Ozone Treatment Modulates Reactive Oxygen Species Metabolism Regulation and Enhances Storage Quality of Kiwifruit During Cold Storage

Abstract

Fresh fruit are highly perishable commodities, facing significant postharvest losses primarily due to physiological deterioration and microbial spoilage. Conventional preservation methods often face limitations regarding safety, residue, and environmental impact. Because of its rapid decomposition and low-residue-impact characteristics, ozone has proven superior as an efficient and eco-friendly solution for preserving fruit quality after harvest. The maturation and aging processes of kiwifruit are closely linked to the involvement of reactive oxygen species (ROS) metabolism. This study aimed to investigate the effects of intermittent ozone treatment (21.4 mg/m³, applied for 0, 1, 3, or 5 h weekly) on ROS metabolism, the antioxidant defense system, and storage quality of kiwifruit during cold storage (0.0 ± 0.5 °C). The results showed ozone treatment slowed the decline in titratable acid (TA) content and fruit firmness, inhibited increases in total soluble solids (TSSs) and weight loss, and maintained the storage quality. Additionally, ozone treatment enhanced the activities of antioxidant-related enzymes. This includes superoxide dismutase (SOD), catalase (CAT), peroxidase (POD), and ascorbate peroxidase (APX). Furthermore, it delayed the reduction in ascorbate (ASA), glutathione (GSH), total phenolic compounds, and flavonoid content, while also preventing the accumulation of ROS and the rise in malondialdehyde (MDA) levels. In summary, the results indicate that ozone treatment enhances the antioxidant capacity of kiwifruit by increasing the structural integrity of cell membranes, preserving the structural integrity of cell membranes, and effectively maintaining the storage quality of the fruit.

1. Introduction

The kiwifruit is a valuable berry that originates from China. It is packed with vitamins, dietary fiber, minerals, and organic acids. Known for its health benefits, it is celebrated for its distinct taste and nutritional content [1]. Studies have demonstrated that kiwifruit possesses various pharmacological activities, including physiological regulatory functions such as antioxidant, anti-inflammatory, and antihypertensive effects [2]. Nevertheless, kiwifruit exhibits distinct respiratory activity, demonstrating considerable vulnerability to postharvest decay. This susceptibility leads to the deterioration of organoleptic properties, nutrient degradation, and a reduction in postharvest longevity [3]. Several treatments have been applied to various kiwifruit varieties to enhance nutritional quality and extend postharvest longevity, including 1-methylcyclopropene (1-MCP) [4], heat treatment [5] electron beam [6], and ozone [7].

The U.S. FDA has sanctioned ozone for utilization in postharvest treatment of agricultural produce since 2001 [8]. Ozone is environmentally safe as it breaks down into oxygen, minimizing the chances of ozone toxicity [9]. Research has shown the optimal ozone dosage significantly boosts antioxidative activity in postharvest produce and suppress the growth of microbes [10]. The impact of ozone treatment on the freshness preservation effect of fruits and vegetables after harvest is related to the concentration of ozone, treatment time, types of fruits and vegetables, and other aspects [11,12,13]. Ozone treatments are primarily administered in two modalities: (1) gaseous ozone may be introduced either continuously or intermittently into the environment where the fruit is stored, or (2) the fruit may undergo washing or immersion in water infused with ozone [14]. Furthermore, intermittent ozone is more scientifically sound and efficient in the process of food storage than other ozone treatment [15]. Studies have demonstrated that intermittent ozone has an excellent preservation effect on strawberries [16], cantaloupe [17], pears [18], cherries [19], and conventional kiwifruit cultivars [20], etc. Nonetheless, the regulatory effects of ozone treatment on ROS metabolism and quality retention in new cultivars like ‘Zhongmi 2’ remain unknown.

