi-drug resistant strain SW2-1;

[0024] Figure 6 Raman spectroscopic images of Escherichia coli before and after nano-ozone water treatment; where A is the standard strain of Escherichia coli ATCC25922, B is the multi-drug resistant strain GK-15, C is the multi-drug resistant strain HZ-130, D is the multi-drug resistant strain SW2-1;

[0025] Figure 7 Flow cytometry apoptosis change diagram of Escherichia coli treated with nano-ozone water;

[0026] Figure 8 Flow cytometry apoptosis change diagram of Salmonella treated with nano-ozone water

[0027] Figure 9 Flow cytometry apoptosis change diagram of Enterococcus treated with nano-ozone water

[0028] Figure 10 Flow cytometry apoptosis change diagram of Campylobacter treated with nano-ozone water Detailed implementation manners

[0029] The various exemplary implementation manners of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, characteristics, and implementation schemes of the present invention.

[0030] It should be understood that the terms described in the present invention are only for describing particular implementation manners and are not used to limit the present invention. Additionally, for the numerical ranges in the present invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any intermediate value within any stated value or stated range, as well as each smaller range between any other stated value or intermediate value within the stated range, is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded from the range.

[0031] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the present invention pertains. Although the present invention only describes preferred methods and materials, any methods and materials similar or equivalent to those described herein can also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials related to the documents. In case of conflict with any incorporated document, the content of this specification shall prevail.

[0032] Without departing from the scope or spirit of the present invention, various improvements and changes can be made to the specific implementation manners of the description of the present invention, which are obvious to those skilled in the art. Other implementation manners obtained from the description of the present invention are obvious to those skilled in the art. The description and examples of the present invention are only exemplary.

[0033] Regarding the terms "comprising", "including", "having", "containing", etc. used herein, they are all open-ended terms, meaning including but not limited to.

[0034] The sources of the strains used in the following examples of the present invention are as follows:

[0035] Escherichia coli: Escherichia coli ATCC 25922 was purchased from ATCC; Escherichia coli GK-15 was isolated from a pig farm of Hunan Xiangcun Gaoke Agriculture Co., Ltd., and was identified as resistant to 3 antibiotics (ampicillin, cefotaxime, ceftazidime); Escherichia coli SW2-1 was isolated from a chicken farm of Shengwang in Liuyang, Hunan Province, and was identified as resistant to 8 antibiotics (ampicillin, tetracycline, florfenicol, sulfisoxazole, cefotaxime, ceftazidime, enrofloxacin, ofloxacin); Escherichia coli HZ-130 was isolated from a pig farm of Hubei Jinlin Purebred Animal Husbandry Co., Ltd., and was identified as resistant to 12 antibiotics (ampicillin, augmentin, spectinomycin, tetracycline, florfenicol, sulfisoxazole, co-trimoxazole, cefotaxime, ceftazidime, enrofloxacin, ofloxacin, meropenem); all of the above were preserved in the Benchmark Laboratory of Veterinary Drug Residues, Huazhong Agricultural University;

[0036] Salmonella: Salmonella CVCC571 was purchased from CVCC; Salmonella B-2 was isolated from a chicken farm of Hexiang Shuimei Ecological Agriculture in Hubei Province, and was identified as resistant to 9 antibiotics (ampicillin, augmentin, ceftazidime, cefotaxime, gentamicin, enrofloxacin, spectinomycin, co-trimoxazole, tetracycline); Salmonella GD-23 was isolated from a chicken-derived strain in the market of Qingyuan City, Guangdong Province, and was identified as resistant to 10 antibiotics (ampicillin, ceftazidime, cefotaxime, enrofloxacin, gentamicin, florfenicol, spectinomycin, co-trimoxazole, tetracycline, ofloxacin); Salmonella JX-18 was isolated from a pig farm of Hunan Xiangcun Gaoke Agriculture Co., Ltd. in Loudi City, Hunan Province, and was identified as resistant to 2 antibiotics (ampicillin, augmentin); all of the above were preserved in the Benchmark Laboratory of Veterinary Drug Residues, Huazhong Agricultural University;

