Micro-nano ozone water saturation and its application in the preservation of fruits and vegetables such as sweet potatoes and lotus seeds

Abstract

This invention discloses micro-nano ozone bubble water and its application in the preservation of fruits and vegetables such as sweet potatoes and lotus seeds. The micro-nano ozone bubble water is prepared by thoroughly mixing gaseous ozone with a liquid medium and then aerating the mixture. The liquid medium is a 1%-3% (v / v) citric acid solution. The prepared micro-nano ozone bubble water exhibits good stability and excellent bactericidal properties in an acidic system, significantly inhibiting *Escherichia coli*, *Staphylococcus aureus*, and pathogens causing sweet potato stem rot and root rot. This technology can effectively extend the storage period of sweet potatoes, tangerines, and lotus seeds, reduce weight loss and decay, and maintain the firmness and nutritional content of fruits and vegetables through sterilization, metabolic regulation, and optimization of the microbial community structure, providing theoretical and technical support for the green preservation of agricultural products.

Description

Technical Field

[0001] This invention belongs to the field of food preservation technology, specifically relating to micro-nano ozone bubble water and its application in the preservation of fruits and vegetables such as sweet potatoes and lotus seeds. Background Technology

[0002] Fresh fruits are rich in water, sugar, various vitamins, organic acids, and minerals, making them an important source of nutrition for the human body. However, fruits are susceptible to mechanical damage and microbial contamination during harvesting, transportation, and storage, leading to a decline in quality and reduced commercial value. Currently, traditional preservation methods mainly rely on low-temperature storage combined with chemical preservatives. While these methods can ensure food safety for a certain period, long-term use may pose potential risks. The overuse of chemical preservatives may accelerate the evolution of drug-resistant bacteria, exacerbating antibiotic resistance problems, and the impact of their degradation products on the ecological environment is equally significant. Therefore, developing a new, natural, safe, efficient, easy-to-operate, and environmentally friendly preservation method is increasingly urgent. Summary of the Invention

[0003] Based on the above problems, this invention provides a micro-nano ozone bubble water and its application in the preservation of fruits and vegetables such as sweet potatoes, tangerines and lotus seeds, providing efficient purification and disinfection technology for the environmental protection industry, food industry, agriculture industry, medical industry and other biological industries, and providing new ideas for clean fermentation technology equipment.

[0004] The first objective of this invention is to provide a method for preparing micro-nano ozone bubble water, which includes the following steps: fully mixing gaseous ozone with a liquid medium to carry out an aeration reaction, wherein the liquid medium is a citric acid solution with a volume ratio of 1%-3%.

[0005] Preferably, the ozone gas is introduced into a micro-nano bubble generator and fully mixed with the liquid medium before undergoing an aeration reaction; the liquid medium is a 2% citric acid solution by volume, with a pH of 2.0-2.5.

[0006] Preferably, a schematic diagram of the micro-nano ozone bubble water preparation device is shown below. Figure 1 As shown, air is used as the gas source, and ozone gas is generated by an ozone generator with an output concentration of 100 mg / L, which is then connected to the air inlet of the vortex micro-nano bubble generator. The inlet and outlet of the micro-nano bubble generator are placed in a sealed liquid container (reaction tank). A 2% citric acid solution (by volume) enters the micro-nano bubble generator through the inlet. The gaseous ozone and the 2% citric acid solution are thoroughly mixed within the micro-nano bubble generator for aeration. The operating parameters of the micro-nano bubble generator are set as follows: inlet pressure 0.3 MPa, aeration flow rate 0.5 L / min, and a volume ratio of 1:5 for mixing gaseous ozone and 2% citric acid solution.

[0007] The second objective of this invention is to provide micro / nano ozone bubble water prepared by the above-described method.

[0008] A third objective of this invention is to provide the application of the aforementioned micro-nano ozone bubble water in antibacterial activity.

[0009] Preferably, the bacteria are Escherichia coli, Staphylococcus aureus, sweet potato stem rot pathogen and / or sweet potato root rot pathogen.

[0010] Preferably, the liquid phase ozone concentration of the micro-nano ozone bubble water is 5-10 mg / L.

[0011] A third objective of this invention is to provide the application of the aforementioned micro-nano ozone bubble water in food preservation.

[0012] Preferably, the liquid phase ozone concentration of the micro-nano ozone bubble water is 1-8 mg / L.

[0013] Preferably, the food is a plant-based food, and the preservation is achieved by maintaining color, inhibiting browning, maintaining firmness, inhibiting decay, reducing water evaporation and weight loss, regulating respiration and metabolic consumption, maintaining soluble solids content, maintaining titratable acid content, inhibiting total phenol loss, inhibiting vitamin C loss, inhibiting starch degradation, inhibiting lignin accumulation, inhibiting malondialdehyde accumulation, inhibiting cellulase activity, inhibiting amylase activity, maintaining peroxidase activity, and / or inhibiting pathogen growth.

[0014] Preferably, the plant-based food is sweet potato, tangerine, or lotus seed.

[0015] Preferably, the sweet potato is a sweet potato infected with sweet potato stem rot pathogen and / or sweet potato root rot pathogen.

[0016] This invention utilizes a gas-liquid dispersion method to prepare micro / nano ozone bubble water, using citric acid as a solubilizer. Under acidic conditions, the solubility, release time, and molecular stability of ozone are superior to those under alkaline conditions, with a slower decomposition rate. Particle size and Zeta potential analysis show that under low pH conditions, H⁺ accumulation generates electrostatic repulsion, resulting in a more uniform bubble structure and improved stability. EPR detection confirms that both pure water and citric acid systems can generate hydroxyl radicals (·OH), while citric acid significantly inhibits ·OH generation intensity, moderately reducing oxidative capacity and ensuring antibacterial effects while avoiding excessive oxidative damage to fruit and vegetable tissues. The effects of different concentrations of micro / nano ozone bubble water on *Escherichia coli* (E. coli) were investigated using broth dilution and plate counting methods. Escherichia coli Staphylococcus aureus ( Staphylococcus aureus ), the pathogen of sweet potato stem rot ( Dickeya dadantiiEch36, Reference: Relationships of preharvest weather conditions and soil factors to susceptibility of sweet potato to postharvest decay caused by Rhizopus stolonifer and Dickeya dadantii GFP markers and colonization patterns of sweet potato stem rot pathogens and sweet potato root rot pathogens ( Fusarium solani-melongenae 24-3, Published Literature: Whole-Genome Sequencing and Comparative Genome Analysis of Fusarium solani-melongenae The antibacterial activity of strain CRI 24-3 from *Causing Fusarium Root and Stem Rot in Sweetpotatoes* was best observed at a concentration of 5 mg / L. Cryo-electron microscopy revealed that after treatment with water containing micro-nano ozone at this concentration, the pathogen's cell membrane exhibited indentation and collapse, resulting in loss of cell integrity and thus effective antibacterial action. In fruit and vegetable preservation applications, the citric acid system moderately reduces the system's oxidizing capacity, which can prolong the antibacterial effect while avoiding excessive oxidative damage to fruit and vegetable tissues caused by strong oxidation.

[0017] Micro-nano ozone bubbling water treatment effectively inhibits the growth of pathogenic microorganisms, reduces the rot rate of sweet potatoes, and significantly affects the respiration intensity and accumulation of various metabolites during storage. Throughout the storage period, samples treated with this process consistently showed better total starch content, hardness, and soluble solids content compared to the control group. The lignin content, α-amylase activity, and cellulase activity of sweet potatoes treated with micro-nano ozone bubbling water exhibited a trend of first increasing and then decreasing, showing significant differences compared to the control group. This research demonstrates that this treatment technology can effectively inhibit the growth and reproduction of sweet potato pathogens while maintaining good storage quality. These results provide a theoretical basis and technical support for developing safe, efficient, and environmentally friendly new methods for preserving fruits and vegetables.

[0018] The tangerine samples treated with micro-nano ozone bubble water showed significantly lower decay and weight loss rates than the control group, and exhibited superior antioxidant capacity. This effectively slowed the fruit decay process and reduced abnormal fluctuations in respiration intensity during storage, thus extending the fruit's shelf life. Compared to the control group, micro-nano ozone bubble water treatment more effectively maintained the content of soluble solids, titratable acids, total phenols, and vitamin C in tangerines, with a significant increase in both vitamin C and total phenol content. This indicates that this treatment technology has a positive effect on improving the nutritional quality of tangerines during storage and can provide an efficient and feasible technical reference for the food preservation field.

[0019] The combination of micro-nano ozone soaking in water and modified atmosphere packaging effectively maintains the color stability of lotus seed peel and delays aging. L * 、 a * 、b * The mixed treatment significantly reduced malondialdehyde (MDA) accumulation and browning, while also decreasing respiration rate, reducing water evaporation and weight loss, stabilizing fruit firmness, and increasing soluble solids and starch content. By regulating starch degradation and sugar balance, it effectively maintained nutritional quality over time. This mixed treatment demonstrated a significant preservation effect on fresh lotus seeds, laying a theoretical foundation for developing safer and more efficient preservation technologies. This research also provides theoretical support for the industrial application of mixed treatment in post-harvest storage of fresh lotus seeds.

[0020] This invention addresses the problem of post-harvest perishability of fruits and vegetables. The prepared micro-nano ozone-infused water (including a combination with a food storage container) exhibits good stability and excellent bactericidal properties in acidic systems (with optimal effect at a concentration of 5 mg / L). This technology can effectively extend the storage period of sweet potatoes, tangerines, and lotus seeds, reduce weight loss and decay, and maintain the firmness and nutritional content of fruits and vegetables through sterilization, metabolic regulation, and optimization of microbial community structure, providing theoretical and technical support for the green preservation of agricultural products. Attached Figure Description

[0021] Figure 1 This is a schematic diagram of a device for preparing micro-nano ozone bubble water (OMNBW).

[0022] Figure 2 The dissolved concentration of ozone in water under different treatment conditions (A) and the ozone exposure during continuous aeration (B) are also considered.

[0023] Figure 3 The values ​​are dissolved ozone concentration (A), ozone exposure (B), ozone half-life (C), cumulative micro / nano bubble size (D), and zeta potential (E) under different pH conditions.

[0024] Figure 4These are the DMPO-OH EPR spectra of micro- and nano-ozone bubbles in water under different solvent solubilization conditions.

[0025] Figure 5 yes F. soani-melongenae 24-3 Micro-nano ozone bubble water under different liquid phase ozone concentrations was cultured in a culture medium for 7 days and... D. dadantii E36 E. coli, Saureus Growth images and antibacterial rates of the culture medium after 24 hours of cultivation under different liquid ozone concentrations.

[0026] Figure 6 yes F. soani-melongenae 24-3 The effects of different liquid phase ozone concentrations on spores and ozone in micro-nano ozone bubble water after 7 days of culture in a culture medium. D. dadantii E36 E. coli, S. aureus Changes in the cell wall of spores in a spore solution cultured in a medium for 24 hours under different liquid ozone concentrations, as observed under a SEM electron microscope.

[0027] Figure 7 Micro-nano ozone water is effective against infection. D. dadantii Ech36 and F. solani-melongenae The effects of 24-3 on the respiration rate (A), firmness (B), decay rate (C) and weight loss (D) of sweet potatoes at the end of storage.

[0028] Figure 8 Micro-nano ozone water is effective against infection. D. dadantii Ech36 and F. solani-melongenae The effects of titratable acid (A), soluble solids (B), MDA (C), and cellulase activity (D) on sweet potato in 24-3.

[0029] Figure 9 Micro-nano ozone water is effective against infection. D. dadantii Ech36 and F. solani-melongenae Effects of α-amylase (A), lignin content (B), peroxidase (C), and total starch content (D) on sweet potato 24-3.

[0030] Figure 10 This describes the changes in the following parameters of tangerines during storage: rot (A), weight loss (B), titratable acid (C), soluble solids (D), total phosphorus (TP) (E), and total vitamin C (VC) (F).