However, ozone decomposes into reactive oxygen species (ROS), like hydrogen peroxide (H₂O₂) and superoxide anions (O²⁻), in aqueous environments. In excess, these ROS can be toxic to plants [21]. Ozone treatment has the potential to induce oxidative stress, characterized by an elevation in ROS, a decrease in intracellular antioxidant levels, a decline in nutritional quality, and a heightened vulnerability to physical harm and bacterial infection [22]. Plants exhibit two fundamental defense mechanisms: enzymatic and non-enzymatic. These systems operate in a synergistic manner to efficiently mitigate excess ROS. The enzymatic ROS-scavenging system includes dismutase (SOD), catalase (CAT), peroxidase (POD) and ascorbate peroxidase (APX). Additionally, non-enzymatic antioxidants, such as ascorbate (ASA) and glutathione (GSH), serve as crucial cellular redox buffers. However, there is a limited number of studies investigating how intermittent ozone treatment modulates ROS metabolism and quality attributes in postharvest kiwifruit.

‘Zhongmi 2’ kiwifruit (Actinidia deliciosa) is a new cultivar developed by the Zhengzhou Fruit Research Institute, Chinese Academy of Agricultural Sciences (CAAS), and has been commercially cultivated in China. Nevertheless, postharvest preservation of ‘Zhongmi 2’ kiwifruit remains unexplored to date. Therefore, this study (1) investigated the effects of ozone treatment on postharvest storage quality of the new cultivar ‘Zhongmi 2’ kiwifruit; (2) revealed the impact of ozone treatment on ROS metabolism in kiwifruit by evaluating both enzymatic and non-enzymatic antioxidant systems related to ROS metabolism. Therefore, this study aims to elucidate the potential of ozone treatment in modulating ROS metabolism and its subsequent effects on storage tolerance of kiwifruit.

2. Materials and Methods

2.1. Fruit Material and Ozone Treatment

The ‘Zhongmi 2’ kiwifruit were harvested from a commercial orchard in Xixia Town, Nanyang City, Henan Province, China, in October 2024. Following harvest, kiwifruits were promptly transported to the laboratory. They were selected by shape, size, and free from mechanical damage (soluble solid content ≥ 6.2%) [23]. Kiwifruit without mechanical damage, uniform in size and maturity, were pre-cooled at (0.0 ± 0.5) °C and used as experimental materials. The kiwifruits were divided into four groups (25 kg each group, approximately 200 kiwifruits) and placed into four sealed boxes equipped with ozone sensors (PR-300YOLED Ozone Sensor, Jinan Keyu Electronic Information Technology Co., Ltd., Jinan, Shandong, China), each with a volume of about 0.4 m³, in a chilly room (Ice temperature experiment pilot library, Daikin Industrial Co., Ltd., Osaka, Japan) maintained at (0.0 ± 0.5) °C. The FL-C08 mobile ozone sterilizer (Feili Electric Technology Co., Ltd., Osaka, Japan) was employed to treat the experimental groups once every seven days at a fumigation mass concentration of 21.4 mg/m³ for durations of 1, 3, and 5 h, while the non-ozone-treated samples served as the control group (CK). The samples were divided into three treatment groups based on ozone exposure duration: T1 (1 h), T2 (3 h), and T3 (5 h). Firmness, weight loss rate, TSS, and TA were measured regularly every 28 days (4 treatment cycles). Each pulp samples were removed from their skins, chopped into smaller pieces, and kept in a cryogenic freezer maintained at −80 °C for future work.

2.2. Determination of Firmness and Rate of Weight Loss

Fruit hardness was assessed using a previously established method with minor modifications [24]. A TA.XT Plus texture analyzer was employed for the measurements (TA.XT Plus, Stable Micro Systems, Godalming, Surrey, UK). Measurements were conducted using a P/2 probe (2 mm diameter) with 1 mm/s penetration velocity to 10 mm depth, with force recorded in newtons (N). The equatorial position of each fruit was selected for measurement.

Record the initial weight of the storage as m₀, weigh the mass at each interval during storage and record it as m₁. Determine weight loss by applying the equation below:

Weight loss rate/% = [(m₀ − m₁)/m₀] × 100%,

(1)

2.3. Determination of TSS and TA Content

Total soluble solids (TSSs), expressed as a percentage (%), were measured from the filtered blend following homogenization with a handheld device that integrates glycolic acid (PALBX/ACID1, ATAGO, Tokyo, Japan) at a temperature of 25 °C. The titratable acid (TA) was tested by diluting the juice 50 times.