[0037] Enterococcus: Enterococcus ATCC29212 was purchased from ATCC; Enterococcus GX-22, LG4-2 and HX-6 were all isolated from chicken farms in Hunan; GX-22 was identified as resistant to 9 antibiotics (doxycycline, enrofloxacin, erythromycin, florfenicol, gentamicin, linezolid, sulfisoxazole, tiamulin, tilmicosin); LG4-2 was identified as resistant to 7 antibiotics (enrofloxacin, erythromycin, florfenicol, gentamicin, sulfisoxazole, tiamulin, tilmicosin); HX-6 was identified as resistant to 8 antibiotics (enrofloxacin, erythromycin, florfenicol, gentamicin, sulfisoxazole, penicillin, tiamulin, tilmicosin); all were preserved in the Benchmark Laboratory of Veterinary Drug Residues, Huazhong Agricultural University;

[0038] Campylobacter: The Campylobacter standard strain NCTC11168 was purchased from the National Collection of Type Culture, Colindale. Campylobacter 1-19 was isolated from Shengwang Chicken Farm in Liuyang, Hunan Province and was identified as resistant to 6 antibiotics (azithromycin, ciprofloxacin, clindamycin, nalidixic acid, telithromycin, tetracycline). Campylobacter 2-3 was isolated from a chicken source sample from Yasheng Food Co., Ltd. in Huangpi District, Wuhan, Hubei Province and was identified as resistant to 3 antibiotics (ciprofloxacin, nalidixic acid, tetracycline). Campylobacter JH-2 was isolated from Huafu Pig Farm in Jinggangshan, Jiangxi Province and was identified as resistant to 8 antibiotics (tetracycline, ciprofloxacin, clindamycin, erythromycin, gentamicin, nalidixic acid, telithromycin, tetracycline). All were preserved in the Veterinary Drug Residue Reference Laboratory of Huazhong Agricultural University.

[0039] Example 1 Preparation of Nano-Ozone Water

[0040] 1. Instrument and equipment: Ultra-fine bubble generator: Model UFB-N4B, purchased from Hirose Holdings Co., Ltd., Japan. Ozone generator: Air-cooled model FG-L50G, purchased from Guangzhou Feige Environmental Protection Technology Co., Ltd.

[0041] 2. Preparation method: Connect water, gas and other pipelines and wiring. Inject 4 L of double-distilled water into the reaction tank (10 mm above the aeration equipment in the reaction tank). Open the oxygen cylinder, confirm that the inlet pressure of the gas is 0.5 MPa, turn on the switch of the ozone generator, press the switch of the gas pressure gauge to adjust the gas pressure regulating valve to ensure that the display on the pressure gauge is within the range of 0.15 - 0.2 MPa, and then tighten the locking nut of the regulating valve. Open the flow regulating valve of the gas flow meter to adjust it to 100 mL / min, discharge the gas at a flow rate of 30 s, confirm the stable discharge of the gas, adjust the gas volume through the regulating valve, and turn on the switch of the ultrasonic oscillator while introducing the gas to obtain nano-ozone water.

[0042] Example 2 Performance Determination of Nano-Ozone Water

[0043] 1. Concentration determination of nano-ozone water

[0044] Determination method: According to the standard CJ / T - 3028.2, the iodometric method is used for the determination of ozone water concentration: Measure 1 mL of potassium iodide solution, pour it into a 500 mL absorption flask, then add a certain volume of the test solution, and then add sulfuric acid solution to make the pH value of the test solution drop below 2.0 and shake well, and let it stand for 5 min. Titrate with 0.1 mol / L sodium thiosulfate standard solution. When the solution turns light yellow, add about 1 mL of starch solution, add starch solution 0.5 mL before the end point of titration, and quickly titrate until the color disappears. Repeat 3 times and record the consumption of sodium thiosulfate standard solution. Finally, calculate the nano-ozone water concentration through the following formula.