[0031] Figure 11 This describes the effect of different treatments on the color change of lotus seeds after harvest.

[0032] Figure 12The effects of different treatments on postharvest lotus seed respiration rate (A), hardness (B), soluble solids (C), total ta (TA) (D), weight loss (E), peroxidase (F), total starch content (G), and malondialdehyde content (H).

[0033] Different letters in the figure indicate significant differences between different treatments. P <0.05). Detailed Implementation

[0034] The following embodiments are further illustrations of the present invention, but not limitations thereof.

[0035] Unless otherwise specified, the experimental methods used in the embodiments of this invention are all conventional methods.

[0036] Unless otherwise specified, all materials and reagents used in the embodiments of this invention are commercially available.

[0037] Soluble starch (analytical grade) was purchased from Tianjin Kemei Chemical Reagent Co., Ltd.; potassium iodide and sodium thiosulfate (both analytical grade) were purchased from Sinopharm Chemical Reagent Co., Ltd.; sulfuric acid (analytical grade) was purchased from Shanghai Suisheng Company. Glacial acetic acid (analytical grade) was purchased from Shandong Keyuan Biochemical Co., Ltd. Microbial culture consumables: hydrolyzed casein peptone (MH) broth, nutrient agar, potato dextrose agar (PDA) and potato dextrose broth (PDB) were purchased from Guangdong Huankai Biotechnology Co., Ltd., and petri dishes were purchased from Beijing Jieke Technology Co., Ltd. Test strains included: *Escherichia coli* (E. coli) long-term stored in our laboratory. Escherichia coli, E. coli ) and Staphylococcus aureus ( Staphylococcus aureus, S. aureus ); and stem rot pathogens originating from the Guangdong Academy of Agricultural Sciences base in Guangzhou, Guangdong Province. (D. dadantii) Ech36) and root rot pathogen ( F. solani-melongenae 24-3).

[0038] Example 1: Preparation and Characterization of Micro / Nano Ozone Bubble Water

[0039] 1. Experimental Methods

[0040] (1) Preparation of micro-nano ozone bubble water by gas-liquid dispersion method

[0041] A schematic diagram of the micro-nano ozone bubble water preparation device is shown below. Figure 1As shown, the ozone generator (HY-006-20A, Guangzhou Dahuan Ozone Technology Co., Ltd.) uses air as the source of ozone gas and connects the generated ozone gas to the air inlet of the vortex micro-nano bubble machine (Huangdao District Weinayan Internet Sales Center), with an ozone output concentration of 100 mg / L. Furthermore, the inlet and outlet of the micro-nano bubble machine are placed in a stainless steel liquid-sealed container (reaction tank) with a height of 26.8 cm, a bottom diameter of 21 cm, and a volume of approximately 10 L. Using tap water as a control, aqueous solutions of glacial acetic acid, citric acid, and sodium bicarbonate with volume ratios of 0.5%, 1%, and 2% were prepared, respectively. The pH of the 2% citric acid solution was 2.1. NaOH was used to adjust the pH of the 2% citric acid solution, setting gradient solutions with pH values ​​of 2.34, 4.48, 6.38, 8.96, and 11.2. Micro-nano ozone bubble water was prepared in aqueous solutions of different concentrations. The operating parameters of the micro-nano bubble machine were set as follows: inlet pressure 0.3 MPa, aeration flow rate 0.5 L / min, and gaseous ozone and test solution were mixed by the micro-nano bubble machine at a volume ratio of 1:5. After aeration for 10 minutes, 20 minutes and 30 minutes, the solution in the reaction tank was shaken and sampled. The samples were placed in sterile centrifuge tubes and stored at 4°C for subsequent detection of liquid ozone concentration, bubble particle size distribution and other indicators.

[0042] (2) Detection of dissolved ozone concentration

[0043] Iodometric titration is a classic method for ozone detection. Its basic principle is based on the redox reaction between ozone (a strong oxidant) and I⁻ ions in a KI solution, generating free iodine. As the free radical concentration increases, the system's color gradually changes from light yellow to deep red. Adding Na₂S₂O₃ standard titrant as a reducing agent converts the generated free iodine into I⁻. A starch indicator is added to determine the endpoint. When the last drop of standard titrant is added, the mixture changes from blue to colorless, indicating that chemical equilibrium has been reached. According to the established kinetic model of the two-step reaction, each mole of ozone requires 2 moles of Na₂S₂O₃, thus accurately determining the ozone concentration in the target sample.

[0044]

[0045] In the formula: C is the concentration of ozone water (mg / L); ANa is the volume of sodium thiosulfate standard solution used (mL); B is the concentration of sodium thiosulfate standard solution (mol / L); V0 is the sampling volume of ozone water (mL).

[0046] (3) Ozone exposure in water containing micro-nano ozone bubbles

[0047] Ozone exposure refers to the total amount of ozone concentration (mol·L⁻¹) in the liquid phase accumulated over a certain period of time. Samples were taken during the aeration process (0 – 30 min) and after aeration was stopped (0 – 24 h), and the relationship curve of liquid phase ozone concentration with time was plotted. The corresponding ozone exposure ∫[O₃]dT was obtained by numerical integration using the integration function of Origin software.

[0048] (4) Determination of particle size and zeta potential of micro- and nano-sized ozone bubbles

[0049] The pH of a 2% citric acid solution was adjusted using NaOH to create a gradient solution with pH values ​​of 2.34, 4.48, and 6.38. Micro-nano ozone bubble water was prepared in these solutions with different pH values. Ozone generated by an ozone generator was introduced into the micro-nano bubble device, which was then immersed in 10 L of water. After reacting for 30 minutes, samples were taken and stored at room temperature for 10 hours. Data acquisition and quantitative analysis were then performed using a nanoparticle size analyzer and a Zeta potential detection device.

[0050] (5) Ozone mass transfer efficiency

[0051] Through a quantitative comparative study of micro- and nano-ozone bubble water under different treatment conditions, the dissolution mass transfer process was characterized by the maximum dissolved ozone concentration and the average ozone solubilization rate in each experimental group. The average ozone solubilization rate is the average rate of increase in dissolved ozone concentration from bubble generation until the dissolved ozone concentration first reaches its peak.

[0052] (6) Determination of hydroxyl radical content

[0053] Micro- and nano-sized ozone bubbles were prepared using water and a 2% citric acid solution. Electron paramagnetic resonance (ESR) technology was employed, with 50 mM 5,5-dimethyl-1-pyrrolidone-N-oxide (DMPO) and 2,2,6,6-tetramethyl-4-piperidinol (TMP) as spin trapping agents, successfully capturing free radicals in the solution and forming stable adducts. The samples were then transferred to a quartz capillary for ESR detection.

[0054] (7) Evaluation of the antibacterial efficacy of micro-nano ozone bubble water

[0055] The effects of micro / nano ozone bubble water (prepared with 2% citric acid by volume) on the efficacy of ozone bubble water were evaluated using the plate count method. E. coli , S. aureus , D. dadantii Ech36The antibacterial activity was determined. First, the sample solution was serially diluted using hydrolyzed casein peptone broth, resulting in ozone concentrations in the liquid phase of 5 mg / L, 2.5 mg / L, 1.25 mg / L, 0.625 mg / L, 0.3125 mg / L, and 0 mg / L, respectively. 10... 6 A bacterial suspension of CFU / mL was added to the diluted sample at a volume ratio of 1:1. After incubation at 37°C for 4 hours, the absorbance (OD) of the sample at 600 nm was measured using a microplate reader. 600 Then, 100 µL of the solution was evenly spread onto the corresponding nutrient agar plates, and these plates were incubated for another 24 hours at the same temperature to promote bacterial growth. In the plate culture experiment, if the observed bacterial cell community number was less than five colony-forming units, this concentration was defined as the minimum bactericidal concentration. The colony-forming units (CFU) method was used. A preliminary evaluation and discussion were conducted based on the bactericidal ratio. The following is the formula for calculating the bactericidal ratio based on the number of colonies on the agar plate:

[0056] Here, 'a' represents the number of live bacteria in the control group, while 'b' represents the number of live bacteria in the sample.

[0057] The effects of micro-nano ozone bubble water (prepared with 2% citric acid by volume) on root rot pathogens were evaluated using the broth dilution method. F. solani-melongenae To achieve the antibacterial efficacy of 24-3), firstly, the fungal hyphae covering the culture dish were washed with sterile water on a clean bench. Next, the mixture was filtered through 400-mesh gauze, and then rinsed with 5000 ml of water. g Centrifuge for 5 minutes. After removing the supernatant, concentrate the solution and adjust the concentration of the spore solution to 1×10⁻⁶ using a hemocytometer. 7 CFU / mL. The concentration was determined using a broth dilution method in a 96-well plate. 200 µL of a solution consisting of PDB medium and micro / nano ozone bubble water was added to the first well of the 96-well plate, with a liquid ozone concentration of 5 mg / L. Subsequently, 100 µL of PDB was added to the remaining wells of the 96-well plate, and the 100 µL mixture from the first well was transferred to the second well, and so on, resulting in liquid ozone concentrations of 5 mg / L, 2.5 mg / L, 1.25 mg / L, 0.625 mg / L, 0.3125 mg / L, and 0 mg / L, respectively. Next, 100 μL of spore suspension (1×10⁻⁶) was added to each well. 7 CFU·mL -1Ensure thorough mixing, then cover with a 96-well plate and incubate at 28°C for 72 hours. Each sample was tested three times. A positive control was prepared by mixing 200 µL of LPDB with 100 µL of spore suspension, while the negative control used only PDB. Sterilized and cooled PDA medium was added to several 5 cm diameter petri dishes. Then, 100 μL of the mixture was extracted from each 96-well plate and inoculated onto PDA plates. All petri dishes were then incubated at 28°C for 7 days. Colony diameters were measured and data recorded. Each treatment was repeated three times. The mycelial growth formula was calculated as follows:

[0058] Where A and B are the average colony diameters of the control group and the treatment group, respectively, and C is the initial diameter of the mycelial disc.

[0059] (8) Scanning electron microscopy (SEM) characterization

[0060] Using a scanning electron microscope, the bacterial membrane damage was comprehensively studied. The bacterial samples treated with micro-nano ozone bubbles in step (7) were fixed in glutaraldehyde solution to ensure morphological stability. They were fully solidified in a dark room and then dehydrated with ethanol according to the standard procedure. The samples were first washed with phosphate buffer and then placed in ethanol environments of 25%, 50%, 75%, 90% and 100% concentrations in ascending order to gradually dehydrate. Finally, a conductive layer was applied to the surface using sputtering gold plating. All strains were observed under a scanning electron microscope and analyzed in detail. The control group of bacteria that did not undergo any treatment also underwent the above pretreatment process simultaneously.

[0061] 2. Experimental Results

[0062] (1) Effect of different solubilizers on the saturated concentration of dissolved ozone

[0063] like Figure 2 As shown in Figure A, the study found that within the 0-20 minute timeframe, the ozone concentration in the liquid phase continuously increased over time. Within the same aeration duration, the ozone solubility of the 0.5%, 1%, and 2% citric acid and 0.5%, 1%, and 2% glacial acetic acid treatment groups was significantly better than that of the 0.5%, 1%, and 2% sodium bicarbonate treatment group and the control group (CK). At 20 minutes of aeration, the 2% citric acid treatment showed the highest gaseous ozone dissolution and the highest liquid-phase ozone concentration, with a significant difference compared to the CK control group. P(≤0.05), but the liquid ozone concentrations in the 0.5%, 1%, and 2% sodium bicarbonate treatment groups were significantly lower than those in the control group (CK). Within 20-30 minutes, the liquid ozone concentrations in the 0.5%, 1%, and 2% citric acid and 0.5%, 1%, and 2% glacial acetic acid treatment groups continuously increased, indicating that the gaseous ozone dissolution in these groups had not reached saturation during this period and could continue to dissolve. In contrast, the 0.5%, 1%, and 2% sodium bicarbonate treatments showed a gradual increase, without significant fluctuations and only fluctuating within a specific range, confirming that ozone dissolution had approached saturation at this point. Notably, the liquid ozone concentration in the CK group began to decrease after 20 to 30 minutes of aeration, indicating that the liquid ozone concentration in the CK group had reached its peak at 20 minutes of aeration and began to gradually decrease after 20 minutes. After 30 minutes of aeration, the saturated ozone concentration in the liquid phase treated with 2% sodium bicarbonate was the lowest, with a peak concentration of 1.4 ± 0.30 mg / L. The saturated ozone concentration in the liquid phase treated with 2% citric acid was the highest, with a peak concentration of 12.84 ± 0.24 mg / L. The comparison of curves from different treatment groups shows that the treatment method significantly affects the saturated ozone concentration in the liquid phase. 0.5%, 1%, and 2% citric acid effectively promoted ozone solubility in water, with 2% citric acid being slightly better than 0.5% and 1% citric acid.

