A chlorine dioxide slow-release agent, its preparation process and application

Abstract

This invention discloses a chlorine dioxide slow-release agent, its preparation process, and its applications. The raw materials include a solid phase and a liquid phase. The solid phase comprises a precursor, a gelling agent, an acidifying agent, a chelating agent, a calcium salt, and a forming agent. The liquid phase comprises lemon oil, an emulsifier, and a solvent. The chlorine dioxide slow-release agent of this invention has a long release time, and its concentration can be controlled within the national standard range. It can be applied to the fields of preservation and sterilization of agricultural and sideline products.

Description

Technical Field

[0001] This invention relates to the field of agricultural product preservation technology, specifically to a chlorine dioxide slow-release agent, its preparation process, and its application. Background Technology

[0002] There are three methods for preserving agricultural products: chemical, physical, and biological. Physical preservation methods have drawbacks such as requiring large-scale equipment and high configuration costs, making them unsuitable for widespread application. Chemical methods pose significant food safety risks. Biological preservation methods require personnel with a background in microbiology to operate the bacterial solutions, the purchase cycle for bacterial strains is long, and activation and cultivation are also time-consuming. Improper storage of activated bacterial strains can easily lead to strain degeneration. Therefore, this method is cumbersome, has poor operability, and is not suitable for large-scale promotion and use.

[0003] Chlorine dioxide is an internationally recognized green and safe disinfectant. It does not react with organic compounds to produce toxic chlorination byproducts and has broad and stable bactericidal activity. It is used as a disinfectant for water, fruits, and vegetables and has been listed as a food additive by the Food and Agriculture Organization. Chlorine dioxide is a promising chemical substance; studies have found that it can reduce and delay the aging of various products, including blueberries, raspberries, strawberries, and longans, including browning and disease. However, chlorine dioxide is reactive; its gaseous form is unfavorable for storage and transportation, high concentrations can easily cause explosions, and it decomposes and oxidizes under heat and light. Its liquid form is extremely unstable and not suitable for long-term storage. Due to its inherent physicochemical properties, chlorine dioxide has certain limitations in practical applications. Therefore, it is necessary to research a slow-release agent that allows for the slow release of chlorine dioxide gas, meeting the requirements for long-term disinfection and sterilization as well as fruit and vegetable preservation. Summary of the Invention

[0004] To address the shortcomings of existing technologies, this invention provides a chlorine dioxide slow-release agent, its preparation process, and its applications. The chlorine dioxide slow-release agent has a long release time, and its concentration can be controlled within national standard ranges. It can be applied to fields such as preservation and sterilization of agricultural and sideline products.

[0005] To achieve the above objectives, the present invention adopts the following technical solution:

[0006] In a first aspect, the present invention provides a chlorine dioxide slow-release agent, the raw materials of which include a solid phase and a liquid phase, wherein the solid phase comprises a precursor, a gelling agent, an acidifying agent, a chelating agent, a calcium salt, and a molding agent, and the liquid phase comprises lemon oil, an emulsifier, and a solvent.

[0007] Preferably, in the solid phase, the mass ratio of the precursor, gelling agent, acidifying agent, chelating agent, calcium ions, and molding agent is (15-25):(1-5):(1-5):(2-5):(0.15-1.3):(1-5), and in the liquid phase, the volume ratio of lemon oil, emulsifier, and solvent is (4-10):(6-10):(60-120). When the mass unit is g, the volume unit is mL.

[0008] In this invention, the amount of calcium ions used is closely related to the amount of sodium alginate and chelating agent used. The molar amount of calcium ions must be controlled to be 75% to 125% of the molar amount of sodium alginate, otherwise the ability of calcium ions to inhibit the release of chlorine dioxide will be affected.

[0009] Optionally, the precursor is selected from one or more of sodium chlorite, calcium chlorite, magnesium chlorite, potassium chlorite, and barium chlorite.

[0010] Optionally, the gelling agent is selected from one or more of xanthan gum, alginate, sodium alginate, sodium carboxymethyl cellulose, polyacrylic acid resin, polyacrylamide resin, and modified starch.

[0011] Optionally, the acidifying agent is selected from one or more of acetic acid, malic acid, oxalic acid, phosphoric acid, citric acid, tartaric acid, arsenic acid, formic acid, and boric acid.

[0012] Optionally, the chelating agent is EDTA.

[0013] Optionally, the calcium ions are selected from one or more of calcium chloride, calcium carbonate, and calcium sulfate.

[0014] Optionally, the molding agent is agar.

[0015] Optionally, the emulsifier is selected from one or more of Span 20, Span 80, poloxamer S30691, poloxamer S30692, and Tween 80. Preferably, the emulsifier is Span 20 and / or Tween 80, and the mass ratio of lemon oil to emulsifier is 6:1.7 mL / g. More preferably, the emulsifier is Span 20 and Tween 80, and the mass ratio of Span 20 to Tween 80 is 1.33:0.37.

[0016] In some embodiments of the present invention, the solvent is water; however, it can also be other solvents commonly used in the art.

[0017] Preferably, the solid-phase raw material of the chlorine dioxide sustained-release agent further includes gluconolactone. More preferably, after adding gluconolactone, the mass ratio of the precursor, gelling agent, acidifying agent, chelating agent, calcium salt, molding agent and gluconolactone is (15-25):(1-5):(1-5):(2-5):(0.15-1.3):(1-5):(1-4).

[0018] Secondly, the present invention provides a preparation process for the chlorine dioxide sustained-release agent, comprising the following steps:

[0019] Mix the gelling agent with solvent 1 to obtain gel A;

[0020] Acidifier, lemon oil, emulsifier and solvent 2 are mixed to obtain emulsion B;

[0021] The chelating agent, calcium ions, fixing agent and solvent 3 are mixed and the pH is adjusted to 6-8 to obtain mixture C;

[0022] Mix gel A and emulsion B, add the precursor, stir until homogeneous, then add mixture C, stir, and freeze to obtain the final product.

[0023] Preferably, the steps further include: mixing gel A and emulsion B, adding the precursor, stirring and mixing evenly, adding mixture C, then adding gluconolactone, stirring and freezing.

[0024] In some embodiments of the present invention, solvent 1, solvent 2 and solvent 3 are all water, and their volume ratio is 75:1.5:46.

[0025] Preferably, the lemon oil has a volume fraction of 6 to 8 parts; the stirring time for mixing gel A and emulsion B and then adding the precursor and stirring until uniform is 30 to 90 minutes; the calcium ions have a mass fraction of 0.3 to 0.51 parts; wherein, when the volume fraction is in mL, the mass fraction is in g.

[0026] The pH adjuster described in this invention can be any commonly used adjuster in the art, such as hydrochloric acid or acetic acid.