[0045] Formula: CO3 = V Na ×C×24000 / V0 (mg / L)

[0046] Where: CO3 --- concentration of nano-ozone water, mg / L; V Na --- dosage of sodium thiosulfate standard solution, mL; C --- concentration of sodium thiosulfate standard solution, mg / L; V0 --- sampling volume of nano-ozone water, mL.

[0047] Result: The initial concentration of nano-ozone water prepared with a gas pressure of 0.5 MPa in combination with a nano-bubble generator for 10 min is 8.0 mg / L.

[0048] 2. Determination of particle size and Zeta potential of nano-ozone water

[0049] Instrument and equipment: Nano particle size and Zeta potential analyzer: Zetasizer Nano ZS type, purchased from Malvern Instruments Ltd., UK.

[0050] Determination method: Take the newly prepared nano-ozone water for detection and record the initial concentration. After standing for 1, 2, and 3 h respectively, take 2 mL of nano-ozone water and add it to the sample cell. Put the sample cell into the instrument, select the liquid measurement medium and set the measurement parameters to 3 cycles. After the screen reading is stable, copy and record the data results.

[0051] Result: The average particle size and Zeta potential of nano-ozone water after standing for 1, 2, and 3 h are shown in Table 1. Within 3 h, the particle size of nano-ozone water is below 10 μm, meeting the particle size level of nano-bubble water.

[0052] Table 1 Particle size and Zeta potential of nano-ozone water

[0053]

[0054] Note: Average particle size and Zeta potential of nano-ozone water after standing for 1, 2, and 3 h.

[0055] 3. Determination of stability of nano-ozone water

[0056] 3.1 Influence of temperature on the stability of nano-ozone water

[0057] Determination method: Fill the newly prepared nano-ozone water into a 300 mL glass bottle with a lid and tighten the lid. Place it in an environment of 4 °C and 25 °C for 80 min respectively. Take samples for concentration determination every 20 min, and repeat 3 times to take the average value as the measurement result of the detection concentration.

[0058] Result: As Figure 1As shown in the figure; to further analyze whether temperature is a significant factor affecting the stability of nano-ozone water, an analysis of variance was carried out, and the results are shown in Table 2.

[0059] Table 2 Analysis of variance table of time-temperature

[0060]

[0061] 3.2 Influence results of initial concentration on the stability of nano-ozone water

[0062] Determination method: Nano-ozone water with initial concentrations of 2.52, 5.04, and 7.44 mg / L was filled in a 300 mL glass bottle with a lid and the bottle cap was tightened, and then placed for 80 min. The concentration was measured by sampling every 20 min, and the average value was taken after repeating 3 times as the measurement result of the detected concentration.

[0063] Results: As Figure 2 shown in the figure; to further analyze whether different placement times are significant factors affecting the stability of nano-ozone water, an analysis of variance was carried out, and the results are shown in Table 3.

[0064] Table 3 Analysis of variance table of time-concentration

[0065]

[0066] Summary: By monitoring the effects of different temperatures and initial concentrations on the stability of nano-ozone water, it was found that in terms of temperature conditions, the storage conditions of normal temperature environment (25 °C) and refrigerated environment (4 °C) within 80 min do not significantly affect the trend of the concentration of nano-ozone water decreasing with time; but within the same time, the amplitude of the concentration of nano-ozone water decreasing with time is significantly affected by different initial concentrations. The higher the initial concentration of nano-ozone water, the faster the concentration decreases within the same time.

[0067] Example 3 Quantitative bactericidal performance experiment of nano-ozone water suspension

[0068] (1) Preparation of bacterial suspension: In a sterile environment, use a cotton swab to scrape the colony and mix it in the pre-prepared sterile PBS, shake well and set aside. Add sterile PBS to the standard turbidimetric tube, put it into the turbidimeter to calibrate the bacterial liquid concentration to 0.5 McFarland units (1×10 8 CFU / mL), and dilute other concentrations as needed. After shaking and mixing evenly, it is the Escherichia coli suspension.