[0064] (2) Effect of different solubilizers on ozone exposure

[0065] like Figure 2 As shown in Figure B, the ozone exposure levels of each group were ranked according to the aeration time. The results showed that the 2% citric acid treatment had the highest exposure, followed by 1% citric acid, 2% glacial acetic acid, 0.5% citric acid, 1% glacial acetic acid, 0.5% glacial acetic acid, control group (CK), 0.5% sodium bicarbonate, and 1% sodium bicarbonate. The 2% sodium bicarbonate treatment group had the lowest exposure. The ozone exposure levels of the 0.5%, 1%, and 2% citric acid and 0.5%, 1%, and 2% glacial acetic acid treatments were consistently higher than those of the CK group, and the differences were statistically significant. P(≤ 0.05) During the entire aeration period, the ozone exposure of the 2% citric acid treatment group increased the fastest, and at the end of aeration, the ozone exposure was much higher than that of other treatment groups. The 2% sodium bicarbonate treatment group showed the slowest increase, remaining at the lowest level throughout, and was lower than other treatment groups at the end of aeration. The ozone exposure of the CK group was consistently higher than that of the 0.5%, 1%, and 2% sodium bicarbonate treatment groups, but lower than that of the 0.5%, 1%, and 2% citric acid and 0.5%, 1%, and 2% glacial acetic acid treatment groups. The results indicate that the 0.5%, 1%, and 2% citric acid and 0.5%, 1%, and 2% glacial acetic acid treatments can effectively increase the ozone exposure in the water, while the 0.5%, 1%, and 2% sodium bicarbonate treatment groups are less effective than the CK group in increasing ozone exposure. The results showed that when the aeration time was 30 minutes, the ozone exposure levels in the 0.5%, 1%, and 2% citric acid treatment groups and the 0.5%, 1%, and 2% glacial acetic acid treatment groups were all higher than those in the control group (CK). Conversely, the ozone exposure levels in the 0.5%, 1%, and 2% sodium bicarbonate treatment groups were all lower than those in the CK group, with the highest ozone exposure level (249 mol·L⁻¹) in the 2% citric acid treatment group. -1 The minimum ozone exposure for 2% sodium bicarbonate is 15.8 mol·L⁻¹. -1 In the 0.5% citric acid treatment and the control group, the ozone exposure was 157 mol·L⁻¹ min, respectively. -1 ·min and 77.2 mol·L -1 The ozone exposure was significantly lower in the 0.5%, 1%, and 2% sodium bicarbonate treatment groups than in the control group (CK), indicating that these three treatments were not conducive to the dissolution and accumulation of ozone in water. In conclusion, the effects of different treatments on ozone exposure varied significantly. P Citric acid (≤ 0.05%), 0.5%, 1%, and 2% can effectively increase ozone exposure, with 2% citric acid being the most effective.

[0066] (3) Effect of different solubilizers on ozone mass transfer efficiency

[0067] Table 1 shows the mass transfer efficiency of micro / nano ozone bubble water treated with different methods. Tables 1 and 1 show that 0.5%, 1%, and 2% citric acid and 0.5%, 1%, and 2% glacial acetic acid significantly increased the peak dissolved ozone concentration. The highest dissolved ozone concentration was achieved with 2% citric acid, reaching 12.84 mg / L, with an average ozone increase rate approximately four times that of the control group (CK). In contrast, the peak dissolved ozone concentrations with 0.5%, 1%, and 2% sodium bicarbonate were significantly lower than those with the CK, approximately 1.82, 1.46, and 2.34 times lower, respectively. After 30 minutes of aeration, the average ozone increase rates with 0.5%, 1%, and 2% citric acid and 0.5%, 1%, and 2% glacial acetic acid were much higher than those with the CK and 0.5%, 1%, and 2% sodium bicarbonate. The 2% citric acid group showed the fastest average ozone increase rate, reaching 0.43 mol·L⁻¹.-1 The next best treatments were 0.32 mol·L⁻¹, with 0.5% citric acid and 1% glacial acetic acid being the next most effective. -1 However, the average ozone increase rate of 2% sodium bicarbonate was the slowest at 0.05 mol·L⁻¹. -1 The concentration of mol·min was significantly lower than that of the control group (0.11 mol·L⁻¹). -1 The results showed that, under three different concentrations (0.5%, 1%, and 2%), the addition of citric acid and glacial acetic acid significantly improved the solubility of ozone in the aqueous phase, thereby effectively enhancing its mass transfer efficiency. Among these, the 2% citric acid treatment group showed the most outstanding performance in promoting ozone mass transfer, with significantly better results than all other treatment groups. In contrast, the 0.5%, 1%, and 2% sodium bicarbonate treatment groups not only failed to have a positive impact on the ozone mass transfer process, but their overall performance was even lower than that of the blank control group (CK) without any added substances.

[0068] (4) Dissolved ozone saturation concentration under different pH conditions

[0069] The effect of different pH conditions on dissolved ozone concentration was investigated, and ozone concentration was determined using the iodometric method. Figure 3 A. The results showed that, under different pH environments, the concentration of liquid ozone exhibited a phased change characteristic with the extension of aeration time. Overall, the initial rate of increase was relatively rapid, followed by a gradual stabilization. Furthermore, as the aeration process continued, the difference in liquid ozone concentration between different pH groups continuously widened. From the start of aeration, in the first 10 minutes, regardless of the pH value, the ozone concentration in the liquid phase steadily increased without any abnormal fluctuations. Between 10 and 20 minutes, the concentration continued to rise in the pH range of 2.34 to 8.96; however, at pH 11.2, the concentration remained relatively stable, essentially entering a plateau. Between 20 and 30 minutes, the ozone concentration in the system with a pH between 2.34 and 6.38 continued to slowly climb; while the groups with pH 8.96 and 11.2 showed no significant fluctuations, indicating that they had reached their dissolution saturation point. Based on the dissolution curves at different pH values, the trend of pH affecting the saturation concentration of liquid ozone shows that at pH 2.34, the saturation concentration reaches 8.2 ± 0.25 mg / L; however, at pH 11.2, the saturation concentration drops to only 1.68 ± 0.24 mg / L. This indicates that the lower the pH, the easier it is for ozone to accumulate in water, showing a clear negative correlation. Conversely, the higher the pH, the lower the liquid ozone concentration. This means that in acidic environments, ozone is more easily dissolved and retained in water; however, in alkaline environments, it inhibits ozone dissolution.

[0070] (5) Ozone exposure under different pH conditions

[0071] Under different pH conditions, ozone exposure levels gradually increased with increasing aeration time. For example... Figure 3 As shown in Figure B, the lower the pH value, the higher the rate of increase and the final value of ozone exposure. It is also evident that ozone exposure gradually decreases with increasing pH value, with the highest ozone exposure at pH 2.24 and the lowest at pH 11.2, showing the slowest increase in ozone exposure. When the aeration time was 30 minutes, the ozone exposure values ​​at pH 2.24, 6.38, and 11.2 were 143.8 mol·L⁻¹. -1 ·min, 101 mol·L -1 ·min, 29.2 mol·L -1 •min. Linear fitting analysis showed a significant linear relationship between ozone exposure and aeration time under different pH conditions. The slope of the linear fitting represented the rate of increase in ozone exposure over time; the higher the slope, the faster the rate of increase. After 30 minutes of aeration, the ozone exposure and time were nearly directly proportional under different pH conditions. The rate of increase in ozone exposure gradually increased with decreasing pH. Specifically, the rates for pH values ​​of 2.24, 6.38, and 11.2 were 6.06, 4.43, and 1.25, respectively. The slope was largest and the rate of increase was fastest at pH value 2.34, while the slope was smallest and the rate of increase was slowest at pH value 11.2. This indicates that low pH conditions significantly increase ozone exposure; the lower the pH, the higher the value and the faster the rate of increase in ozone exposure. High pH conditions decrease ozone exposure. Therefore, low pH conditions have a highly efficient promoting effect on ozone mass transfer.

[0072] (6) Ozone mass transfer efficiency under different pH conditions

[0073] The mass transfer efficiency of micro / nano ozone bubbles prepared at different pH values ​​was studied, as shown in Table 2. With increasing pH, the peak dissolved ozone concentration gradually decreased, with the highest dissolved ozone concentration (8.2 ± 0.25 mg / L) observed at pH 2.34. -1 The lowest dissolved ozone concentration was 1.68 ± 0.24 mg L at a pH of 11.2. -1 The average ozone increase rate at pH 2.23 was approximately four times that at pH 11.2, and the average ozone increase rate decreased with increasing pH. The fastest average ozone increase rate (0.273 mol·L⁻¹) was observed at pH 2.34. -1 At a pH of 11.2, the average ozone increase rate was the slowest, at 0.056 mol·L⁻¹. -1In summary, the lower the pH value, the higher the peak concentration of dissolved ozone and the faster the average ozone generation rate. High pH conditions reduce both the peak concentration and average ozone generation rate, thus decreasing the mass transfer efficiency of ozone in water. Therefore, ozone solubility is affected by different pH values, with low pH conditions promoting ozone dissolution in water.

[0074] (7) Ozone half-life under different pH conditions

[0075] Further investigation was conducted into the decay process of dissolved ozone concentration in water under different pH conditions. For example... Figure 3 As shown in Figure C, the higher the pH value, the shorter the half-life of dissolved ozone. Furthermore, the dissolved ozone concentration shows a continuous decreasing trend over time after aeration stops, with the rate of decrease accelerating in the early stages compared to the later stages. pH has a significant impact on ozone dissolution and stability: under acidic conditions (especially pH=2.34), the initial dissolved ozone concentration is the highest, and the rate of decay over time is the slowest, maintaining a high level even after 24 hours. As pH increases, the initial ozone concentration gradually decreases, and the decomposition rate significantly accelerates. Under strongly alkaline conditions (pH=11.2), ozone decomposes almost rapidly. These results confirm that a low-pH acidic environment can effectively inhibit ozone decomposition and significantly improve the stability of the micro / nano ozone bubble water system. Specifically, the longest ozone half-life (6.68 hours) is observed at pH 2.34 (close to the half-life of 6.83 hours at pH 2.1 with 2% citric acid); the shortest half-life is observed at pH 11.2. The overall dissolved ozone concentration is higher in low-pH environments than in high-pH environments. The above phenomena are closely related to the differences in ozone solubility in water. Ozone solubility is much higher in low-pH environments than in high-pH environments, exhibiting a higher peak concentration and a faster rate of concentration increase. In summary, the lower the pH value of water, the better the solubility and stability of ozone in water, and the longer its half-life; conversely, high-pH environments accelerate ozone decomposition, thereby shortening its half-life.