[0027] Preferably, after mixing gel A and emulsion B and adding the precursor, the mixture is first stirred at 35-45°C and then at 0-4°C to ensure uniform mixing.

[0028] Preferably, the chlorine dioxide slow-release agent is a gel type.

[0029] Thirdly, the present invention provides the application of the chlorine dioxide slow-release agent or the chlorine dioxide slow-release agent obtained by the preparation process in the preservation, disinfection and sterilization of agricultural and sideline products.

[0030] In the application of the chlorine dioxide slow-release agent, under the same quality conditions, dividing the chlorine dioxide slow-release agent into several portions is more effective than using a single portion of the slow-release agent.

[0031] Preferably, the application specifically includes: maintaining titratable acid content, maintaining soluble solids content, reducing weight loss, improving decay and hardness, inhibiting relative conductivity, improving color difference, delaying malondialdehyde increase, inhibiting lipoxidase activity, inhibiting polyphenol oxidase, inhibiting peroxidase, and reducing pathogens.

[0032] Preferably, the application specifically refers to the antibacterial effect of chlorine dioxide on brown rot fungi, yeasts, and Escherichia coli. More specifically, the application refers to the inhibition of brown rot fungi by chlorine dioxide.

[0033] More preferably, the concentration of chlorine dioxide is ≥0.025 mmol / L. Further, the concentration of chlorine dioxide is ≥0.05 mmol / L.

[0034] The beneficial effects of this invention are:

[0035] This invention provides a chlorine dioxide nanoemulsion sustained-release agent. The raw materials include a solid phase and a liquid phase. The solid phase comprises a precursor, a gelling agent, an acidifying agent, a chelating agent, a calcium salt, and a molding agent. The liquid phase comprises lemon oil, an emulsifier, and a solvent. The calcium salt inhibits chlorine dioxide release during storage. The chelating agent binds to calcium ions to form a complex, inhibiting chlorine dioxide release. The emulsifier encapsulates the aqueous acidifying agent, causing it to slowly precipitate. Lemon oil resists the oxidizing effect of chlorine dioxide. A molding agent is added to shape the product. For better molding results, glucono delta-lactone can be added to assist in the final product molding. The raw materials work together to extend the sustained-release time of the agent. This invention provides a sustained-release agent with a long release time and a concentration that can be controlled within national standards. Measurements of sodium chlorite residue show high initial consumption followed by a slower consumption rate, with high data fitting (all values ​​above 0.9). This chlorine dioxide nanoemulsion sustained-release agent has a release time of approximately one month.

[0036] The present invention has found that when Span 20 and Tween 80 are used as emulsifiers, the emulsification effect is better, the emulsion is transparent and without layering after uniform mixing, and the HLB is 10.

[0037] In the preparation process of the chlorine dioxide slow-release agent of the present invention, gel A, emulsion B and mixture C are prepared respectively. In the preparation of mixture C, the pH needs to be adjusted to dissolve the chelating agent and promote the binding of the chelating agent with calcium ions. If the pH is too acidic or too alkaline, the chelating agent will not dissolve or will not play a chelating role. Furthermore, when adding the precursor after mixing gel A and emulsion B, the temperature needs to be strictly controlled. Stirring at 35-45°C first is to improve the emulsification effect of the emulsifier, and then stirring at 0-4°C is to reduce the consumption of sodium chlorite.

[0038] This invention measures and analyzes indicators such as titratable acid, soluble solids, weight loss rate, decay index, firmness, electrical conductivity, color difference, and malondialdehyde (MDA) during the post-harvest storage of Longquan peaches. The results show that, compared with the control group (CK), chlorine dioxide slow-release agent treatment can reduce the decay index, weight loss rate, and MDA content of the fruit, and better maintain the fruit firmness, soluble solids, color difference, titratable acid content, electrical conductivity, PPO, and POD. Overall, the preservation effect of chlorine dioxide slow-release agent treatment is better.

[0039] Some agricultural products, such as peaches, are susceptible to fungal infections during post-harvest storage due to their soft tissues and thin skins. Brown rot fungus is the main pathogen causing post-harvest soft rot in peaches, and this disease is the most destructive after harvest. It is highly likely to occur during storage, transportation, sales, and consumption, causing rapid fruit decay and rendering the fruit inedible, resulting in significant economic losses. Escherichia coli is one of the main sources of food contamination, and its consumption can easily cause diarrhea, vomiting, and other gastrointestinal symptoms. Yeast can rapidly multiply by utilizing nutrients on the surface of the peel and at wound sites, thus consuming nutrients at the wound site and covering the entire wound surface. Bacteria accelerate the decay and spoilage of peaches, making them unsuitable for storage and transportation. This invention selects three common bacteria in the storage and transportation of agricultural products—brown rot fungus, yeast, and Escherichia coli—and uses a solid-state reverse culture method to investigate the inhibitory effect of chlorine dioxide on these three microorganisms. The inhibitory effect of chlorine dioxide on these three microorganisms is verified by measuring indicators such as bacterial plaque diameter, protein and DNA content, and dehydrogenase activity. Experimental results showed that chlorine dioxide inhibited the growth of brown rot fungi, yeast, and Escherichia coli. After treatment with the same concentration of chlorine dioxide, the colony diameter of brown rot fungi was significantly smaller than that of yeast and Escherichia coli, indicating that chlorine dioxide had the best bactericidal effect on brown rot fungi. Bacterial survival rates showed the same trend: the higher the chlorine dioxide concentration, the faster the bacterial survival rate decreased, demonstrating the strong bactericidal effect of chlorine dioxide. After chlorine dioxide treatment, the conductivity, extracellular protein, and DNA content of the three strains increased, and the MDA content of all three strains increased significantly. Low doses of chlorine dioxide caused a sharp decrease in bacterial dehydrogenase activity, while high doses reduced dehydrogenase activity to below 40%, indicating that chlorine dioxide has a strong inhibitory effect on bacterial metabolic enzymes. These findings provide a theoretical basis for the widespread application of chlorine dioxide as a safe and non-toxic antibacterial agent. Attached Figure Description

[0040] Figure 1 The diagram shows the chlorine dioxide sustained-release agent prepared according to the present invention.