[0069] (2) Identification of neutralizer: The neutralizer is a phosphate buffer solution containing 10 g / L sodium thiosulfate. The diluent is prepared by adding 0.40 g of tryptone and 3.40 g of sodium chloride to 400 mL of distilled water, stirring with a glass rod until completely dissolved, adjusting the pH value to 7.0 ± 0.2 (20 °C), autoclaving at 121 °C for 15 min, and storing at 4 °C for later use.

[0070] To investigate whether the neutralizer can effectively neutralize ozone water (concentration: 1.98 mg / L) without affecting Escherichia coli and its recovery culture, the following six groups of experiments were set up: Group 1: 4.5 mL of nano-ozone water + 0.5 mL of bacterial suspension; Group 2: 0.5 mL of the mixture (4.5 mL of nano-ozone water + 0.5 mL of bacterial suspension) + 4.5 mL of neutralizer; Group 3 (to observe whether the neutralizer inhibits bacteria): 4.5 mL of neutralizer + 0.5 mL of bacterial suspension; Group 4 (to observe the effect of neutralization products): 4.5 mL of neutralization products (4.5 mL of nano-ozone water + 0.5 mL of neutralizer) + 0.5 mL of bacterial suspension; Group 5 (bacterial count control group): 4.5 mL of diluent + 0.5 mL of bacterial suspension; Group 6: 0.5 mL of diluent + 0.5 mL of neutralizer + 0.5 mL of bacterial culture medium (broth medium). The experiment was repeated 3 times.

[0071] (3) Suspension quantitative bactericidal experiment: Test group: Pipette 5 mL of bacterial suspension into a test tube, then add 45 mL of nano-ozone water with a specified concentration, mix well and let it stand for a specified reaction time. This is sample solution 1. Then pipette 0.5 mL of sample solution 1 into a test tube containing 4.5 mL of neutralizer, shake well, and after reacting for 10 min, perform viable bacteria culture and counting. Positive control group: Repeat the above operation using sterile PBS instead of nano-ozone water. Each group of experiments needs to be repeated 3 times, record the viable bacteria concentration (CFU / mL) of each group of experiments, and calculate according to the following formula:

[0072] Calculation of killing rate: Calculation of logarithm of killing value: KL = lg(N0) - lg(N X )

[0073] Where: η --- Bacterial killing rate, %; N0 --- Viable bacteria concentration of the positive control group, CFU·mL -1 ; N X --- Viable bacteria concentration of the experimental group, CFU·mL -1 ; KL --- Logarithm of killing value.

[0074] (4) Bactericidal performance test of Escherichia coli standard strain: With the concentration of nano-ozone water as the variable, the operation process is the same as that of the "suspension quantitative bactericidal experiment". Take the newly prepared nano-ozone water and dilute it by different multiples and conduct synchronous concentration determination to determine its concentrations to be 0.42, 1.12, 1.98, 4.14, and 8.48 mg / L respectively. The preparation of the Escherichia coli bacterial suspension is the same as that of the "bacterial suspension preparation" to determine the reduction effect of nano-ozone water at different concentrations on the Escherichia coli standard strain.

[0075] With different treatment times as variables, the operation process is the same as that of the "suspension quantitative bactericidal experiment". Use nano-ozone water with a concentration of 2.04 mg / L to conduct a bactericidal experiment on Escherichia coli. The treatment times of nano-ozone water are 3, 10, 30, and 60 min respectively to determine the influence of different treatment times on the reduction effect of the Escherichia coli standard strain.

[0076] (5) Experiment on the killing effect of nano-ozone water on Escherichia coli with different drug resistance phenotypes: Take the newly prepared nano-ozone water and dilute it by different multiples and conduct synchronous concentration determination to determine its concentrations to be 5.28, 3.24, 1.32, and 0.72 mg / L respectively. The bacterial solution concentration is set to 1.0×10 8 CFU / mL, the treatment time is 10 min, and Escherichia coli ATCC 25922, GK-15, SW2-1, and HZ-130 are respectively experimented according to the steps of the "suspension quantitative bactericidal experiment" to determine the difference in the reduction effect of nano-ozone water at different concentrations on clinically isolated Escherichia coli with sensitivity and different drug resistance phenotypes.