[0076] (8) Particle size and potential of micro-nano ozone bubble water

[0077] To investigate the influence mechanism of pH on the stability of micro / nano ozone bubbles in water, this invention systematically analyzed the particle size distribution of micro / nano ozone bubbles under different pH conditions. Figure 3(D) Sodium hydroxide tablets were used to adjust the pH of the water, and the cumulative particle size distribution of each group of bubbles was measured 10 hours after aeration was stopped. The results showed that under different pH conditions, the cumulative particle size distribution of micro- and nano-bubbles exhibited a trend of "rapid increase followed by a gradual plateau," and the bubble size gradually decreased with increasing pH. When the pH increased from 2.27 to 6.67, the bubble size decreased from approximately 90 μm to approximately 40 μm; as the particle size increased, the rate of increase in the cumulative particle size distribution gradually slowed down. This indicates that pH is a key factor in regulating the particle size distribution of micro- and nano-ozone bubbles.

[0078] To further elucidate the regulatory mechanism of pH on the stability of micro / nano ozone bubble water, this invention measured the Zeta potential under different pH systems. Figure 3 E). Under different pH conditions, the Zeta potential of micro- and nano-bubbles showed a significant decreasing trend overall. When the pH value increased from 2.27 to 6.67, the Zeta potential decreased from a positive value of 8.43 mV to a negative value of -0.66 mV. In low-pH aqueous solutions, hydrogen ions (H⁺) enriched at the gas-liquid interface alter the bubble interface characteristics, causing the surface of the generated micro- and nano-bubbles to carry a positive charge. Thus, the surface charge characteristics of the bubbles and the stability of the system are highly dependent on pH conditions. As the pH value gradually increases, the Zeta potential of micro- and nano-ozone bubbles continuously decreases: under strongly acidic conditions (pH = 2.27), the Zeta potential is approximately +8.94 mV, the bubble surface carries a positive charge, the absolute value of the Zeta potential is the largest, the electrostatic repulsion between bubbles is stronger, and the system stability is optimal; while at pH = 6.67, the Zeta potential approaches the isoelectric point, the net charge on the bubble surface is almost zero, the electrostatic repulsion between bubbles is weakest, and aggregation is very likely to occur, resulting in the worst system stability. The results in summary indicate that a strongly acidic environment with pH = 2.27 is more conducive to maintaining the stability of the micro-nano ozone bubble system.

[0079] (9) Electron paramagnetic resonance analysis

[0080] Electron paramagnetic resonance (EPR) technology was used to detect hydroxyl radicals (·OH) in a micro / nano ozone bubble water system using DMPO as a spin trapping agent. Figure 4As shown, typical DMPO-OH quartet characteristic peaks were observed in both pure water (H2O) and citric acid (C6H8O7) media systems, confirming that micro- and nano-ozone can effectively activate and generate •OH radicals in both systems. Comparison of spectral line intensities and integrated areas reveals that the DMPO-OH signal in the pure water system is significantly stronger than that in the citric acid system. This indicates that citric acid can effectively weaken the strong oxidative excitation effect of ozone by adjusting the system's pH, moderately reducing the generation and accumulation of •OH radicals within the system. A moderate level of •OH radicals is beneficial in preventing excessive oxidative stress damage to fruit and vegetable tissues, which is of great significance for maintaining the integrity of fruit and vegetable cells and improving the safety of composite preservation treatments.

[0081] (10) Evaluation of the antibacterial effect of micro-nano ozone bubble water

[0082] Gradient concentration experiments were conducted to determine the effects of micro-nano ozone bubbles on water. E. coli , S. aureus , D. dadantii E36 and F. soani-melongenae The antibacterial effect of 24-3 was investigated to determine its effective concentration range for inhibiting the aforementioned pathogens. Results are as follows: Figure 5 As shown, the number of colonies on the plates gradually increases as the ozone concentration decreases. In the treatment with a liquid ozone concentration of 5 mg / L, no obvious colonies were observed in the culture dishes of any of the test strains, indicating that there was almost no colony growth in any treatment group at this concentration. In the treatment with a liquid ozone concentration of 2.5 mg / L… F. soani- melongenae Slight mycelial growth was observed at 24-3. D. dadantii E36 showed a small number of bacterial colonies growing, while E. coli, S. aureus Colony growth began to appear, with the number of colonies all exceeding 5, whereas under other concentration conditions... F. soani- melongenae 24-3 D. dadantii E36 E. coli, Saureus Colonies grew on all samples, with higher concentrations at lower concentrations. At an ozone concentration of 0 mg / L, almost all bacterial strains grew abundantly on the plates, forming dense colonies that covered the entire petri dish. These results demonstrate the beneficial effects of micro-nano ozone-infused water on… F. soani-melongenae The minimum bactericidal concentration for 24-3 is 5 mg / L. D. dadantii The minimum bactericidal concentration of E36 is 2.5 mg / L, while E. coli and Saureus The minimum bactericidal concentration was 5 mg / L. The results showed that the bactericide had good stability and a certain bactericidal effect. Based on the number of colonies, the bactericidal rate of antibacterial activity was plotted. Compared with the control group, as the concentration of liquid ozone decreased, the bactericidal rate of the experimental group... E. coli, S. aureus The number of bacterial colonies gradually increased. No bacterial growth was observed on plates with a liquid ozone concentration of 5 mg / L, indicating that micro-nano ozone bubble water has a positive effect on bacterial growth. E. coli, S. aureus All have effective bactericidal effects. Different concentrations of micro-nano ozone in water have varying effects on... S. aureus The sterilization rates were 100%, 89.9%, 89%, 68.6%, and 51%, respectively. E. coli The sterilization rates were 100%, 95.50%, 89.60%, 57.90%, and 28.60%, respectively. D. dadantii The sterilization rates of E36 were 99.57%, 98.93%, 95.84%, 79.61%, and 7.93%, respectively. F. soani-melongenae The sterilization rates of 24-3 were 95.84%, 82.77%, 77.96%, 37.09%, and 13.93%, respectively, which further demonstrates the effectiveness of micro-nano ozone water ionization. E. coli, S. aureus、D. dadantii E36 F. soani-melongenae 24-3 has a significant antibacterial effect.

[0083] (11) Antibacterial mechanism of micro-nano ozone water bubbling

[0084] Scanning electron microscopy (SEM) was used to examine water treated with micro-nano ozone. E. coli , S. aureus , D. dadantii Ech36 and F. soani-melongenae The cell structure of 24-3 cells was observed. Figure 6 In the untreated (0 mg / L) control group, all microorganisms retained their original morphology. E. coli It has a typical rod shape and a smooth surface; S. aureus They are spherical and arranged in a regular pattern; D. dadantii Ech36 cells are plump and have intact cell walls; F. soani-melongenae The 24-3 spores were plump and densely structured. After treatment with 5 mg / L micro-nano ozone solution, the cell walls of all microorganisms suffered irreversible damage. E. coli The bacterial cells shrink and lyse, and the integrity of the cell wall is destroyed. S. aureus The cells visibly collapsed and ruptured, leaking intracellular material and causing bacterial inactivation; D. dadantii Ech36 cells were twisted and disintegrated, with a large number of cell fragments appearing; F. soani-melongenae 24-3 spore walls shrank and ruptured, exposing the internal structure. These results indicate that treatment with micro-nano ozone bubble water caused bacterial cell membranes to rupture or break down, resulting in the loss of their normal structure and function. Micro-nano ozone bubble water exhibited significant destructive effects on Gram-negative bacteria, Gram-positive bacteria, and fungal spores, demonstrating excellent broad-spectrum antibacterial properties.

[0085] Example 2: Study on the effect of micro-nano ozone water on post-harvest preservation and mechanism of action of sweet potato

[0086] 1. Experimental Methods

[0087] (1) Sweet potato preservation experiment

[0088] The micro-nano ozone bubble water prepared with 2% citric acid in Example 1 was selected, and the micro-nano ozone bubble water was diluted with a volume ratio of 2% citric acid aqueous solution to a liquid phase ozone concentration of 2 mg / L.

[0089] Select sweet potatoes of similar maturity, size, and shape, and use a concentration of 10... 7 CFU / mL D. dadantii Ech36 and spore concentration of 10 7 CFU / mL F. solani-melongenae Soak the sweet potatoes for 3 minutes at 24-3°C, allow them to air dry at room temperature, and then soak them in a solution of micro-nano ozone at a concentration of 2 mg / L for 3 minutes. After air drying, store the sweet potatoes at room temperature (25±2°C, relative humidity 60±5%). A CK1 group (infected group) was set up. D. dadantii Ech36), Group T1 (infected) D. dadantii Ech36 sweet potatoes were treated with 2 mg / L micro-nano ozone in water), and CK2 group (infected) F. solani-melongenae 24-3) and T2 group (infected) F. solani-melongenae Sweet potatoes aged 24-3 were treated with 2 mg / L micro-nano ozone solution in water. Each group was replicated four times. After 0, 5, 10, 15, 20, and 30 days, changes in respiration, weight loss, firmness, soluble solids, and titratable acidity were measured. At each time point, 3-5 sweet potato samples were randomly selected and stored at -80°C using liquid nitrogen flash freezing for subsequent enzyme activity assays.

[0090] (2) Measurement of respiratory intensity

[0091] The tests were conducted using a Li-6262CO2 / H2O analyzer (LI-COR Inc., Lincoln, NE, USA). Before the actual measurement, the instrument should be turned on and preheated for at least 15 minutes until it stabilizes. The prepared sample is then placed in a 2.5 L sealed plastic container with a venting tube attached. The tube is connected to the instrument interface, and the system is started to begin measurement. For each measurement, after the data stabilizes, the CO2 concentration is recorded after 3 minutes. This process is repeated every minute until all sampling points are reached.

[0092]

[0093] In this formula, t represents the time when the CO2 concentration is recorded, i.e., min;

[0094] At time t1, C1 represents the concentration of CO2 inside the closed container, which is nmol·mol⁻¹. -1 ;

[0095] At time t2, C2 represents the concentration of CO2 inside the closed container, which is nmol·mol⁻¹. -1 ;

[0096] V represents the capacity L of the closed container;

[0097] M represents the molar mass of CO2 in g·mol⁻¹ -1 ;

[0098] V0 represents the molar volume of CO2 at a specific temperature (L·mol). -1 ;

[0099] m represents the weight of the sweet potato in kg.

[0100] (3) Hardness determination: Three sweet potatoes were randomly selected from each treatment group and tested using a GY-64 hardness tester manufactured by Adeberg Instruments Co., Ltd. The experimental data were recorded. The arithmetic mean was used to represent the characteristics and distribution of the population.

[0101] (4) Determination of rot rate: Starting from the 20th day of storage, 30 sweet potato samples were selected for rot rate determination. The disease was graded according to the diameter of the lesions: Grade 0, no symptoms; Grade 1, lesion diameter ≤ 10 mm; Grade 2, 10 mm < lesion diameter ≤ 20 mm; Grade 3, 20 mm < lesion diameter ≤ 30 mm; Grade 4, lesion diameter > 30 mm. The rot rate was only counted for Grade 0 and Grade 1 samples. The calculation formula is as follows:

[0102] N0 represents the number of grade 0 sweet potatoes (without lesions), and N1 represents the number of grade 1 sweet potatoes (lesion diameter ≤ 10 mm).

[0103] (5) Determination of weight loss rate: The weight loss rate of sweet potatoes was measured by weighing, and the weight of sweet potatoes was measured by an electronic balance with an accuracy of 0.1. The calculation formula is as follows:

[0104] In the formula, a is the weight of the sweet potato before storage, in g; b is the weight of the sweet potato on day N of storage, in g.

[0105] (6) Determination of soluble solids (TSS) and titratable acid (TA): Three sweet potato samples were randomly selected from each group, ground, filtered through four layers of gauze, and homogenized. The homogenate was measured using an ATAGO portable fruit saccharimeter (model PAL-BX, Japan). To ensure the accuracy and reliability of the data, each step was repeated three times, and the arithmetic mean of the three measurements was taken as the result.

[0106] (7) Determination of malondialdehyde (MDA) content: Use the malondialdehyde (MDA) content detection kit (Beijing Box Biotechnology Co., Ltd., Beijing, China). For specific operating procedures, please refer to the instructions for use of the kit.