[0041] Figure 2 This is a fitting graph of sodium chlorite residue in experimental groups 1-6 of Example 5;

[0042] Figure 3 This is a fitting graph of sodium chlorite residue in experimental groups 7-12 of Example 5;

[0043] Figure 4 This is a fitting graph showing the residual sodium chlorite content in experimental groups 13-17 of Example 5;

[0044] Figure 5 Figure showing the effect of different chlorine dioxide gelling agents on the titratable acid content of peaches;

[0045] Figure 6Figure showing the effect of different chlorine dioxide gelling agents on the soluble solids content of peaches;

[0046] Figure 7 Figure showing the effect of different chlorine dioxide gelling agents on the weight loss rate of peaches;

[0047] Figure 8 Figure showing the effect of different chlorine dioxide gel treatments on the decay index of peaches;

[0048] Figure 9 Figure showing the effect of different chlorine dioxide gelling agents on the hardness of peaches;

[0049] Figure 10 Figure showing the effect of different chlorine dioxide gelling agents on the relative conductivity of peaches;

[0050] Figure 11 Figure showing the effect of different chlorine dioxide gel treatments on the malondialdehyde content of peaches;

[0051] Figure 12 Figure showing the effect of different chlorine dioxide gel agents on peach lipid peroxidase;

[0052] Figure 13 Figure showing the effect of different chlorine dioxide gel treatments on polyphenol oxidase in peaches;

[0053] Figure 14 Figure showing the effect of different chlorine dioxide gel treatments on peroxidase in peaches;

[0054] Figure 15 The graph shows the effect of different concentrations of chlorine dioxide treatment on the survival rate of three bacteria.

[0055] Figure 16 The graph shows the effect of different concentrations of chlorine dioxide treatment on the conductivity of three bacteria.

[0056] Figure 17 The graph shows the effect of different concentrations of chlorine dioxide treatment on the protein content of three bacteria.

[0057] Figure 18 The effect of different concentrations of chlorine dioxide treatment on the DNA content of three bacteria is shown in the figure.

[0058] Figure 19 The effect of different concentrations of chlorine dioxide treatment on the MDA content of three bacteria is shown in the figure.

[0059] Figure 20 Figure 1 shows the effect of different concentrations of chlorine dioxide treatment on the dehydrogenase activity of three bacteria.

[0060] Figure 21 This is a scanning electron microscope image of bacteria after treatment with 0.1 mmol / L chlorine dioxide for 16 h. Detailed Implementation

[0061] To enable those skilled in the art to better understand the technical solution of the invention, the invention will be further described in detail below with reference to specific embodiments.

[0062] Example 1: Preparation of Chlorine Dioxide Slow-Release Agent

[0063] This embodiment provides a preparation process for a chlorine dioxide sustained-release agent, including the following steps:

[0064] Mix 2g of sodium alginate with 75mL of water to obtain gel A;

[0065] Dissolve 2g of tartaric acid in 1.5mL of water by sonication, then add it in small amounts several times to 6mL of lemon oil, and add an emulsifier to obtain emulsion B;

[0066] Dissolve calcium chloride in 1 mL of water to obtain a calcium chloride solution. Pipette the calcium chloride solution into an EDTA solution (2.92 g dissolved in 20 mL of water). Adjust the pH to 7.0 with acetic acid. Then add 1.875 g of agar and 25 mL of water. Mix to obtain mixture C.

[0067] Mix gel A and emulsion B, then add 18g of sodium chlorite. Stir at 35-45℃ and then at 0-4℃ until homogeneous. Add mixture C and stir. Finally, add 1.8g of glucono delta-lactone and stir. Freeze at -18℃ to obtain the final product.

[0068] The prepared chlorine dioxide slow-release agent is shown in the figure below. Figure 1 As shown.

[0069] Example 2: Screening of Emulsifier Types

[0070] Regarding the preparation process described in Example 1, the types of emulsifiers used were screened:

[0071] Take 6 portions of 6 mL lemon oil and add 0.5 g Span 20, 0.5 g Span 80, 0.5 g Poloxamer S30691, 0.5 g Poloxamer S30692, and 0.5 g Tween 80 respectively. Mix well and let stand for 6 hours. Observe the color change and measure the layer height. The results are shown in Table 1.

[0072] Table 1 Results of emulsifier selection

[0073]

[0074] As shown in Table 1, Span 20 and Tween 80 can be mixed evenly with lemon oil without separation. However, Span 80 and lemon oil show some uneven mixing. Furthermore, the two types of poloxamer are immiscible with lemon oil or cannot be mixed evenly. Therefore, Span 20 and / or Tween 80 are preferred as emulsifiers.

[0075] Example 3 Screening of Single Emulsifier Dosage

[0076] Dosage screening was performed on the single emulsifiers obtained in Example 2:

[0077] While maintaining a consistent amount of 6 mL lemon oil, 0.5 g, 1 g, 1.5 g, 1.7 g, and 2 g of Span 20 were added respectively, mixed thoroughly, and allowed to stand for 6 hours before the separation height was measured. The results are shown in Table 2.

[0078] Table 2. Screening Results of Span 20 Dosage

[0079]

[0080] As shown in Table 2, different amounts of Span 20 can be mixed evenly with lemon oil, but after standing for a while, layering occurs. The layering is most severe when 1g of Span 20 is added, reaching 0.52cm, while the layering height is the least when 1.7g of Span 20 is added, at 0.32cm. Therefore, the preferred amount of Span 20 is 1.7g.

[0081] Example 4 Screening of Mixed Emulsifier Dosage

[0082] The dosage of the emulsifiers obtained in Example 2 when used in combination was further screened:

[0083] Using a single emulsifier can produce a uniform mixture, but after a period of time, stratification will occur. Therefore, the amount of emulsifier was adjusted according to the HLB value. While maintaining the same amount of 6 mL of lemon oil, two emulsifiers were added, mixed evenly, and left to stand for 6 hours to observe the changes. The results are shown in Table 3.

[0084] Table 3 Screening of Mixed Emulsifier Dosage

[0085]

[0086] As can be seen from Table 3, when HLB is 9 and 9.5, the emulsion is milky in color and separates into layers after being mixed evenly. When HLB is 10, the emulsion is transparent and separates into layers after being mixed evenly. Therefore, the preferred dosage of Span 20 is 1.33g and the content of Tween 80 is 0.37g.

[0087] Example 5: Beckman Design-Response Surface Methodology Optimization of Preparation Process

[0088] After determining the preparation process based on the process described in Example 1 and the emulsifier screened in Example 4, three factors were investigated: stirring time of sodium chlorite, amount of lemon oil, and amount of calcium ions added. The experimental design is shown in Table 4.

[0089] Table 4 Box-Behnken Experimental Design

[0090]

[0091] Table 5. Results of the Box-Behnken experiment

[0092]

[0093]

[0094] Experimental methods:

[0095] The chlorine dioxide sustained-release agent prepared using the above 17 sets of parameters was used as a sample for testing. The residual NaClO2 in the nanoemulsion sustained-release agent was determined by iodometric titration. NaClO2 reacts with potassium iodide in an acidic environment, reducing chlorite ions to chloride ions, and the solution turns brown. Sodium thiosulfate is then used to titrate elemental iodine, reducing it to colorless iodide ions, and the brown color disappears. The sodium chlorite content can be determined based on the sodium thiosulfate consumption rate. A mixture of 0.5g sample, 25mL distilled water, 2g potassium iodide, 15mL distilled water, and 5mL 10mol / L HCl was reacted in the dark for 10min. The solution was then titrated with 50mL water and sodium thiosulfate until a light yellow color was reached. 1mL of starch indicator was added, and titration continued until the deep blue color disappeared. The readings were recorded, and each group was repeated three times.