[0077] (6) Data analysis: The identification results of the neutralizer are shown in Table 4. The number of colonies in the first group and the sixth group are both 0, less than the number of colonies in the second group; the number of colonies in the second group is less than the number of colonies in the third, fourth, and fifth groups, and the differences among these three groups are not obvious, and the error rate is within 15%. This neutralizer formulation meets the requirements for neutralizers in the disinfectant standard.

[0078] The results of the suspension quantitative bactericidal experiment of nano-ozone water conducted under two control variables of different concentrations of nano-ozone water and different treatment times are shown in Figure 3 A and B of. It can be seen from Figure 3 A and B of that the disinfection effect of nano-ozone water is related to the concentration of ozone water, and the higher the concentration of nano-ozone water, the higher the bactericidal rate and the killing logarithm value; when the concentration of nano-ozone water is 1.98 mg / L, the 4 treatment times of the Escherichia coli suspension and nano-ozone water all play a bactericidal role, and the bactericidal rate is greater than 90%, and the killing logarithm value is greater than 1. Therefore, time is not an influencing factor for the disinfection effect of nano-ozone water; similarly, there is no significant difference in the disinfection effect of nano-ozone water on Escherichia coli with different drug resistance phenotypes ( Figure 3 C of).

[0079] Colony statistical results of neutralizer identification in Table 4

[0080]

[0081] Example 4 Detection of the effect of nano-ozone water on biofilm formation ability by microplate reader

[0082] (1) Bacterial incubation: According to the method of Example 3, prepare the bacterial suspensions of Escherichia coli ATCC 25922, GK-15, SW2-1 and HZ-130, as well as nano-ozone water respectively. Select 45 mL of nano-ozone water with a concentration of 1.82 mg / L and incubate it with 5 mL of bacterial suspension with a concentration of 1.0×10 8 CFU / mL for 10 min to obtain a mixed solution of nano-ozone water and bacterial suspension, which is used as the experimental group (ONW). Replace the mixed solution of nano-ozone water and bacterial suspension with fresh broth culture medium as the negative control group (Negative control), and replace nano-ozone water with broth medium as the control group (control).

[0083] (2) Sample treatment: Pipette 100 μL of each sample into a 96-well plate. Add 100 μL of LB liquid medium to each well of the 96-well plate in advance, and culture it in a constant temperature incubator at 37 °C for 24 h, 48 h, and 72 h. Take out the 96-well plates cultured at different time points from the constant temperature incubator, remove the bacterial liquid with a pipette gun, add 200 μL of sterile phosphate buffer and wash 3 times, add 0.1% crystal violet staining solution and stain for 30 min, then wash twice with phosphate buffer, then place it in an oven to dry for 20 min, and then add 200 μL of 95% ethanol, and use a microplate reader to detect its absorbance value (OD 600 ) under the condition of 600 nm. The experiment was repeated 3 times.

[0084] According to the measured OD 600 value, the formation of the bacterial strain biofilm is divided into the following 4 categories: Use the OD 600 value of the negative broth group as OD C to evaluate the biofilm formation ability. OD>4OD C indicates strong biofilm formation, 2OD C <OD≤4OD C indicates medium biofilm formation, OD C <OD≤2OD C indicates weak biofilm formation, OD≤OD C indicates no biofilm formation.

[0085] (3) Data analysis: The results are shown in Figure 4 . It can be seen from Figure 4 that with the increase of the culture time, the biofilm formation amount of Escherichia coli also increases. At 72 h, the OD600 The OD values are all greater than those at 24 h 600 values. The biofilm formation amounts of the strains after being treated with nano-ozone water for 72 h are all lower than those of the control group to varying degrees. The reduction values of nano-ozone water for OD 600 show significant differences. According to the data results, it can be concluded that under the treatment of nano-ozone water, Escherichia coli can reduce the biofilm formation amounts of the strains with biofilm formation.