2.4. Determination of MDA Content and the Cell Membrane Permeability

The quantification of malondialdehyde (MDA) levels was conducted utilizing TBA chromogenic method as outlined by Liu et al. [25]. The results were reported in mmol•g⁻¹.

The method developed by Chen et al. [26] to determine conductivity. The conductivity (P₀) of the deionized water containing small disks of kiwifruit flesh (1 cm in diameter) was measured after 1 h immersion. Subsequently, the conductivity (P₁) of the extract was measured again after 15 min in a boiling water bath (HWS-24, Yiheng, Shanghai, China). The permeability of the cell membrane was determined using the equation below:

Cell membrane permeability/% = (P₀/P₁) × 100%,

(2)

2.5. Determination of Total Phenolic and Flavonoid Content

The total contents of phenolic and flavonoids were quantified spectrophotometrically employing a method that has been previously validated [27]. A weight of 1 g of fruit pulp was measured and combined with a small quantity of pre-chilled 1% HCl-methanol. The mixture was crushed and blended in an ice bath before being placed into 10 mL graduated test tubes. The extract was then incubated at 4 °C for 20 min, with occasional shaking. Following this, its absorbance was recorded at 280 and 325 nm.

2.6. Determination of O²⁻, H₂O₂, VC, and GSH Content

The O²⁻⋅content was measured using the method outlined by Venkatachalam et al. [28] with some minor adjustments. A sample of tissue weighing 2 g was mixed with 6 mL chilled potassium phosphate buffer (50 mmol/L, pH 7.8) and subsequently placed on ice. The resulting mixture was centrifuged at 5000× g for 15 min at 4 °C. Following centrifugation, 1 mL supernatant was mixed with 0.9 mL potassium phosphate buffer and 0.1 mL of hydroxylamine hydrochloride, and the mixture was incubated for 30 min at 25 °C. After that period, 1 mL of this solution was combined with 1 mL 3-aminobenzenesulphonic acid and 1 mL 1-naphthylamine, and the mixture was permitted to incubate for 20 min at 25 °C. The absorbance was measured at 530 nm using a spectrophotometer (Evolution 201, Thermo Scientific, Waltham, MA, USA). Additionally, sample tissue (1 g) was prepared to measure H₂O₂ content using the method outlined by Prochazkova et al. [29]. All results were reported in mmol•g⁻¹.

One gram of kiwifruit pulp was added to five mL 50 g/L TCA solution. The sample was ground while submerged in an ice bath and then filtered. Grind and filter after calibration. Add 1 mL extract to a test tube, then incorporate 1 mL TCA, and measure absorbance at 532 nm. The vitamin C (VC) content was showed as mg•100 g⁻¹.

The technique outlined by Su, Z.H et al. [30] was employed for measuring GSH. A 1.0 g portion of fruit was measured, and then 1.0 mL of a trichloroacetic acid solution at a concentration of 50 g/L (which includes 5 mmol/L EDTA-Na₂) that had been pre-chilled to 4 °C was added. After grinding in an ice bath, centrifuge at 8000 rpm for 20 min to obtain the supernatant. The absorbance was quantified spectrophotometrically at 412 nm. The decrease in GSH levels per gram of fruit was assessed by examining the absorbance readings against a standard curve. GSH content was showed as mmol•g⁻¹.

2.7. Determination of Enzyme Activities Related to ROS Metabolism

SOD activity was measured using the nitroblue tetrazolium method [31]. One gram pulp was added to five mL of 50-mmol phosphate-buffer solution and homogenized in an ice bath. The blended mixture was placed into a centrifuge tube and spun for 20 min at 4 °C and 8000 rpm. The top layer of the solution obtained served as the enzyme extract. The reaction mixture was made up of 54.0 mL 14.5 mmol/L methionine, along with 2.0 mL each of three different solutions of ethylenediaminetetraacetic acid, one at 3 µmol/L and two at 2.0 µmol/L. Additionally, it included 2 mL of 2.25 mmol/L azurotetrazole and 2 mL of 75 µmol/L riboflavin. An amount of 3.0 mL reaction solution was combined with 50 µL enzyme extract and illuminated under a fluorescent lamp at approximately 25 °C and 11 W for 10 min. The absorbance was recorded at 560 nm. One unit of SOD activity (U) is defined as the amount required to achieve a 50% reduction in the photoreduction of nitroblue tetrazolium. All results were quantified and reported in U•g⁻¹.