[0096] The results of the Box-Behnken experiment are shown in Table 5.

[0097] Results analysis:

[0098] The retention rate of NaClO2 in the slow-release agent decreased as NaClO2 was consumed. In the early stage of the experiment, the sodium chlorite content in the slow-release agent was high, resulting in a rapid reaction with tartaric acid and rapid consumption of sodium chlorite. As time progressed, the consumption rate of sodium chlorite gradually decreased. Because the slow-release agent was consumed rapidly in the early stage and the rate of decrease slowed down in the later stage, the residual amount of sodium chlorite resembled a quadratic function model. The relationship between the residual amount of sodium chlorite and time was fitted using the model: Y = AX 2 +BX+C, where A is the coefficient of the quadratic term in the fitted equation, B is the coefficient of the linear term in the fitted equation, and C is the constant of the fitted equation. The fitting results are shown in Table 6 and... Figures 2-4 As shown.

[0099] from Figures 2-4It can be seen that the curves of each group have basically the same trend. The changes are fast in the early stage of the experiment and slow in the later stage. Among all the fitting graphs, the curves of groups 9 and 11 changed relatively slowly in the early stage.

[0100] Table 6. Table of coefficients for regression equations of fitted curves

[0101]

[0102]

[0103] As shown in Table 6, the coefficients of determination for all 17 fitted equations are greater than 0.9, and the significance level is P < 0.05, indicating that the quadratic equation model is suitable for the analysis of sodium chlorite residues, and this model can be used to analyze the results.

[0104] Example 6: Application of chlorine dioxide gelling agent in the preservation of peaches

[0105] Experimental Design:

[0106] 1. Sample processing and index determination of Longquan peaches

[0107] The peaches used in the experiment were harvested from Longquanshan in Chengdu. They were picked when they were 80% ripe, and fruits with similar color, size, and no mechanical damage or pests were selected (fruits were 80% ripe, with similar color and size, and no wounds). After harvesting, the peaches were cooled by fans and divided into 8 groups (4 groups for weight loss rate and decay index, and 4 groups for other index tests), with 18 fruits in each group. After grouping, the fruits were placed in 10L cardboard boxes. The control group (CK) did not contain a slow-release agent, while the other boxes contained chlorine dioxide slow-release agent. Box A contained a complete piece of slow-release agent and was designated as treatment group A. Box B contained slow-release agent divided into 2 equal parts and was designated as treatment group B. Box C contained slow-release agent divided into 3 equal parts and was designated as treatment group C. The fruits were stored at an ambient temperature (25℃, 84% humidity), and the indexes were measured every 2 days, with each measurement performed in triplicate.

[0108] 2. Titrizable acidity (TA) determination of Longquan peaches

[0109] Peel the sample, homogenize 50g of pulp in 50mL of deionized water for 2 minutes, mix with 150mL of distilled water, filter, take 5mL of filtrate, add 2 drops of 1% phenolphthalein, titrate with 0.1mol / L sodium hydroxide solution until the solution turns light pink and does not fade in 30s, the endpoint is recorded, record the volume of sodium hydroxide titrant used, repeat three times, the result is calculated based on the amount of malic acid, the conversion factor is 0.067.

[0110] 3. Soluble solids

[0111] The soluble solids (TSS) content of peach fruit was determined using a PAL-1 handheld saccharimeter and expressed as a percentage (%).

[0112] 4. Weightlessness rate

[0113] The weighing method was adopted, that is, the fruit was weighed before storage and recorded as A1, and the fruit was weighed once every 2 days during storage and recorded as A2. The weight loss rate = (A1-A2) / A1×100%.

[0114] 5. Decay Index

[0115] The fruit pulp rot index is divided into 6 levels: Level 1 has no rot, Level 2 indicates 1-2 rotten spots, Level 3 indicates 2-4 rotten spots, Level 4 has 1 / 4-1 / 3 rotten area, Level 5 has 1 / 3- rotten area, and Level 6 has a rotten area greater than 1 / 2.

[0116] 6. Hardness

[0117] Ten fruits were taken for each test, and the firmness of each fruit was measured at three locations. The test data were recorded, and the average value and standard deviation were calculated. The average value represents the firmness of the fruit.

[0118] 7. Relative conductivity measurement

[0119] The conductivity was measured according to the literature. 3g of fruit pulp was soaked in 30mL of distilled water and left to stand for 1 hour. The initial conductivity was measured using a conductivity meter and recorded as R1. After boiling in a water bath for 10 minutes and cooling to room temperature, the conductivity was measured again and recorded as R2. Each treatment group was measured three times and the average value was taken. The conductivity of distilled water was R.

[0120] Relative conductivity = [(R1-R) / (R2-R)]*100%

[0121] 8. Determination of color difference in peaches

[0122] Take 2g of fruit peel, chop it, extract it with 5mL of 50% and 100% ethanol by ultrasonic extraction for 5 minutes, centrifuge at 12000r / min for 5 minutes, collect the supernatant, and perform full-wavelength scanning.

[0123] 9. Determination of malondialdehyde content

[0124] Sample extraction: Take 1g of sample, add 5mL of 100g / L TCA solution, grind into a homogenate, centrifuge at 10000r / min for 20min at 4℃, collect the supernatant, and store at low temperature for later use.

[0125] The samples were analyzed using the thiobarbituric acid method. 2.0 mL of supernatant (with 2.0 mL of 100 g / L TCA solution added to the control blank tube instead of the extraction buffer) and 2.0 mL of 0.67% TBA were mixed, boiled in a water bath for 20 min, cooled, and then centrifuged once more. The OD of the supernatant was then measured. 450 OD 532 and OD 600 The absorbance value at the specified wavelength was recorded, and the result was repeated three times.

[0126] 10. Lipoxidase (LOX)

[0127] Sample extraction: Weigh 5g of sample, homogenize with 5mL of extraction buffer at 4℃, and centrifuge at 12000r / min for 30min at 4℃.

[0128] Sample determination: Mix 2.75 mL of 0.1 mol / L, pH 5.5 acetate-sodium acetate buffer and 50 μL of 0.1 mol / L linoleic acid solution, incubate at 30°C for 10 min, then add 200 μL of extraction supernatant. Zero the solution with distilled water and measure the OD. 234 The absorbance value at the specified wavelength was recorded, and the result was repeated three times.

[0129] 11. Extraction and determination of polyphenol oxidase (PPO) activity

[0130] Extraction buffer: Weigh 340 mg PEG6000, 4 g polyvinylpyrrolidone, and 1 mL Triton X-100, and bring the volume to 100 mL with 0.1 mol / L, pH 5.5 acetate-sodium acetate buffer.