[0086] Example 5 Observe the effects of nano-ozone water on the morphology and internal structure of Escherichia coli

[0087] 1. Observe the morphological changes of Escherichia coli after being treated with nano-ozone water by field emission scanning electron microscopy

[0088] (1) Bacterial incubation: Scrape an appropriate amount of bacteria from the solid medium, wash and vortex mix them with sterile PBS, and repeat the operation 3 times to remove impurities such as sugars, salts, and metabolites in the solid medium. Use 45 mL of nano-ozone water with a concentration of 8 mg / L to incubate with 5 mL of Escherichia coli ATCC25922, GK-15, HZ-130, and SW2-1 bacterial suspensions with a concentration of 1.0×10 8 CFU / mL for 10 min to obtain a mixed solution of nano-ozone water and bacterial suspension, which is used as the experimental group (ONW). Use broth medium to replace nano-ozone water to treat the strains, which is used as the control group (Control).

[0089] (2) Sample treatment: Take samples from each group, centrifuge them at 4000 r / min for 20 min, and take about 0.1 mL of the bacterial precipitate. Add glutaraldehyde fixative and place it under refrigerated conditions for 4 - 24 h. Wash it 3 times with sterile PBS, and then wash the bacteria successively with 30%, 50%, and 70% ethanol, and place it at room temperature for 20 min. Finally, pre-cool the bacterial precipitate in an environment of -80 °C for 0.5 - 4 h, then cool it at a temperature of -10 - 50 °C, and then put it into a vacuum (11.3 - 13 Pa) freeze dryer for freeze-drying for 6 - 24 h. Use a field emission scanning electron microscope to observe the changes in the external three-dimensional structure of Escherichia coli treated with nano-ozone water, and take pictures to record the obtained image results.

[0090] (3) Image observation: The results are shown in Figure 5, where A is E. coli ATCC25922, B is E. coli GK-15, C is E. coli HZ-130, and D is E. coli SW2-1. It can be seen that the bacterial membrane of E. coli is broken after nano-ozone water treatment. According to the control group, ATCC25922 presents a uniform long rod-shaped structure, and the surface is relatively smooth and round, and the bacteria are three-dimensional and full, indicating that the bacterial membrane of E. coli ATCC25922 is intact. After treatment with nano-ozone water, large areas of wrinkles and ruptures appear on its surface, the three-dimensional structure of the bacteria changes significantly, and the external morphology is deformed and rugged. After treatment with nano-ozone water, the morphology changes significantly, the content of the substance is lost, the outside of the bacteria becomes concave and shriveled, and the bacterial membrane is destroyed, leading to the death of the bacteria.

[0091] 2. Transmission electron microscopy and room temperature ultrathin section electron microscopy observation of the morphological changes of Escherichia coli after nano-ozone water treatment

[0092] (1) Bacterial incubation: Scrape an appropriate amount of bacteria from the culture medium, wash with sterile PBS, vortex and mix, and repeat the operation three times. Select 45 mL of nano-ozone water with a concentration of 5.08 mg / L and mix with 5 mL of nano-ozone water with a concentration of 1.0×10 8 The suspension of E. coli ATCC25922, GK-15, SW2-1 and HZ-130 with CFU / mL was incubated for 10 min as the experimental group (ONW). The broth medium was used to replace the nano-ozone water treatment strains as the control group (Control).

[0093] (2) Sample processing: Each group took samples and added them to glutaraldehyde fixative solution and let them stand for 4 hours before centrifugation to allow the bacteria to adsorb on the copper mesh. Sodium phosphotungstate negative staining was performed for 30 seconds, and the solution was blotted until the copper mesh was completely dry. The samples were added to glutaraldehyde fixative solution and let stand for 4 hours before centrifugation and ultrathin sectioning was performed at room temperature. The changes in the appearance and internal structure of Escherichia coli caused by nano-ozone water were observed using a KV3000 transmission electron microscope, and the images were recorded.