POD activity was conducted utilizing the specified methodology [32]. One gram pulp was mixed with 5 mL 50 mmol/L phosphate buffer and blended in an ice bath. The blended mixture was then centrifuged for 20 min at 4 °C and 8000 rpm. The upper layer of the solution was gathered as the enzyme extract. In the test tubes, 2 mL 0.1 mol/L acetate buffer, 1 mL 0.25% guaiacol solution, 0.05 mL the enzyme extract, and 0.1 mL 0.75% H₂O₂ were added in a specific sequence. Samples were thoroughly mixed at 25 °C for 5 min. The absorbance values were taken at 470 nm. A single unit of POD activity (U) is defined as the quantity that produces a change in absorbance of 0.01 per minute at a wavelength of 470 nm. All results were quantified and reported in U•g⁻¹.

CAT activity was conducted using the approach described by Yang, R et al. [33]. 1 g pulp was mixed with 5 mL 50 mmol/L phosphate buffer and blended in an ice bath. The resulting homogenized solution was placed in a centrifuge tube and spun for 20 min at 4 °C and 8000 rpm. The upper layer of the solution obtained served as the enzyme extract. Subsequently, 0.1 mL enzyme extract was mixed with 2.9 mL 20 mmol/L H₂O₂, while distilled water was used as reference. Absorbance readings at 240 nm were taken every 15 s, yielding six data points, with each measurement conducted three times. The unit of CAT activity (U) was established as a reduction in absorbance of 0.01 per minute. All results were quantified and reported in U•g⁻¹.

APX activity was conducted using UV spectrophotometry (Evolution 201, Thermo Scientific, Waltham, MA, USA). Frozen kiwifruit samples (5 g) were combined with 5 mL of a 0.1 mol/L potassium phosphate (pH7.5), which contained 0.1 mmol/L ethylenediaminetetraacetic acid (EDTA), 1 mmol/L AAA, and 2% crosslinked polyvinylpyrrolidone. The mixture was crushed while in an ice bath and subsequently moved to a centrifuge tube. The reaction system comprised 2.6 mL of reaction buffer, 0.3 mL 2 mmol/L H₂O₂, and 0.1 mL enzyme extract. The amount of enzyme required to achieve a 0.01 change in absorbance at 290 nm per minute per gram pulp was characterized as one unit of APX activity. All results were quantified and reported in U•g⁻¹.

The activity of lipoxygenase (LOX) was assessed utilizing the methodology outlined by Fernández et al. [34] with slight modifications. Frozen kiwifruit samples weighing 3 g were combined with 3 mL 0.1 mol/L phosphate buffer while maintained on ice. The sample was subjected to centrifugation at 12,000× g for a duration of 10 min at 4 °C. The liquid obtained after centrifugation was called enzyme extract. The extract exhibits a change of 1 ΔOD234 nm/min per gram, defining one LOX activity unit (U•g⁻¹).

2.8. Statistical Analysis

All data are expressed as the mean ± standard deviation (SD) from three independent biological replicates. The analysis was conducted using one-way ANOVA with IBM SPSS Statistics software (version 19.0). Significance was assessed at the 5% level (p < 0.05) through Duncan’s multiple range test at each time point, with the 1% level (p < 0.01) used to highlight particularly robust findings. Diagrams were generated using Origin 2021 (Origin Lab Corporation, Northampton, MA, USA).

3. Results and Discussion

3.1. Effect of Ozone Treatment on Firmness, Weight Loss, TSS, and TA Content

Figure 1 depicts the variations in firmness, TSS, TA, and weight loss among the treatment groups (T1, T2, T3) in comparison to the CK group. Fruit flesh firmness is a crucial measure for evaluating the storage quality, and slowing down the softening process is a useful strategy for prolonging their shelf life [35]. Figure 1A illustrates that the firmness of kiwifruit was 2.35 N at the beginning, and it demonstrated a declining trend as the length of storage was prolonged. After 56 days of storage, T1–T3 groups collectively demonstrated a significant inhibition of kiwifruit firmness compared to CK (p < 0.05). Among these, T2 group showed the most pronounced effect, with a firmness of 1.12 N in the end, which was 65.44% higher than CK. This improvement can be attributed to ozone’s capacity to hinder the function of enzymes like polygalacturonase and pectin methylesterase that degrade cell walls, thereby delaying the softening of pulp [36].