[0131] PPO and POD sample extraction: Weigh 5g of sample, homogenize with 5mL of extraction buffer at 4℃, and centrifuge at 12000r / min for 30min at 4℃.

[0132] PPO was measured using the catechol method: 4.0 mL of acetate-sodium acetate buffer (50 mmol / L, pH 5.5), 0.1 mL of supernatant, 1.0 mL of 50 mmol / L catechol solution, and distilled water was used for zeroing. The OD value was then measured. 470 The absorbance value at the specified wavelength was recorded, and the result was repeated three times.

[0133] 12. Extraction and determination of peroxidase (POD) activity

[0134] POD was measured using the guaiacol method. 2.8 mL of 25 mol / L guaiacol, 0.2 mL of 0.5 mol / L guaiacol, and 0.4 mL of sample extract were used, with the zeroing setting adjusted to distilled water. 470 The absorbance value at the specified wavelength was recorded, and the result was repeated three times.

[0135] 13. Colony determination

[0136] The total number of colonies was determined by plate counting. A 0.5 cm section of the decayed part was taken, chopped with sterile scissors, and placed in 10 mL of sterile physiological saline. The mixture was shaken and stirred until homogeneous. The mixture was then diluted to different concentrations of bacterial suspension. 0.1 mL of each suspension was spread onto a PDA and incubated at 37°C for 2-3 days before counting.

[0137] Results analysis:

[0138] 1. Effect on the titratable acid content of peaches

[0139] like Figure 5 As shown, the titratable acid content is inversely proportional to the storage time; the titratable acid content decreases with increasing storage time, with the CK group showing the largest decrease. On day 6, the CK group decreased to 0.37%, while groups A, B, and C were 3.86%, 3.91%, and 4.08%, respectively, showing a smaller decrease in the treatment groups. Subsequently, the titratable acid content continued to decrease. By day 10, the titratable acid content in the CK group was 3.12%, a decrease of 16%; the content in group A was 3.46%, a decrease of 10.62%; and the content in group B was 3.51%, a decrease of 10.23%, with no significant difference (P>0.05). The titratable acid content in group C was 3.69%, a decrease of 9.6%, with a significant difference (P<0.05). Chlorine dioxide may effectively maintain the content of titratable acid by preventing the conversion of organic acids into soluble sugars and reducing respiration, indicating that the rapid release of chlorine dioxide can effectively slow down the decline in the titratable acid content of Longquan peaches after harvest and maintain a good flavor.

[0140] 2. Effects on soluble solids in peaches

[0141] Soluble solids refer to all compounds dissolved in water, including sugars, acids, vitamins, and minerals. In fruits and vegetables, the content of soluble solids and sugars is directly proportional to the weight of the fruit or vegetable and is an important indicator of quality. The content of soluble solids directly affects the taste of peaches. Soluble solids gradually accumulate as a respiration substrate, releasing energy until the peach is fully ripe. Continuous nutrient depletion leads to a decline in peach quality and a shortened post-harvest shelf life. Changes in soluble solids content are shown in the diagram. Figure 6 As shown, soluble solids exhibited a trend of first increasing and then decreasing. The initial increase may be due to starch hydrolysis into monosaccharides. After reaching its maximum value, soluble solids began to decrease, possibly due to a reduced respiration rate. After 2 days of storage, group A had the lowest soluble solids content, while group B had the highest. After 6 days of storage, group B had the highest soluble solids content at 10.86%, followed by group A. At the end of storage, the CK group had a content of 10.55%, which was 4%, 9%, and 18% lower than groups A, B, and C, respectively.

[0142] 3. Impact on the quality loss rate of peaches

[0143] Peaches are highly susceptible to rapid water loss, leading to fruit shrinkage. Postharvest weight loss is primarily due to water loss and nutrient consumption via transpiration and respiration. The weight loss rate of Longquan peaches varies as follows: Figure 7As shown in the figure, the weight loss rate of Longquan peaches is directly proportional to the number of storage days; that is, the longer the storage time, the higher the weight loss rate. In the first four days, the weight loss rate of Longquan peaches changed little. Between the fourth and sixth days, the weight loss rate increased significantly, increasing by 1.81%, 1.69%, 1.62%, and 1.13% respectively compared to the fourth day. The weight loss rates of the chlorine dioxide treatment groups were significantly lower than those of the control group (CK), indicating that chlorine dioxide can reduce the effect of moisture in Longquan peaches during storage, with the control group showing the best effect. Current research has found that chlorine dioxide can delay softening by reducing fruit weight loss and slowing down metabolism.

[0144] 4. Impact on the decay index of peaches

[0145] Depend on Figure 8 It can be seen that the decay index of Longquan peaches increased in all groups during storage. Compared with the control group (CK), the decay index of the chlorine dioxide treatment group was significantly lower (P<0.05). On the 10th day, the decay index of the CK group rose to 0.55, and the decay situation further aggravated, basically losing its commercial value. The decay indices of groups A, B, and C were 10%, 15.67%, and 21.45% lower than those of the CK group, respectively, indicating that chlorine dioxide treatment can reduce the decay rate of Longquan peaches.

[0146] 5. Effect on the firmness of peaches

[0147] The decrease in firmness is due to cell wall degradation caused by increased activity of endogenous autolytic enzymes in the cell wall; enzymatic hydrolysis of the cell wall and hydrolysis of pectinase by pectinase, which tightly binds the fruit cells, lead to cell dispersion and softening of the pulp. The effects of different treatments on the firmness of Longquan peaches are as follows: Figure 9 As shown, the firmness of the fruit tissue decreased throughout the storage period, with group B exhibiting the highest firmness on day 2 of storage. This firmness was 14.8%, 10.01%, and 5.76% higher than groups CK, A, and C, respectively. At the end of storage, the firmness of group CK was 3.63, while the firmness of groups A, B, and C were 3.97, 4.33, and 4.63 kg / cm², respectively. 2 The firmness of the fruit treated with chlorine dioxide was significantly higher than that of the control group, indicating that chlorine dioxide treatment can significantly reduce the decrease in the firmness of peaches. Therefore, chlorine dioxide gas has a certain effect on improving the firmness of Longquan peaches.

[0148] 6. Effect on the electrical conductivity of peaches

[0149] During fruit senescence, apoptosis and nutrient degradation severely impair the normal function of the cell membrane, making it more permeable. A higher relative conductivity indicates a greater degree of damage to cell membrane integrity, and therefore can be used to evaluate the freshness of peaches. Figure 10As shown, the relative conductivity of Longquan peaches is directly proportional to the storage time; the longer the storage time, the greater the relative conductivity. The relative conductivity of the treated Longquan peaches was lower than that of the control group. On the 10th day of storage, the difference in relative conductivity between the control group and the CK group was the greatest, and the difference was significant between the two groups on days 4-6 (p<0.05). Therefore, chlorine dioxide treatment can effectively inhibit the increase in the relative conductivity of Longquan peaches.