[0107] (8) Determination of cellulase activity: Cellulase (CL) activity assay kit (Beijing Box Biotechnology Co., Ltd., Beijing, China) was used for the assay. The specific operation steps are as per the kit instructions.

[0108] (9) Determination of α-amylase activity: The α-amylase activity assay kit (Beijing Box Biotechnology Co., Ltd., Beijing, China) was used. The operation was strictly carried out in accordance with the product instructions to obtain stable and reliable experimental data.

[0109] (10) Determination of lignin content: The lignin content was determined using a lignin content detection kit (Beijing Box Biotechnology Co., Ltd., Beijing, China). The specific operating steps are described in the kit instructions.

[0110] (11) Determination of peroxidase (POD) activity: Peroxidase detection kit (Jiangsu Greens Biotechnology Co., Ltd., Suzhou, Jiangsu, China) was used for the determination. The specific operation steps are as per the kit instructions.

[0111] (12) Determination of starch content: After extraction with 80% ethanol solution by shaking, the supernatant (for soluble sugar determination) and precipitate (for starch determination) are separated by centrifugation. Under acidic conditions, starch is decomposed into monosaccharides. The addition of anthrone reagent results in the formation of a blue-green furfural derivative with a noticeable absorption peak near 620 nm. The change in absorbance is measured using a spectrophotometer, and the starch content is calculated based on the standard curve. The starch content detection kit (Beijing Box Biotechnology Co., Ltd., Beijing, China) is used for testing. Specific operating procedures can be found in the kit's instruction manual.

[0112] 2. Experimental Results

[0113] (1) Effect of micro-nano ozone water treatment on sweet potato respiration rate

[0114] like Figure 7 As shown in A, vaccination D.dadantiiAfter Ech36, the respiration intensity of the samples fluctuated significantly in the early stage of storage, generally showing a trend of "first decreasing and then increasing". From day 0 to day 15 of storage, the respiration intensity of both CK1 and T1 decreased, reaching its lowest value on day 15, with a significant difference between the two. P ≤ 0.05), with the T1 treatment showing a more significant decrease. From day 15 to 30, respiration rates in both treatments began to recover, with CK1 showing a greater increase. Throughout the storage period, the T1 treatment maintained a consistently low respiration rate, indicating its positive effect on disease suppression and physiological trait regulation. Infected by fungi ( F. solani-melongenae The respiration intensity of sweet potatoes (24-3) during storage generally showed a trend of first decreasing and then increasing. In the first 15 days, both CK2 and T2 showed an initial slight increase, followed by a gradual decrease, reaching their lowest point on day 15. From day 15 to day 30, the respiration intensity significantly increased, and the difference between the two groups continued to widen. At the end of the experiment, the respiration intensity of CK2 was significantly higher than that of T2. P The levels were ≤ 0.05, and remained consistently high, indicating that T2 treatment effectively inhibited pathogen growth and its impact on host physiological metabolism. The results suggest that micro-nano ozone bubble water treatment has an effect on… D. dadantii Ech36 and F. solani-melongenae The growth of 24-3 has a significant inhibitory effect and can effectively extend the storage time of sweet potatoes.

[0115] (2) Effect of micro-nano ozone water treatment on the hardness of sweet potatoes

[0116] like Figure 7 As shown in Figure B, the sweet potato tissue gradually softens with prolonged storage. (Infection) D. dadantii After Ech36, the firmness of sweet potatoes showed a significant decreasing trend, with the decrease being particularly pronounced in the first 5 days, after which the rate of decrease slowed down. At the end of storage, there was a statistically significant difference between CK1 and T1. P (≤ 0.05%), sweet potatoes in group T1 consistently maintained a high firmness level, indicating that this treatment had a positive delaying effect on inhibiting the pathogenicity of the pathogen. Infecting pathogen F. solani- melongenae After 24-3 days, the hardness of sweet potatoes showed a rapid decrease in the early stage and a slow recovery in the later stage. The decrease in hardness was significant in the first 5 days, and continued to decrease until day 30, but at a slower rate. During the 20-30 day storage period, there was a significant difference in hardness between the treatment group and the control group. P ≤ 0.05). Experimental data showed that T2 maintained a high hardness level throughout the storage period, indicating that this method has significant application value in inhibiting sweet potato root rot and delaying fruit softening. The results also showed that sweet potatoes treated with micro / nano ozone bubble technology exhibited a significantly reduced rate of hardness decline during storage, and pathogens were effectively controlled. D. dadantii Ech36 and F. solani-melongenae The damage to the tissue structure of sweet potatoes caused by 24-3 was effectively inhibited, thereby significantly improving the quality of sweet potatoes during storage.

[0117] (3) Effect of micro-nano ozone water treatment on sweet potato rot rate

[0118] The effect of micro-nano ozone water treatment on the rot rate of sweet potatoes infected with two pathogens, such as Figure 7 As shown in C. Inoculate after 20 days of storage. D. dadantii Ech36 and F. soani-melongenae The decay rates of the control groups (CK1 and CK2) in the 24-3 group were 4.5% and 5.5%, respectively. After treatment with 2 mg / L micro-nano ozone solution, the decay rates of the corresponding treatment groups (T1 and T2) significantly decreased to 1.0% and 2.0%. After 30 days of storage, the decay rates of CK1 and CK2 further increased to 7.8% and 8.8%, respectively, while the decay rates of T1 and T2 remained stable at 4.4%. It is worth noting that... F. soani-melongenae The degree of decay caused by 24-3 infection was slightly higher at all time points than D.dadantii Ech36. The antibacterial mechanism of micro-nano ozone bubble water lies in its ability to release reactive oxygen species, disrupting the cell membrane integrity of pathogens and interfering with the activity of key intracellular enzymes, thereby inhibiting pathogen growth and reproduction. Experimental results show that this micro-nano ozone bubble water treatment significantly slows down sweet potato rot caused by two specific pathogens. With prolonged storage, the rot rate continued to rise, reflecting the continuous proliferation and spread of pathogens within the sweet potato tissue; however, compared to the control group, the increase in rot rate in the treated group was more gradual, further confirming the effective inhibitory effect of micro-nano ozone bubble water on pathogen activity.

[0119] (4) Effect of micro-nano ozone water treatment on the weight loss rate of sweet potatoes

[0120] like Figure 7 As shown in D, in infection D. dadantii In Ech36 sweet potatoes, the weight loss rate gradually increased with prolonged storage time, with slow growth in the initial stage (0-10 days) and a significant acceleration in the later stage (10-30 days). Experimental results showed a significant difference between the T1 group and the CK1 group. P The fact that the weight loss rate of group T1 remained low throughout the storage period (≤ 0.05%) confirms that the treatment can inhibit pathogens. D. dadantii Ech36 causes disease in sweet potatoes. Sweet potatoes are infected. F. solani- melongenaeAfter 24-3 days, the weight loss rate of sweet potatoes continued to increase during storage. The control group CK2 showed a faster rate of weight loss in the early stages, exceeding that of the T2 group; while the weight loss rate of the T2 group showed a gradually decreasing trend. Statistical analysis indicated that the difference between the CK2 and T2 groups reached a significant level at the end of storage. P The weight loss rate of group T2 was ≤ 0.05%, and the weight loss rate of group T2 was consistently lower than that of group CK2 throughout the entire storage period. The results indicate that the weight loss rate of sweet potatoes treated with micro-nano ozone bubbles was significantly reduced during storage, and the rate of moisture evaporation was effectively slowed down.

[0121] (5) Effects of micro-nano ozone bubble water treatment on soluble solids and titratable acidity of sweet potato

[0122] like Figure 8 As shown in A, infection D. adantii The acidity variation pattern of sweet potatoes in Ech36 was as follows: CK1 initially decreased, then increased, and then decreased again, reaching its lowest point on day 5. It rebounded on day 20 and continued to rise until the end of storage, where fluctuations increased. T1 generally showed a trend of initially decreasing steadily and then slowly recovering. It remained at a low level for the first 15 days, reaching its lowest point on day 20, and then gradually recovered over time. Compared to CK1, T1 reduced the rate of acidity accumulation and peak concentration in disease-infected plants. P ≤ 0.05), indicating that the intervention improved the overall quality characteristics of the fruit. Infection F. solani-melongenae The acidity of sweet potatoes (24-3) during storage showed that CK2 acidity initially decreased, then slightly increased, and then increased again, reaching its minimum on day 5. T2 acidity initially decreased and then increased, reaching its lowest value on day 10. From day 0 to 5 of storage, CK2 acidity was lower than T2. ​​From day 5 to 30 of storage, CK2 acidity remained higher than T2. ​​After day 15, the difference between CK2 and T2 acidity gradually increased with storage time, becoming significant at the end of storage. P (≤ 0.05), indicating that the T2 treatment significantly reduced the infection rate of sweet potato root rot pathogen. This demonstrates the effectiveness of the T2 treatment in reducing the infection rate of root rot pathogen. F. solani-melongenae 24-3. This reduces the acidity of sweet potatoes, minimizing fluctuations in acidity during storage. In conclusion, micro-nano ozone bubbling in water can reduce the acidity of sweet potatoes infected with both pathogens, alleviate drastic acidity fluctuations during storage, and improve the quality of pathogen-infected sweet potatoes.

[0123] D. dadantii Ech36 and F. solani-melongenae 24-3 Effects on the accumulation pattern of soluble solids during sweet potato tuber storage, such as Figure 8As shown in Figure B, overall, the soluble solids content of the control group CK1 exhibited a fluctuating pattern of "decline-rise-decline again" with storage time; while the treatment group T1 showed a trend of "decline-rise-decline again-rise again". During storage days 0-5, the soluble solids content of both CK1 and T1 decreased; from storage days 5-10, the content of both groups increased. During storage days 10-30, CK1 showed an initial increase followed by a decrease, reaching a peak on day 20 and then continuously declining; T1 showed an initial decrease followed by an increase. At the end of storage, the content of T1 was significantly higher than that of CK1. Throughout the entire storage period, the soluble solids content of the T1 group was consistently higher than that of CK1, indicating that micro-nano ozone bubble water treatment can effectively slow down the degradation rate of soluble solids during sweet potato storage. F. solani-melongenae The variation pattern of soluble solids content in sweet potatoes infected with this pathogen (24-3) showed that throughout the storage period, the soluble solids content of sweet potatoes infected with this pathogen generally showed a trend of first decreasing and then increasing. During storage days 0-20, there was no significant difference in soluble solids content between the control group CK2 and the treatment group T2. P The soluble solids content (SSD) was >0.05%, and the trends were highly consistent, all reaching their lowest value on day 5. At the end of storage, the SSD content in group T2 was slightly higher than that in group CK2, and the SSD content in group T2 remained higher than that in the control group throughout the entire storage period. These results indicate that T2 treatment can effectively delay the degradation process of soluble solids. In summary, micro-nano ozone bubble water treatment can effectively delay the decrease in soluble solids content in sweet potatoes infected with both pathogens, and the regulatory effect in the later stages of treatment is better than that in the earlier stages.