[0094] (3) Image observation: Results are shown in Figure 5 It can be seen that the bacterial structure of ATCC25922 was destroyed after being treated with nano-ozone water, and the standard bacillus morphology was lost. GK-15 and SW2-1 may have vacuoles inside the cells due to the loss of content substances. The main change of HZ-130 is that there is obvious lysis on the cell surface, showing a trend of content substance loss. Further use of room temperature ultrathin section transmission electron microscopy to observe the changes in the internal structure of the strains shows that vacuoles can be seen in the middle of the four strains after being treated with nano-ozone water, and the electron density is significantly reduced compared with the control group, indicating that nano-ozone water has caused direct damage to the intracellular substances of Escherichia coli.

[0095] Example 6 Verification of the Effect of Nano-Ozone Water on Escherichia coli at the Molecular Level of Cellular Substances Using Laser Confocal Raman Spectroscopy

[0096] (1) Cell culture: Scrape an appropriate amount of bacteria from the solid medium, wash and vortex mix with sterile PBS, and repeat the operation 3 times. Select 45 mL of nano-ozone water with a concentration of 8.0 mg / L and incubate it with 5 mL of Escherichia coli ATCC25922 and GK-15 bacterial suspensions with a concentration of 1.0×10 8 CFU / mL for 10 min to obtain a mixed solution of nano-ozone water and bacterial suspension, which is used as the experimental group (NBW). At the same time, set up a control group (Control), and replace the nano-ozone water treatment with broth medium for the strains.

[0097] (2) Sample treatment: Drop 2 μL of the bacterial solution onto a glass slide coated with aluminum foil paper and air-dry it at room temperature. Single-cell Raman spectra are obtained using a laser confocal micro-Raman spectrometer equipped with a 532 nm Nd:YAG laser and a 600 g / mm grating. After placing the air-dried bacterial sample on the stage, use a 50× objective lens with a numerical aperture of 0.50 to observe single cells and obtain Raman signals. The integration time for each single-cell Raman spectrum is 35 s, and the laser power is 100%.

[0098] (3) Image observation: The results are shown in Figure 6 . It can be seen that in the Raman spectrum of the standard strain ATCC, the 1547 cm -1 peak is the N-H bending vibration peak, and this characteristic absorption peak belongs to nucleic acid-related substances. The Raman peak at 1458 cm -1 is assigned to the contribution of the biomolecule peptidoglycan, which belongs to the characteristic peak of the cell wall. The 1338 cm -1 peak is the ATP characteristic peak, and the 968 cm -1 peak is the polysaccharide characteristic peak, which belongs to the characteristic peak of the main components in the cell membrane. In the Raman spectrum after using nano-ozone water, except that the characteristic peak of ATP is slightly more obvious, other peaks are significantly weaker, indicating that the addition of nano-ozone water can damage the structure of the strain, including the substances that may be contained in its cell membrane and cell wall. Similarly, in the Raman spectra of the other 3 strains of Escherichia coli, the positions and intensities of the Raman spectral peaks of the main components in the strains are basically the same as those of the standard strain, indicating that the main components and their contents in the strains are basically the same. In summary, nano-ozone water can weaken the nucleic acids, cell membrane and cell wall components, and the metabolite ATP in the cell contents of the strain, and cause the death of the strain under their combined action.

[0099] Example 7 Effect of Nano-Ozone Water on Bacterial Apoptosis

[0100] Test strains: Escherichia coli ATCC 25922, GK-15, SW2-1, and HZ-130;

[0101] Salmonella CVCC571, B-2, GD-23, and JX-18;

[0102] Enterococcus ATCC29212, GX-22, LG4-2, and HX-6;

[0103] Campylobacter NCTC11168, 1-19, 2-3, and JH-2.