[0150] 7. The effect on color difference of peaches

[0151] During storage, the color of Longquan peaches changed, becoming darker with longer storage time. Table 7 shows the color changes after 100% ethanol extraction. The absorbance value was directly proportional to the storage time. The absorbance value of the control group (CK) was higher than that of the treatment group, indicating that the peaches in the CK group were darker. This is because chlorine dioxide can prevent the formation of ethylene from methionine, destroying existing ethylene and delaying fruit senescence and decay. Three absorption wavelengths appeared on the second day, indicating changes in the Longquan peaches. Afterward, only two absorption wavelengths were observed, mainly around 265 and 310 nm. However, at the end of storage, the absorption wavelength increased, possibly due to changes in certain components of the Longquan peaches. Table 8 shows the color changes after 50% ethanol extraction, with two absorption wavelengths, approximately around 262 and 310 nm. The same increase in absorption wavelengths was observed at the end of storage.

[0152] Table 7. Effects of 100% ethanol extraction on the color of Longquan peaches in different treatment groups.

[0153]

[0154] Table 8. Effects of 50% ethanol extraction on the color of Longquan peaches in different treatment groups.

[0155]

[0156]

[0157] 8. Effect on malondialdehyde content in peaches

[0158] The effects of different treatments on malondialdehyde in Longquan peaches, such as Figure 11 As shown, the MDA content continued to increase during storage, and chlorine dioxide treatment could delay the increase of MDA content in the fruit. After 8 days of storage, the MDA content of the treated group was significantly lower than that of the control group (P<0.05). At the end of storage, the MDA content of the control group reached its maximum value (4.39 nmol / g), which was 10.2% higher than group A, 18.7% higher than group B, and 21.46% higher than group C.

[0159] 9. Effects on lipooxidase (LOX) in peaches

[0160] like Figure 12 As shown, LOX enzyme activity initially increased and then decreased during storage. The enzyme activity in the control group reached its peak of 2.76ΔOD on day 6. 234 / min·g, while the enzyme activity in the C treatment group was 2.32ΔOD. 234 The enzyme activity in the CK group was significantly lower than that in the control group (CK group). By day 6 of storage, the LOX content decreased in all groups. On day 10, the enzyme activity in the CK group was 2.35 ΔOD. 234 The concentration of chlorine dioxide was 20.21% higher than that of the C treatment group. Chlorine dioxide can effectively inhibit the increase of LOX enzyme activity in peach fruit, showing good antioxidant effect and extending the shelf life of peaches.

[0161] 10. Effects on polyphenol oxidase (PPO) in peaches

[0162] Polyphenol oxidase (PPO) is a major enzyme that catalyzes the oxidation of endogenous polyphenols in fruits and vegetables to produce melanin, thus causing enzymatic browning. The changes in polyphenol oxidase (PPO) levels in peaches under different treatment groups are shown below. Figure 13 As shown, PPO activity increased with prolonged storage. The figure also shows that, except for the second day, the PPO enzyme activity in the treatment group was lower than that in the control group. On day 4, the enzyme activity in the control group was 5.1 ΔOD. 420 / min·g, treatment group A had a ΔOD of 4.76. 420 / min·g, treatment group B had a ΔOD of 4.33. 420 / min·g, C treatment group was 4.27ΔOD 420 / min·g, on day 10, the activities of the CK group and each experimental group were 6.12, 5.71, 5.34, and 5.01ΔOD, respectively. 420 / min·g. Therefore, the C treatment group can effectively inhibit PPO enzyme activity, which may be because high concentrations of chlorine dioxide effectively inhibit PPO.

[0163] 11. Effects on peroxidase (POD) in peaches

[0164] Low POD enzyme activity can inhibit browning of the fruit pulp. Figure 14 It can be seen that during storage, the POD activity of Longquan peaches generally showed a trend of first increasing and then decreasing, with the maximum POD activity occurring on the second day. The POD activity of the CK group was 44.82ΔOD. 470 / min·g, which was 1.59 times that of day 0. After 4 days of storage, the POD activities of Longquan peaches in groups A, B, and C were 39.42, 40.1, and 42.22ΔOD, respectively. 470The POD activity was significantly higher than that of the control group (P<0.05), possibly due to the strong oxidizing effect of chlorine dioxide and the senescence of the Longquan peach itself, which stimulated the Longquan peach and led to an increase in its POD activity. From the 4th day, the POD activity of each group decreased, with the treatment group being higher than the CK group, indicating that chlorine dioxide treatment has an inhibitory effect on the POD enzyme activity of Longquan peach.

[0165] 12. Effect on the total bacterial count of peaches

[0166] Peach fruits are susceptible to pathogen infection during harvesting and transportation. Chlorine dioxide, with its strong oxidizing properties, easily penetrates cell membranes. Increased cell membrane permeability is due to chlorine dioxide altering the conformation of outer membrane proteins and lipids, oxidizing amino acids, particularly tyrosine, thereby affecting microbial protein synthesis and metabolism, thus reducing the increase of pathogens. The effects of different treatments on the bacterial colony count of Longquan peaches are shown in Table 9: CK group > A group > B group > C group. This means that at the same concentration, a larger release area results in a better bactericidal effect. The rapid release of chlorine dioxide gas triggers strong oxidation in a short time, thereby killing microorganisms on the surface.

[0167] Table 9. Effects of different treatments on the total bacterial count of Longquan peaches.

[0168]

[0169] Note: - indicates no colonies on the plate surface, + indicates that the number of colonies on the plate is too high to count.

[0170] Example 7: Inhibitory effect of chlorine dioxide on brown rot fungi, yeast and Escherichia coli

[0171] Experimental Design:

[0172] Escherichia coli BNCC336902 (E. coli) and yeast JC2636 (Yeast) were both provided by the Microbiology Laboratory of the Sichuan Provincial Experimental Teaching Center for Basic Biomedical Research, Chengdu University.

[0173] 1. Isolation and revival of brown rot fungi

[0174] A small amount of rotten parts were collected from diseased Longquanshui peaches and serially diluted with sterile water. 0.1 mL of the diluted solution was placed on sterile PDA solid medium, spread evenly, and incubated at 25°C for 1 day. Single colonies were selected based on colony morphology and transferred to fresh sterile medium for further incubation. This process was repeated three times. Morphological and PCR verification confirmed the presence of *Brachys thunbergii*, a brown rot fungus. The strain was stored on PDA slant at 4°C. Brown rot fungus, yeast, and *Escherichia coli* were picked using an inoculation loop and inoculated into 100 mL of sterile LB liquid medium, respectively. The medium was incubated at 25°C, 28°C, and 37°C with shaking at 100 rpm for 24 hours. The concentration was adjusted to 10... 6 CFU / mL, take 50 μL of bacterial suspension and place it in the center of a PDA plate, spread it evenly, and incubate the petri dishes at 25℃, 28℃ and 37℃ for 1 day for later use.