[0124] (6) Effect of micro-nano ozone bubble water treatment on malondialdehyde content in sweet potatoes

[0125] like Figure 8 As shown in C, micro-nano ozone bubble water treatment for infection D. dadantii The MDA content in sweet potatoes (T1) treated with Ech36 gradually increased with prolonged storage time. Sweet potatoes without micro / nano ozone water treatment (CK1) showed a rapid increase in MDA content from 16.3 nmol / g to 28.5 nmol / g within 0-5 days of storage, with a continued increase from 5-30 days, reaching a peak of 39.7 nmol / g on day 30. In contrast, the MDA content in T1 remained between 16.3 nmol / g and 16.1 nmol / g within 0-5 days of storage, gradually increasing from 5-30 days, reaching a peak of 30.3 nmol / g on day 30. T1 showed significantly lower MDA content than CK1 at all time points. P ≤ 0.05). For infection F. solani-melongenaeIn sweet potatoes stored for 24-3 days, the MDA content generally showed a continuous upward trend. In the CK2 group, the MDA content rapidly increased from 16.3 nmol / g to 33.1 nmol / g during storage from day 0 to day 15, reaching a peak of 40.6 nmol / g on day 30. In contrast, the MDA content in the T2 treatment increased from 16.3 nmol / g to 28.1 nmol / g during storage from day 0 to day 15, significantly lower than that of CK2, reaching a peak of 33.0 nmol / g on day 30. The MDA content in the T2 treatment was consistently lower than that in the control group. P (≤ 0.05). Experimental results showed that micro-nano ozone bubble water treatment could significantly reduce the accumulation of MDA in sweet potatoes induced by these two pathogens, maintaining a low MDA content in the treatment group throughout the storage period, while the control group showed a continuous increase, especially with a significantly accelerated accumulation rate in the later stages of storage. This treatment effectively delayed the quality deterioration of sweet potatoes during storage by reducing cell membrane lipid peroxidation damage and protecting cell structural integrity.

[0126] (7) Effect of micro-nano ozone bubble water treatment on sweet potato cellulase activity

[0127] like Figure 8 As shown in D, infection D. dadantii In Ech36 sweet potatoes, cellulase activity in the CK1 group continuously increased from day 0 to day 10 of storage, peaking at 680.8 U / g on day 10. It then began to decline from day 10 to day 20, reaching a minimum of 459.5 U / g on day 30. Cellulase activity in the T1 group peaked at 475.2 U / g on day 10 of storage, then began to decline, reaching 158.3 U / g on day 20 and 248.2 U / g on day 30. Compared to CK1, cellulase activity in the T1 group was significantly lower. P ≤ 0.05). Infection F. solani-melongenae In sweet potatoes aged 24-3, the cellulase activity in group CK2 continued to increase from day 0 to day 10 of storage, reaching a peak of 984.0 U / g on day 10, which was significantly higher than that in group T2. P ≤ 0.05); decreased rapidly from day 10 to day 30, dropping to 439.2 U / g on day 30, but still significantly higher than T2 ( P ≤ 0.05). Cellulase activity in group T2 increased rapidly from day 0 to day 10 of storage, peaking at 815.7 U / g on day 10, and then declined, decreasing to 141.8 U / g on day 30. Compared with CK2, cellulase activity in T2 was significantly reduced ( P (≤ 0.05), the activity peak decreased significantly. In summary, micro-nano ozone bubble water treatment can significantly inhibit the activity peak. D. dadantii Ech36 and F. solani-melongenae24-3 The activity of cellulase in sweet potatoes infected by two pathogens slowed down the degradation rate of cell wall components by the pathogens. Cellulase is a key pathogenic factor for pathogens to overcome the physical barriers of plants. Downregulation of its activity helps maintain the integrity of cell wall structure and delays the process of tissue softening and decay.

[0128] (8) Effect of micro-nano ozone bubble water treatment on sweet potato α-amylase

[0129] like Figure 9 As shown in Figure A, the activity of sweet potato α-amylase generally showed a trend of first increasing and then decreasing. Among them, infection... D. dadantii In Ech36 sweet potatoes, the α-amylase activity of CK1 rapidly increased from 4.6 U / g to 15.4 U / g during storage days 0 to 5, remained at 15.6 U / g during storage days 5 to 15, and rapidly decreased during storage days 15 to 30, dropping to 4.4 U / g on day 30. In the T1 group, the activity continuously increased during storage days 0 to 20, reaching a peak of 15.2 U / g on day 20, and gradually decreased from day 20 to 30, showing a significant difference from the control group. P ≤ 0.05). The results showed that micro-nano ozone bubble water treatment significantly inhibited the sharp decline in α-amylase activity and delayed starch degradation. Infection F. solani-melongenae In sweet potatoes of variety 24-3, the α-amylase activity in group CK2 increased from 4.6 U / g to 15.7 U / g within 0-15 days, peaking on day 15, and then rapidly decreased from day 15 to day 30 of storage, dropping to 4.5 U / g on day 30. In group T2, the activity gradually increased within 0-20 days, peaking at 12.0 U / g on day 20, and then gradually decreased from day 20 to day 30 of storage, reaching 11.6 U / g on day 30, significantly higher than the control group. P (≤ 0.05). This indicates that the treatment group delayed the peak activity of α-amylase, thus slowing down the starch degradation process.

[0130] (9) Effect of micro-nano ozone water treatment on lignin content in sweet potato

[0131] Depend on Figure 9 As shown in B, the effects of micro-nano ozone bubble water treatment on the lignin content of sweet potatoes infected with different pathogens vary. For infections... D. dadantiiIn Ech36 sweet potatoes, the lignin content in CK1 increased from 42.1 mg / g to 64.3 mg / g during storage days 0 to 10, then decreased to 44.2 mg / g, before rapidly increasing to 73.1 mg / g, and then decreasing to 27.6 mg / g during storage days 20 to 30. In group T1, the lignin content continuously decreased during storage days 0 to 15, peaked at 51.7 mg / g during storage days 15 to 20, and then rapidly decreased during storage days 20 to 30. Overall, the lignin content in T1 was consistently lower than that in CK1. P ≤ 0.05). For infection F. solani-melongenae In sweet potatoes of variety 24-3, the lignin content in CK2 rapidly increased from day 0 to day 10 of storage, reaching a peak of 63.9 mg / g, and gradually decreased to 21.8 mg / g from day 10 to day 30. T2 rapidly increased from day 0 to day 5 of storage, reaching 80.8 mg / g on day 5, and then began to decline, dropping to 16.7 mg / g on day 30, significantly lower than the control group. P (≤ 0.05). In summary, micro-nano ozone bubble water treatment can significantly reduce the cellulase activity of two pathogens infecting sweet potatoes, inhibit excessive lignin accumulation, thereby delaying the lignification process and helping to maintain the good taste and storage quality of sweet potatoes.

[0132] (10) Effect of micro-nano ozone bubble water treatment on peroxidase activity in sweet potato

[0133] like Figure 9 As shown in C, infection D. dadantii In sweet potatoes of Ech36, the POD activity of CK1 increased from 47.4 ΔOD during storage from day 0 to day 30. 470 / (g·min) continued to rise to 126.4 ΔOD 470 / (g·min); while the T1 treatment resulted in 47.4 ΔOD during storage from day 0 to day 30. 470 / (g·min) rapidly increased to 102.1 ΔOD 470 / (g·min), significantly lower than the control group ( P ≤ 0.05). This indicates that micro / nano bubble water treatment can inhibit the increase of POD activity. Infection F. solani-melongenae In sweet potatoes aged 24-3, the POD activity of CK2 increased from 47.4 ΔOD on day 0 to day 5 of storage. 470 / (g·min) rapidly increased to 97.5 ΔOD 470 / (g·min), then gradually increased, reaching 116.0 ΔOD on the 30th day of storage. 470 / (g·min). T2 decreased slightly from day 0 to day 5 of storage, then showed a continuous upward trend from day 5 to day 30 of storage, reaching 113.4 ΔOD on day 30 of storage.470 / (g·min), the POD activity in group T2 was significantly lower than that in the control group. In summary, micro-nano ozone bubble water treatment can significantly inhibit the increase of POD activity in sweet potatoes infected by the two pathogens and effectively alleviate oxidative stress.

[0134] (11) Effect of micro-nano ozone bubble water treatment on sweet potato starch content

[0135] like Figure 9 As shown in Figure D, micro-nano ozone bubble water treatment affected the total starch content of sweet potatoes infected with different pathogens, showing an overall trend of first increasing and then decreasing. D. dadantii In Ech36 sweet potatoes, the starch content in CK1 increased from 54.8 mg / g to 76.3 mg / g from day 0 to day 15 of storage, and then gradually decreased from day 15 to day 30, dropping to 51.7 mg / g on day 30. Meanwhile, T1 increased from 54.8 mg / g to a peak of 93.3 mg / g from day 0 to day 15 of storage, significantly higher than the control group. P (≤ 0.05), showing a gradual decreasing trend during storage from 15 to 30 days, dropping to 70.1 mg / g on the 30th day. Infection F. solani-melongenae In sweet potatoes of variety 24-3, the starch content in CK2 increased from 54.8 mg / g to 68.6 mg / g from day 0 to day 15 of storage, and then gradually decreased from day 15 to day 30, dropping to 51.2 mg / g on day 30. Meanwhile, T2 increased from 54.8 mg / g to a peak of 85.6 mg / g from day 0 to day 15 of storage, significantly higher than the control group (…). P The concentration of ≤ 0.05 mg / g showed a gradual decreasing trend during storage from 15 to 30 days, decreasing to 69.0 mg / g on day 30, which was significantly different from the control group. P (≤ 0.05). The results showed that micro-nano ozone bubble water treatment could effectively maintain a high starch content in the later stage of storage and delay the degradation rate of sweet potato starch under the infection of the two pathogens.

[0136] Example 3: Study on the Preservation Effect of Micro-Nano Ozone Bubbling Water on Satsuma Mandarins

[0137] 1. Experimental Methods

[0138] (1) Pretreatment of tangerines

[0139] Micro-nano ozone bubble water prepared with 2% citric acid in Example 1 was selected, and the micro-nano ozone bubble water was diluted with 2% citric acid aqueous solution by volume to liquid phase ozone concentrations of 1.44 mg / L, 2.88 mg / L, and 6.8 mg / L.

[0140] Satsuma mandarins with similar maturity, size, and shape were selected, with imidacloprid and imidazole as positive controls and water as a blank control. The mandarins were soaked for 3 minutes in water, 2% citric acid solution (v / v), imidacloprid solution (277.5 mg / L), imidazole solution (338.35 mg / L), and micro-nano ozone solution (1.44 mg / L, 2.88 mg / L, and 6.8 mg / L), followed by natural drying at room temperature. The dried mandarins were stored at room temperature (25±2℃, 60±5% relative humidity), and rot rate, weight loss, soluble solids, and titratable acidity were tested on days 0, 5, 10, 20, and 30. Each treatment was replicated four times, with five mandarins randomly selected at each time point, flash-frozen in liquid nitrogen, and stored at -80℃ for subsequent enzyme activity and other indicator detection.

[0141] (2) Data measurement

[0142] The decay rate was determined statistically. The standard for fruit decay rate included localized decay, water leakage, and mold growth, all of which were counted in the number of decayed fruits. The number of decayed Satsuma mandarins under different treatments was calculated using the following formula:

[0143] Among them, R i Let N represent the number of rotten fruits counted on day i, and N be the total number of fruits in the sample.

[0144] The determination of weight loss rate, soluble solids, and titratable acid shall be performed in accordance with the method of Example 2.

[0145] The total phenol content was determined using a total phenol (TP) assay kit (China, Jiangsu, Edison Biotechnology Co., Ltd.). For specific operating procedures, please refer to the kit's instruction manual.

[0146] Vitamin C content was determined using a Vitamin C Content Assay Kit (Nanjing Jiancheng Biotechnology Institute, China). For detailed operating procedures, please refer to the kit's instruction manual.

[0147] 2. Experimental Results

[0148] (1) Effect of micro-nano ozone bubble water treatment on the decay rate of tangerines

[0149] like Figure 10 As shown in Figure A, with the continuous progression of storage time, the decay rate of Satsuma mandarin fruits in all groups showed a gradual upward trend, especially in the CK and citric acid treatment groups, where the decay rate increased sharply from day 10. During the same storage period, the decay rate of fruits in the CK and citric acid treatment groups was significantly higher than that of the other treatment groups. On day 30, there were significant differences in the measured indicators between the CK group and other treatment groups (…).P (≤ 0.05) The decay rate of the CK group was consistently higher than that of other groups, reaching 65.3%. The decay rates of the groups treated with imidacloprid, imidacloprid, and micro-nano ozone bubble water at concentrations of 1.44 mg / L, 2.88 mg / L, and 6.8 mg / L were 41.0%, 48.0%, 45.0%, 32.7%, and 41.3%, respectively, while the decay rate of the citric acid treatment group was 60.0%. The results indicate that micro-nano ozone bubble water can effectively slow down the decay of tangerines and extend their shelf life.