[0104] Test method: Mix 45 mL of nano-ozone water with concentrations of 2, 4, and 8 mg / L respectively with 5 mL of the test strain bacterial solution with a concentration of 1.0×10 8 CFU / mL and treat for 10 min. At the same time, use pure water (without ozone) as a control.

[0105] Measurement method: Centrifuge the treated bacterial solution at 3000 r / min for 5 min, aspirate the supernatant, and leave about 50 μL of the culture solution to avoid aspirating the cells. Then add about 1 mL of PBS pre-cooled at 4°C, resuspend the cells, and transfer them into a 1.5 mL centrifuge tube, and repeat twice to leave the precipitate. Resuspend the cells with 250 mL of Binding Buffer diluted with deionized water, and then add 400 μL of PBS. When staining, add 5 μL of Annexin V-FITC and 10 μL of 20 μg / mL PI staining agent according to the need in the dark. After mixing, incubate in the dark at room temperature for 15 min, filter it into a flow cytometry tube, and analyze it using a flow cytometer.

[0106] Results: Results Figures 7 - 10 are shown. In the figure, the horizontal axis represents the fluorescence intensity of the Annexin V-FITC dye on the cell membrane, and the vertical axis represents the fluorescence intensity of the PI dye in the cell nucleus. Each point represents an identifiable cell particle, and the density of the points reflects the bacterial concentration corresponding to the fluorescence intensity. The Q1, Q2, Q3, and Q4 quadrants respectively represent fragmented cells FITC(-) / PI(+), apoptotic cells FITC(+) / PI(-), broken cells FITC(+) / PI(+), and intact cells FITC(-) / PI(-). Q4→Q3→Q2→Q1 indicates that the degree of cell damage gradually deepens, successively undergoing the processes of cell membrane damage, esterase inactivation, and DNA cleavage. From Figures 7 - 10 it can be seen that the apoptosis of bacteria increases with the increase of nano-ozone water, and the apoptosis of drug-resistant bacteria is no different from that of standard sensitive strains.

[0107] The embodiments described above are only descriptions of the preferred embodiments of the present invention, and do not limit the scope of the present invention. Without departing from the design spirit of the present invention, various deformations and improvements made by those of ordinary skill in the art to the technical solutions of the present invention shall fall within the protection scope determined by the claims of the present invention.

Claims

1. A preparation method of nano-ozone water, characterized in that Using an ultra-fine bubble generator and an ozone generator, set the inlet pressure of oxygen to 0.4 - 0.6 MPa, control the gas pressure gauge of the ozone generator within the range of 0.15 - 0.2 MPa, set the flow regulating valve of the gas flow meter to 80 - 120 mL / min, and discharge the gas at a flow rate of 30 s; while introducing the gas, perform ultrasonic treatment to prepare the nano-ozone water.

2. Use of the nano-ozone water prepared by the preparation method according to claim 1 in the preparation of a bacterial disinfection product, characterized in that, The bacterial disinfection product has the function of inhibiting and / or killing foodborne pathogenic bacteria; the foodborne pathogenic bacteria include Escherichia coli.

3. The application according to claim 2, wherein The foodborne pathogenic bacteria also include Campylobacter, Salmonella, and Enterococcus.

4. The application according to claim 2, wherein The concentration of the nano-ozone water is not less than 2 mg / L.

5. A bacteria disinfection product, characterized in that, The active ingredient in the bacterial disinfection product is the nano-ozone water prepared by the preparation method described in claim 1.

6. Use of the nano-ozone water prepared by the preparation method according to claim 1 in the preparation of a bacterial lysate, characterized in that, The bacterial lysate has the function of destroying the bacterial cell membrane; the bacteria include Escherichia coli.

7. The application according to claim 6, wherein The bacteria also include Campylobacter, Salmonella, and Enterococcus.

8. The application according to claim 6, characterized in that, The concentration of the nano-ozone water is not less than 8 mg / L.

9. A bacterial lysate, characterized in that, The active ingredient in the bacterial lysate is the nano-ozone water prepared by the preparation method described in claim 1.

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