[0175] 2. Antibacterial activity assay

[0176] Using a sterile punch, take a piece of mycelial cake from item "1" and invert it onto the center of a PDA plate. Accurately weigh chlorine dioxide solid and prepare 10 mL of chlorine dioxide solutions of 0.0125, 0.025, 0.05, and 0.1 mmol / L respectively with distilled water. Pour each 10 mL solution into a 50 mL beaker. Place the beaker and petri dish in a 10 L plastic box and incubate at 25℃, 28℃, and 37℃ for 16 h, respectively. Remove the petri dish and continue incubation until the colony diameter of the control group is greater than 2 / 3 of the petri dish diameter, then end the incubation. Use the untreated mycelial cake as the control group.

[0177] (1) Colony diameter determination

[0178] After the culture was completed, the diameter of the bacterial plaque in each group was measured three times using a vernier caliper and the average value was taken.

[0179] (2) Survival rate determination

[0180] After colony culture, scrape off a plaque of the same size (approximately 4 cm in diameter) with a knife, place it in a test tube, disperse it evenly with 20 mL of sterile PBS, and measure the absorbance at a wavelength of 600 nm. Compare the absorbance value with that of the blank control group, and calculate the survival rate lg(N) using the following formula. t / N0):

[0181] lg(N t / N0)=lg(A t / A0)

[0182] Where, N t N0 and A represent the bacterial counts in the chlorine dioxide treatment group and the control group, respectively. t A0 and A0 represent the absorbance values ​​of the bacterial suspensions in the chlorine dioxide treatment group and the blank group, respectively.

[0183] (3) Conductivity measurement

[0184] Take the bacterial plaques from section "2" after the culture is completed, disperse the sample in sterile water and place it in a beaker. Measure the conductivity using a conductivity meter and record the value as A1. After measurement, boil all samples in a water bath for 15 minutes, cool, and measure the conductivity again, recording the value as A2. Calculate the conductivity:

[0185] Conductivity (%) = A1 / A2 × 100

[0186] 3. Protein and DNA content determination

[0187] The colonies cultured after reverse inoculation were washed with PBS and centrifuged at 12000 rpm for 30 min; the supernatant was collected and the OD was measured using a UV spectrophotometer. 260 and OD 280 DNA content is expressed as the absorbance at a wavelength of 260 nm. Protein content is then calculated.

[0188] Protein content (mg / mL) = 1.45 × OD 280 -0.74×OD 260

[0189] 4. MDA determination

[0190] After reverse inoculation, the colonies were washed with PBS, and then 1.5 mL of the supernatant was taken and 3 mL of 10% trichloroacetic acid (w / v) was added. The mixture was centrifuged at 4℃ and 4500 r / min for 30 min. 2 mL of the supernatant was taken and 4.5 mL of 0.67% thiobarbituric acid (w / v) was added. The mixture was boiled in a water bath for 10 min, centrifuged, and the absorbance was measured at 532 nm. The content was calculated according to the MDA standard curve.

[0191] 5. Dehydrogenase activity assay

[0192] After reverse inoculation, the colonies were washed with PBS, 6 mL of bacterial culture was taken, 2 mL of Tris-HCl (pH 8.8) solution and 2 mL of TTC-glucose solution were added, and the mixture was heated in a water bath at 37°C for 2 h. Then, 7.5 mL of toluene and glacial acetic acid solution were added respectively. After standing at room temperature for 1 h, the absorbance was measured at a wavelength of 485 nm. Bacteria that were not treated with chlorine dioxide were used as negative controls.

[0193] 6. Bacterial morphology observation

[0194] Take the bacteria cultured under item "2", treat with chlorine dioxide for 16 h, take the bacterial cake with a 6 mm sterile punch, disperse it in sterile distilled water, centrifuge at 8000 r / min for 5 min; discard the supernatant, add 2 mL of 2.5% glutaraldehyde (w / v), and place at 4℃ for 12 h; perform gradient elution, and after the sample is naturally dried, spray with gold, load the sample, and observe the morphology under a scanning electron microscope.

[0195] 7. Statistical Analysis

[0196] Sample measurements were repeated three times, and results are expressed as mean ± standard deviation. Graphs were generated using Origin 2021 software, and statistical analysis was performed using SPSS 26.0 software. A p-value ≤ 0.05 was considered statistically significant between groups.

[0197] Results analysis:

[0198] 1. Antibacterial activity

[0199] (1) Results of colony diameter measurement

[0200] The colony diameter results are shown in Table 10. There was a negative correlation between chlorine dioxide concentration and colony diameter; that is, the higher the concentration, the smaller the colony diameter. After treatment with the same concentration of chlorine dioxide, the colony diameters of brown rot fungi were significantly smaller than those of yeast and Escherichia coli, indicating that chlorine dioxide had the best bactericidal effect on brown rot fungi.

[0201] Table 10 Effect of chlorine dioxide treatment on colony diameter

[0202]

[0203] ae indicates that the same strain showed significant differences after treatment with different concentrations of chlorine dioxide (P<0.05).

[0204] (2) Survival rate determination results

[0205] Bacterial survival rates showed the same trend, such as Figure 15 As shown, with increasing chlorine dioxide concentration, the survival rates of brown rot fungi, yeast, and Escherichia coli gradually decreased. Among them, the logarithmic value of brown rot fungi decreased the fastest, decreasing by 1.73 log CFU / mL, while the survival rates of yeast and Escherichia coli decreased by 1.26 and 1.03 log CFU / mL, respectively, indicating the strong bactericidal effect of chlorine dioxide. When the concentration was ≥0.025 mmol / L, the survival rate of brown rot fungi was significantly lower than that of yeast and Escherichia coli (P<0.05), and when the concentration was ≥0.05 mmol / L, the survival rate of yeast was significantly lower than that of Escherichia coli (P<0.05).

[0206] (3) Conductivity Measurement Results

[0207] The conductivity measurement results are as follows Figure 16 As shown, the conductivity of the bacterial culture medium increased continuously with increasing chlorine dioxide concentration, indicating that different concentrations of chlorine dioxide caused varying degrees of damage to the cell membranes of brown rot fungi, yeast, and Escherichia coli, with higher concentrations resulting in greater cell membrane damage. This suggests that high-concentration chlorine dioxide solutions can significantly disrupt the cell structure of these three bacteria, damaging cell integrity, increasing cell membrane permeability, and allowing intracellular substances to leak out of the cell. When the chlorine dioxide concentration was ≤0.0125 mmol / L, there was no significant difference in conductivity among the three strains; as the chlorine dioxide concentration increased to 0.025 mmol / L, the conductivity order was brown rot fungi > yeast > Escherichia coli.