[0150] (2) Effect of micro-nano ozone bubble water treatment on the weight loss rate of tangerines

[0151] like Figure 10 B. The weight loss rate of the control group (CK) and the citric acid treatment group increased more rapidly than that of other treatment groups. From the 20th day of storage, the weight loss rate of the fruit in the CK group and the citric acid treatment group began to rise sharply. By the end of the storage period, the weight loss rate of the CK group was as high as 7.05%, while the weight loss rate of the citric acid treatment group was 8.17%. The weight loss rates of the treatment groups treated with imidacloprid, 1.44 mg / L, 2.88 mg / L, and 6.8 mg / L micro-nano ozone bubble water were 3.8%, 5.01%, 4.09%, 4.13%, and 3.99%, respectively. The results showed that micro-nano ozone bubble water treatments at concentrations of 1.44 mg / L, 2.88 mg / L, and 6.8 mg / L could effectively slow down moisture loss and reduce weight loss during storage. The effect of the 6.8 mg / L micro-nano ozone bubble water treatment group was comparable to that of the imidacloprid treatment group.

[0152] (3) Effects of micro-nano ozone bubble water treatment on soluble solids and titratable acid in tangerines

[0153] like Figure 10D. The soluble solids content of Satsuma mandarins showed a fluctuating trend with prolonged storage time, initially decreasing slightly, then increasing, and finally continuously decreasing again. The decrease was more significant in the later stages of storage (after day 20). During storage, there were significant differences between the control group (CK) and the treatment groups treated with imidacloprid, imidacloprid, and micro-nano ozone bubble water at concentrations of 1.44 mg / L, 2.88 mg / L, and 6.8 mg / L (P ≤ 0.05). Within the same storage period, the soluble solids content of the CK and citric acid treatment groups was consistently lower than that of the imidacloprid, imidacloprid, and micro-nano ozone bubble water treatment groups at concentrations of 1.44 mg / L, 2.88 mg / L, and 6.8 mg / L. Specifically, on day 30 of storage, the soluble solids content of the CK and citric acid treatment groups were 10.34% and 11.34%, respectively, while those of the imidacloprid, imidacloprid, and micro-nano ozone bubble water treatment groups were significantly lower. The soluble solids content of the micro-nano ozone bubble water treatment group remained more stable, with corresponding values ​​of 11.43%, 11.27%, 11.83%, 12.14%, and 12.43%, respectively. Compared with the control group, the treatment with imidacloprid, 1.44 mg / L, 2.88 mg / L, and 6.8 mg / L of micro-nano ozone bubble water all inhibited the decrease in soluble solids content. The effect of the micro-nano ozone bubble water treatment group with a concentration of 6.8 mg / L was slightly better than that of the treatment with imidacloprid, 1.44 mg / L, and 2.88 mg / L of micro-nano ozone bubble water.

[0154] like Figure 10C. Throughout the storage period, the titratable acid content of fresh lotus seeds in both the CK and treatment groups did not show a stable upward or downward trend, but rather exhibited fluctuating characteristics. The decrease in titratable acid content in the treatment groups treated with imidacloprid, imidacloprid, and micro-nano ozone water at concentrations of 1.44 mg / L, 2.88 mg / L, and 6.8 mg / L was slightly smaller than that in the CK and citric acid treatment groups. On the 30th day of storage, the titratable acid concentrations in the treatment groups treated with imidacloprid, imidacloprid, and micro-nano ozone water at concentrations of 1.44 mg / L, 2.88 mg / L, and 6.8 mg / L were 0.56%, 0.55%, 0.59%, 0.47%, and 0.63%, respectively. The titratable acid concentrations in the CK group and the citric acid treatment group were 0.57% and 0.50%, respectively; among them, the titratable acid concentration in the micro-nano ozone water treatment group with a concentration of 6.8 mg / L exceeded 0.6%, demonstrating a significant advantage in preservation. The concentrations in the other treatment groups were all below 0.6%. Studies have found that water treatment with 6.8 mg / L micro-nano ozone can effectively reduce the loss of titratable acids in tangerines. Taking all factors into account, treatment with imidacloprid, imidacloprid, and micro-nano ozone at concentrations of 1.44 mg / L, 2.88 mg / L, and 6.8 mg / L effectively delayed the gradual decrease in soluble solids and the subsequent loss of titratable acid components in tangerines over extended storage time. In particular, compared to the control (CK), the 6.8 mg / L micro-nano ozone water treatment group showed slightly better results than the other treatment groups.

[0155] (4) Effect of micro-nano ozone bubble water treatment on total phenol content of saccharide

[0156] like Figure 10 As shown in Figure E, the total phenolic content of the mandarin orange pulp showed a continuous decreasing trend throughout the process. When the storage time was 1 to 5 days, treatment with 6.8 mg / L micro-nano ozone water effectively maintained the total phenolic content in the pulp. When the storage time was 10 to 30 days, the total phenolic content of both the treatment group and the control group gradually decreased, while the total phenolic content of the 6.8 mg / L micro-nano ozone water treatment group remained higher than that of the other treatment groups and the control group throughout the storage process. After the storage process was completed, there was no significant difference in total phenolic content between the treatment group and the control (CK). P(≤ 0.05). Throughout the storage process, the total phenolic content decreased most slowly in the 6.8 mg / L micro-nano ozone bubble water treatment group, while the total phenolic content in the 1.44 mg / L, 2.88 mg / L micro-nano ozone bubble water treatment groups was generally slightly lower than that in the 6.8 mg / L treatment group. However, the decrease in total phenolic content in the CK and citric acid treatment groups was significantly greater than that in the 6.8 mg / L treatment group. The results indicate that 6.8 mg / L micro-nano ozone bubble water treatment can effectively inhibit the decrease in total phenolic content in Satsuma mandarin orange pulp and maintain the stability of its phenolic substances.

[0157] (5) Effect of micro-nano ozone bubble water treatment on VC content of tangerines

[0158] like Figure 10 During storage days 1 to 30, the vitamin C content in the 6.8 mg / L micro-nano ozone bubble water treatment group was consistently slightly higher than that in other treatment groups and the control group, with a relatively smaller decrease. However, during storage days 20 to 30, the decrease in vitamin C in the CK and citric acid treatment groups was significantly greater than that in the 6.8 mg / L micro-nano ozone bubble water treatment group (P ≤ 0.05). By the end of the storage period, the vitamin C content in the pulp of the control group and the citric acid treatment group was significantly lower than that in the imidacloprid and imidacloprid treatment groups, while the content in the micro-nano ozone bubble water at concentrations of 1.44 mg / L, 2.88 mg / L, and 6.8 mg / L was also lower. The study found that treatment with imidacloprid, imidacloprid, and micro-nano ozone bubble water at concentrations of 1.44 mg / L, 2.88 mg / L, and 6.8 mg / L can effectively maintain the vitamin C content in the pulp of Satsuma mandarins. Among these, the 6.8 mg / L micro-nano ozone bubble water treatment was slightly more effective than the other treatment groups.

[0159] Example 4: Study on the Preservation and Mechanism of Fresh Lotus Seeds by Micro-Nano Ozone Illuminated Water

[0160] (1) Pre-treatment of fresh lotus seeds

[0161] The micro-nano ozone bubble water prepared with 2% citric acid in Example 1 was selected, and the micro-nano ozone bubble water was diluted with a volume ratio of 2% citric acid aqueous solution to a liquid phase ozone concentration of 3 mg / L.

[0162] Select the best lotus seeds from fresh lotus pods, removing those with significant differences in size, shape, and maturity. Soak the seeds in 3 mg / L micro-nano ozone bubble water for 3 minutes, then allow them to air dry naturally at room temperature (25±2℃, relative humidity 60±5%). Then, package them into small modified atmosphere packaging boxes (5.5 cm long, 4.4 cm wide, and 2 cm high) with four circular openings (each 1 cm in diameter) according to a standard of 300 g per box. Storage experiments were conducted at 4℃. This invention included a CK group (pretreated with water and packaged in a modified atmosphere packaging box), a T1 group (pretreated with micro-nano ozone bubble water and packaged in a modified atmosphere packaging box), and a T2 group (each packet of ethylene blocking agent 1-MCP was dipped in an appropriate amount of water and placed in a modified atmosphere packaging box containing fresh lotus seeds. The 1-MCP used in the experiment was purchased from Zhongke Youguo (Guangzhou) Biotechnology Co., Ltd.). Each group was replicated four times. On days 0, 3, 5, 10, 15, 20, and 30, the color parameters, breathing intensity, hardness value, weight loss rate, soluble solids content, and total acid content were measured. Simultaneously, the corresponding samples were collected, flash-frozen in liquid nitrogen, and stored at -80℃ for subsequent analysis.

[0163] (2) Data measurement

[0164] Colorimetry was measured using a CHROMA METER CR-400 colorimeter. Ten lotus seeds were randomly selected from each treatment group. To minimize measurement error, the surface of each lotus seed was divided into three regions, with a fixed spacing between the measurement points. The a value was read one by one. * b * L * The values ​​are then averaged. Sample images were taken using a Sony ZVE10 digital camera.

[0165] The respiration rate, stiffness, weight loss, soluble solids, titratable acid and total starch content were determined according to the method in Example 2.

[0166] Peroxidase activity was measured using a peroxidase activity assay kit (China, Beijing, Box Biotechnology Co., Ltd.). For specific operating procedures, please refer to the kit's instruction manual.

[0167] The malondialdehyde (MDA) content was determined using a MDA content detection kit (China, Suzhou, Jiangsu, Greens Biotechnology Co., Ltd.). Specific operating procedures can be found in the kit documentation.

[0168] 2. Experimental Results

[0169] (1) Effect of mixed treatment on color change of fresh lotus seeds

[0170] like Figure 11The control group showed obvious browning on the 3rd day of storage, the T1 group showed slight browning on the 10th day, and the T2 group showed slight browning on the 5th day; by the 20th day of storage, most of the lotus seeds in the control group had browned. L * , a * , b * It is a commonly used indicator to characterize changes in the color of a sample. L * This represents the brightness of the fruit peel; a higher value indicates better brightness. (CK vs. T1 and T2 groups) L * The values ​​all showed a downward trend; among them, the CK group showed a significant decrease in lotus seed brightness starting from the 3rd day of storage, accompanied by obvious yellowing. During the storage period from the 3rd to the 30th day, the control group and the treatment group... L * The values ​​show significant differences ( P ≤ 0.05). The rate of brightness decrease in groups T1 and T2 was significantly slower than that in CK. b * The values ​​reflect the lotus seeds' preference within the yellow-blue color family: higher values ​​in the positive range indicate increased yellowness, while lower values ​​in the negative range indicate a deeper blue. During the storage period from day 3 to day 30, the control group and groups T1 and T2... b * The values ​​generally showed a gradual decreasing trend. On the 3rd day of storage, the control group and the T2 treatment group... b * The values ​​show significant differences ( P ≤ 0.05); during the period from day 5 to day 30 of storage, CK and groups T1 and T2... b * The differences in values ​​all reached a significant level ( P ≤ 0.05). a * The value is mainly used to characterize the reddish-green hue of fresh lotus seeds. a * The changes in the values ​​reflect the lotus seeds' preference within the red-green color spectrum: higher values ​​in the positive range indicate a deeper red hue; lower values ​​in the negative range indicate a more intense green hue. During storage periods of 3 to 30 days, as the storage time increased, the color of fresh lotus seeds gradually transitioned from the harvested green to brown. The treatment group and the control group showed... a * The values ​​all showed a continuous upward trend starting from the third day of storage, compared to the control group. a * The value was consistently lower than that of groups T1 and T2 during the storage period from day 3 to day 30. The control group and the T1 and T2 treatment groups...a * The differences in values ​​all reached a significant level ( P (≤ 0.05). In summary, the micro-nano ozone water treatment can effectively slow down the color deterioration process of fresh lotus seeds during post-harvest storage and reduce the post-harvest quality decline caused by color changes.