[0208] 2. Protein and DNA content

[0209] The results of the determination of the protein content leaked by bacteria in the culture medium are as follows: Figure 17 As shown, after chlorine dioxide treatment, the protein content exuded by brown rot fungi, yeast, and Escherichia coli was positively correlated with the chlorine dioxide concentration. At most concentrations, the protein content order was brown rot fungi > yeast > Escherichia coli (P < 0.05). The extracellular protein content of brown rot fungi was significantly higher than that of yeast and Escherichia coli. The initial concentration of brown rot fungi was 0.017 mg / L. After treatment with 0.05 mmol / L chlorine dioxide, the protein content increased to 0.07 mg / L, which was 3.1 times that of the control group. Treatment of yeast and Escherichia coli with the same concentration of chlorine dioxide (0.05 mmol / L) increased the protein content by 68.84% and 59.31%, respectively, compared to the control group.

[0210] Results of extracellular DNA content assay as follows Figure 18As shown, the extracellular DNA content of bacteria gradually increased with increasing chlorine dioxide concentration. At low concentrations of chlorine dioxide, the DNA content was low, and the change was minimal when the concentration was below 0.025 mmol / L. Subsequently, the content increased rapidly with increasing concentration; the content of brown rot bacteria increased from 2.27 μg / mL to 26.58 μg / mL, and that of yeast increased from 1.6 μg / mL to 19.67 μg / mL. The DNA content of *E. coli* treated with 0.1 mmol / L chlorine dioxide was 5.4 times that of the control group. When the chlorine dioxide concentration reached 0.025 mmol / L, the DNA content of brown rot bacteria increased sharply; when the chlorine dioxide concentration reached 0.05 mmol / L, the extracellular DNA of yeast began to rise rapidly, while the DNA of *E. coli* showed a steady increase. When the chlorine dioxide concentration was 0.05 mmol / L, the DNA content of the three bacteria was in the order of brown rot bacteria > Escherichia coli > yeast (P < 0.05); when the gas concentration increased to 0.1 mmol / L, the DNA content was in the order of brown rot bacteria > yeast > Escherichia coli, and the extracellular DNA concentration of brown rot bacteria was significantly higher than that of the other two bacteria (P < 0.05).

[0211] 3. MDA content

[0212] Results of bacterial MDA content determination are as follows Figure 19 As shown, the intracellular MDA content of bacteria gradually increased with the increase of chlorine dioxide concentration. Under most concentrations, the MDA content was in the order of brown rot fungi > yeast > Escherichia coli. When the concentration was ≥0.0125 mmol / L, the MDA produced by brown rot fungi was significantly higher than that of yeast and Escherichia coli (P<0.05), indicating that chlorine dioxide had a greater effect on brown rot fungi and showed a better inhibitory effect.

[0213] 4. Dehydrogenase activity

[0214] Dehydrogenase activity assay results are as follows Figure 20 As the concentration of chlorine dioxide increased, the dehydrogenase activities of the three bacteria decreased rapidly. At a chlorine dioxide concentration of 0.0125 mmol / L, the enzyme activity of *E. coli* was significantly higher than that of *Brachys rubrum* and yeast (P < 0.05), indicating that the respiratory metabolic capacity of *E. coli* was less hindered, suggesting that chlorine dioxide had a relatively weak destructive effect on *E. coli* enzymes. When the chlorine dioxide concentration increased to 0.025 mmol / L, the enzyme activities of the three strains were *E. coli* > yeast > *Brachys rubrum* (P < 0.05). When the chlorine dioxide concentration was ≥ 0.05 mmol / L, the dehydrogenase activities of the three strains continued to decrease, with no significant differences between groups (P > 0.05), indicating that high concentrations of chlorine dioxide can inactivate the dehydrogenases of different bacteria, thereby causing bacterial death.

[0215] 5. Bacterial morphological changes

[0216] SEM scan results are as follows Figure 21 As shown, the blank group had intact morphology, a relatively smooth surface, and almost no wrinkles, with a clean SEM background, indicating no content effusion. In the treatment group treated with 0.1 mmol / L chlorine dioxide, some of the three bacteria underwent morphological changes, with shriveled, rough, and sunken surfaces, damaged cell membranes, and blurred boundaries. Multiple aggregates and exudates were visible under the microscope. Among them, the brown rot fungus showed the most severe deformation, with a severely collapsed morphology and a large amount of exudate, which is consistent with the inactivation effect.

[0217] The above are merely preferred embodiments of the present invention. It should be noted that the above preferred embodiments should not be considered as limitations on the present invention, and the scope of protection of the present invention should be determined by the scope defined in the claims. For those skilled in the art, several improvements and modifications can be made without departing from the spirit and scope of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A preparation process for a chlorine dioxide sustained-release agent that inhibits brown mold, yeast, and Escherichia coli, characterized in that, Includes the following steps: Mix the gelling agent with water to obtain gel A; An acidifier, lemon oil, emulsifier, and water are mixed to obtain emulsion B, wherein the emulsifier is Span 20 and Tween 80, and the mass ratio of Span 20 to Tween 80 is 1.33:0.37; the mass ratio of lemon oil to emulsifier is 6:1.7 mL / g. Mix the chelating agent, calcium ions, agar and water, and adjust the pH to 6-8 to obtain mixture C; Mix gel A and emulsion B, add the precursor, stir until homogeneous, add mixture C, stir, then add gluconolactone, and freeze to obtain the final product. The gelling agent is selected from one or more of xanthan gum, alginate, sodium alginate, sodium carboxymethyl cellulose, and modified starch; the acidifying agent is selected from one or more of acetic acid, malic acid, citric acid, and tartaric acid; and the precursor is sodium chlorite. The mass ratio of the precursor, gelling agent, acidifying agent, chelating agent, calcium ions, and agar is (15-25):(1-5):(1-5):(2-5):(0.15-1.3):(1-5); the volume fraction of the lemon oil is 8 parts; the stirring time for mixing gel A and emulsion B with the precursor and stirring until uniform is 60 minutes; the mass fraction of the calcium ions is 0.51 parts; where the volume fraction is in mL, the mass fraction is in g.

2. The preparation process of the chlorine dioxide slow-release agent according to claim 1, characterized in that, After mixing gel A and emulsion B, add the precursor, stir at 35-45°C and then at 0-4°C to ensure uniform mixing.

3. The application of a chlorine dioxide slow-release agent obtained by the preparation process described in claim 1 or 2 in the preservation, disinfection and sterilization of agricultural and sideline products.

4. The application according to claim 3, characterized in that, The specific application is the use of chlorine dioxide to inhibit the growth of brown rot fungi, yeasts, and Escherichia coli.

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