[0171] (2) Effect of mixed treatment on the respiration rate of fresh lotus seeds

[0172] like Figure 12 As shown in Figure A, the respiration rate of fresh lotus seeds generally exhibited a "first increase, then decrease" trend throughout the storage process. The respiration rate of fresh lotus seeds reached its peak on the 3rd day of storage, with the respiration rates of T1 and T2 significantly higher than those of the control group (CK). This indicates that T1 and T2 can temporarily enhance the respiration intensity of fresh lotus seeds. From the 5th to the 30th day of storage, the respiration rate of lotus seeds in all groups showed a gradual decreasing trend. Compared with the T1 and T2 treatments, the decrease in respiration rate in the control group was more significant, and the difference between the CK and the treatment groups reached a significant level. P ≤ 0.05); while there was no significant difference in the decrease in respiratory rate between the T1 and T2 groups ( P >0.05). The above results indicate that the combined treatment of micro-nano ozone bubble water altered the respiratory and metabolic patterns of lotus seeds: In the early stages of storage, the respiration rate of the treatment groups (T1, T2) showed a transient increase and was significantly higher than that of the control group (CK group). With prolonged storage, the respiration rate of lotus seeds in the CK group decreased significantly due to tissue deterioration and metabolic collapse, while the respiratory metabolism of the treatment groups remained at a stable level, significantly higher than that of the CK group. This reflects that the treatment effectively delayed the physiological decline process of lotus seeds. Micro-nano ozone bubble water treatment can effectively delay the decrease in respiration rate during the storage of fresh lotus seeds, reduce nutrient consumption, thereby delaying the post-harvest senescence process of lotus seeds and extending their storage and shelf life.

[0173] (3) Effect of mixed treatment on the hardness of fresh lotus seeds

[0174] like Figure 12 As shown in Figure B, with prolonged storage, the firmness of fresh lotus seed pulp in the control group and the T1 and T2 treatment groups all showed a trend of first increasing and then decreasing. The firmness of lotus seeds in the CK group peaked on day 15 (4.14 kg / cm²); the T1 group also peaked on day 15, but slightly higher than the control group, at 4.33 kg / cm²; while the T2 group peaked on day 20, at 3.93 kg / cm². Throughout the entire storage period, the firmness differences between CK and T2 were significant (…). P ≤ 0.05). During the storage period of 15 to 30 days, the differences between the CK group and the T1 group were also significant ( P(≤ 0.05). Overall, the fluctuation range of flesh firmness in T1 and T2 treatments was significantly lower than that in the control group. By day 30, at the same time point, T1 had the highest firmness, followed by T2, and CK had the lowest. Both treatments were effective in delaying flesh softening. The results indicate that micro-nano ozone water immersion treatment can effectively delay the decrease in the firmness of fresh lotus seed flesh and better maintain its original crisp and tender texture.

[0175] (4) Effects of mixed treatment on soluble solids and titratable acidity of fresh lotus seeds

[0176] like Figure 12 C. With prolonged storage time, the soluble solids content in lotus seeds of both the control and treatment groups showed an overall trend of first increasing, then decreasing, and then increasing again. All groups reached their peak on day 30 of storage, with the TSS content in the control group (CK) increasing significantly more than that in the treatment groups (T1 and T2). Under the same storage time conditions, the TSS content of the control and treatment groups showed a clear gradient difference, with CK consistently having the highest content, followed by T1, and T2 having the lowest. Specifically, the TSS content of CK gradually increased from day 3 of storage, reaching 17.27% on day 30; the TSS content growth rates of T1 and T2 were relatively slower, reaching 11.47% and 11.17% respectively on day 30. Statistical results showed that within the 3-30 day storage period, the TSS content differences between T1, T2, and CK were significant (…). P ≤ 0.05). For example... Figure 12 D. Throughout the storage period, the acidity of fresh lotus seeds showed a continuous upward trend with the extension of storage time. By the 30th day of storage, the acidity difference between the CK group and the T1 and T2 groups reached a significant level. P ≤ 0.05), the increase in CK acidity was significantly higher than that of T1 and T2. P (≤0.05), CK titratable acidity was 2.57%, T1 acidity was 1.71%, and T2 acidity was 1.85%. The results showed that both T1 and T2 treatments could effectively inhibit the rate of increase in soluble solids and acidity of fresh lotus seeds, and slow down the changes in their internal components. The effect of T1 was slightly better than that of T2, indicating that micro-nano ozone water can delay the maturation and aging process of fresh lotus seeds by regulating their metabolism.

[0177] (5) Effect of mixed treatment on weight loss rate of fresh lotus seeds

[0178] like Figure 12 As shown in Figure E, the weight loss rate of fresh lotus seeds showed a gradually increasing trend throughout the storage period. The weight loss in the control group was the most significant, considerably higher than that in the treatment group, and the increase in weight loss rate significantly exceeded that at T1 and T2, reaching its peak on day 30 of storage. Within the first 3 days to day 30 of storage, the differences between the control and treatment groups were significant. P≤ 0.05), but the difference between T1 and T2 is not significant ( P >0.05). During the same storage period, the weight loss rate of the control group was consistently higher than that of T1 and T2, but there was no significant difference in weight loss rate between T1 and T2. On day 30 of storage, the weight loss rate of the control group was as high as 21.4%, while the weight loss rate of fresh lotus seeds in the T1 group was relatively low, at only 6.52%, and the weight loss rate of fresh lotus seeds in the T2 group on day 30 was 6.62%. The results showed that the weight loss rate of lotus seeds in both the T1 and T2 treatment groups was significantly lower than that in the control group ( P (≤ 0.05). This indicates that micro-nano ozone bubble water treatment can effectively inhibit the moisture loss of fresh lotus seeds during storage, significantly reduce their weight loss rate, delay weight loss, and further slow down the decline in quality during storage.

[0179] (6) Effect of mixed treatment on peroxidase activity in fresh lotus seeds

[0180] like Figure 12 As shown in Figure F, during the storage of fresh lotus seeds, the peroxidase (POD) activity in both the control and treatment groups generally showed a trend of first decreasing, then increasing, and then decreasing again. The POD activity in groups T1 and T2 was higher than that in the control group throughout the storage period. However, during storage days 15 to 20, the peroxidase activity in the T1 and T2 treatment groups was significantly higher than that in the control group. P ≤ 0.05). However, the difference between T1 and T2 was not significant. On day 30 of storage, the peroxidase activity of T1 reached 2584 U / g, the activity of T2 was 3884 U / g, and the peroxidase activity of CK was 2338 U / g, while the activity of T2 was significantly higher than that of the CK group ( P (≤0.05). In summary, both T1 and T2 treatments effectively maintained the peroxidase activity of fresh lotus seeds during storage, slowing down the rapid decline in activity. The results of this experiment show that micro-nano ozone bubble water treatment, by maintaining a high level of peroxidase activity in lotus seed tissues, enhances the fruit's own antioxidant capacity, reduces membrane lipid peroxidation damage, and thus effectively delays the post-harvest senescence process of fresh lotus seeds. This has a significant effect on maintaining post-harvest quality and extending storage shelf life.

[0181] (7) Effect of mixed treatment on total starch content of fresh lotus seeds

[0182] like Figure 12As shown in Figure G, the starch content of fresh lotus seeds generally showed a trend of first increasing and then gradually decreasing throughout the storage period, reaching a peak on day 15. During the storage period from day 3 to day 30, the starch content of treatments T1 and T2 was consistently higher than that of the control group. However, during the storage period from day 3 to day 15, the starch content of T2 generally exceeded that of T1. However, during the storage period from day 20 to day 30, the rate of decrease in starch content of T1 was slower than that of T2. During the storage period from day 3 to day 30, there was a significant difference between the control group (CK) and treatments T1 and T2. P (≤ 0.05). During the same storage period, T1 had the highest starch content, followed by T2, while CK had the lowest. On day 30 of storage, the starch contents of T1 and T2 were 169.842 mg / g and 136.266 mg / g, respectively, while CK was 110.382 mg / g. The results show that micro-nano ozone bubble water treatment can effectively slow down the rate of decline in the total starch content of fresh lotus seeds, significantly inhibit the degradation of total starch during storage, maintain lotus seed quality, reduce starch degradation, maintain a high starch level, preserve its original nutritional value, and delay the nutrient loss and aging process of fresh lotus seeds.

[0183] (8) Effect of mixed treatment on malondialdehyde content in fresh lotus seeds

[0184] like Figure 12 H, with the extension of storage time, the malondialdehyde content in the CK group increased the most rapidly, while the growth rate of T1 and T2 was significantly slower and always lower than that of the control group. Among them, the growth rate of T1 group was less than that of T2 group. On the 30th day of storage, there was a significant difference between the two treatment groups. P ≤ 0.05). Under the same storage conditions, the malondialdehyde (MDA) content in the control group remained at the highest level, followed by T2, while the concentration in T1 was the lowest. At day 30 of storage, the MDA content in the control group was 22.6 nmol / g, in the T1 group it was 18.5 nmol / g, and in the T2 group it was 20.6 nmol / g. During the storage period from 5 to 30 days, the differences between the control and treatment groups were all highly significant (≤ 0.05). P (≤ 0.05). The results show that micro-nano ozone bubble water treatment can effectively inhibit the increase of malondialdehyde content in fresh lotus seeds during storage, reduce the degree of cell membrane lipid peroxidation, reduce the degree of membrane system damage, and thus delay the aging and quality deterioration process of fresh lotus seeds.

[0185] Table 1. Ozone mass transfer efficiency under different conditions after 30 minutes of aeration

[0186] Table 2. Ozone mass transfer efficiency after 30 minutes of aeration under different pH conditions.

Claims

1. A method for preparing micro / nano ozone bubble water, characterized in that, The process includes the following steps: thoroughly mixing gaseous ozone with a liquid medium for an aeration reaction, wherein the liquid medium is a 1%-3% citric acid solution by volume.

2. The method according to claim 1, characterized in that, The gaseous ozone is introduced into the micro-nano bubble generator and fully mixed with the liquid medium to carry out an aeration reaction; the liquid medium is a 2% citric acid solution by volume and has a pH of 2.0-2.

5.

3. Micro-nano ozone bubble water prepared by the method of claim 1 or 2.

4. The application of the micro-nano ozone bubble water as described in claim 3 in antibacterial activity.

5. The application according to claim 4, characterized in that, The bacteria are Escherichia coli, Staphylococcus aureus, sweet potato stem rot pathogen and / or sweet potato root rot pathogen.

6. The application of the micro-nano ozone bubble water according to claim 3 in food preservation.

7. The application according to claim 6, characterized in that, The liquid phase ozone concentration of the micro-nano ozone bubble water is 1-8 mg / L.

8. The application according to claim 6, characterized in that, The food is a plant-based food, and the preservation method is achieved by maintaining color, inhibiting browning, maintaining firmness, inhibiting decay, reducing water evaporation and weight loss, regulating respiration and metabolic consumption, maintaining soluble solids content, maintaining titratable acid content, inhibiting total phenol loss, inhibiting vitamin C loss, inhibiting starch degradation, inhibiting lignin accumulation, inhibiting malondialdehyde accumulation, inhibiting cellulase activity, inhibiting amylase activity, maintaining peroxidase activity, and / or inhibiting pathogen growth.

9. The application according to claim 8, characterized in that, The plant-based foods mentioned are sweet potatoes, tangerines, or lotus seeds.

10. The application according to claim 9, characterized in that, The sweet potato mentioned is a sweet potato infected with sweet potato stem rot pathogen and / or sweet potato root rot pathogen.

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