Post-harvest weight loss in kiwifruit is primarily attributed to water transpiration and nutrient depletion. Significant weight loss results in the wrinkling of the fruit’s outer skin, adversely affecting its appearance and quality [37]. As illustrated in Figure 1B, weight loss rate in kiwifruit progressively increased over time during the storage period, with T1–T3 groups exhibiting a smaller rate of increase compared to CK. During the storage period, CK experienced a markedly higher weight loss compared to the three groups that received ozone treatment (p < 0.05), with the T2 group demonstrating the most effective results, as its mass loss rate was much less than CK (p < 0.01). This occurrence could be linked to the generation of ROS as a result of ozone breakdown, which directly impacts the stomatal function of the pericarp. ROS leads to an increase in Ca²⁺ concentration in the cytoplasm, causing anions and K⁺ to flow out and resulting in a decrease in the expansion pressure of defense cells. Consequently, this results in the closing of the epidermal stomata in fruits and vegetables, which restricts transpiration and decreases water loss. These findings align with Vlassi et al. [38], which demonstrated that 0.3 ppm ozone reduced weight loss in table grapes.

The TSS content serves as a direct indicator of the ripeness and quality of fruits, and it is a key factor in assessing storage durability and quality of kiwifruit. The TA content of kiwifruit, which consists of quinic acid, malic acid, and citric acid, decreased continuously during storage due to respiration and sugar metabolism processes [39]. The effects of ozone on TSS and TA during storage are illustrated in Figure 1C,D. Kiwifruit exhibited an increase in TSS and a decrease in TA as storage period lengthened. The differences in TSS content were significant at 56 days. The TSS levels in CK were notably greater than treated groups (p < 0.01), suggesting that ozone slowed the process of starch degradation to TSS, thereby inhibiting an increase in TSS content. The TA content differed significantly at 56 days. The CK group showed a notably reduced TA content in comparison to treated groups (p < 0.01). The results show that ozone notably postponed the rise in TSS and the decline in TA in kiwifruit. Luo’s research indicated that kiwifruit treated with 79.44 parts per million of ozone gas for one hour over 7 days maintained high levels of TA content [40].

3.2. Effect of Ozone Treatment on MDA Content, Cell Membrane Permeability and LOX Activity

MDA content is a key marker for fruit ripening and aging, with elevated levels indicating greater lipid peroxidation in cell membranes [41]. Figure 2A demonstrates the impact of ozone on the MDA levels in kiwifruit. Throughout the storage period, MDA levels gradually increased. At the end, the MDA levels in CK were found to be considerably higher in comparison to T2 (p < 0.05). The results show that ozone inhibited the gathering of MDA, thereby delaying fruit senescence. This finding aligns with Yan’s study, which demonstrated that ozone treatment at 45 mg/m³ significantly reduced MDA content in kiwifruit [42].

Cell membrane permeability can indicate the level of harm to cell membrane. An increase in cell membrane permeability signifies a greater degree of damage and a decline in the functionality of the cytoplasmic membrane [39]. Figure 2B demonstrates the impact of ozone on cell membrane permeability in kiwifruit. During the storage, cell membrane permeability gradually increased. At the end, cell membrane permeability in CK was markedly greater than observed in the T1–T3 groups (p < 0.05). These observations are similar to Wang et al. [7], who discovered that ozone treatment (1 mg/L, 10 min) significantly reduced cell membrane permeability in kiwifruit.

LOX is an essential enzyme that facilitates the peroxidation of membrane lipids and the production of ROS during the degradation process. An elevation in LOX activity is associated with the production of MDA and the hastening of fruit senescence. LOX can oxidize unsaturated fatty acids, compromising the integrity of fruit cell membranes and hastening senescence [43]. Figure 2C demonstrates that LOX activity in kiwifruit increased during storage, with CK consistently exhibiting much greater LOX activity than T1–T3 groups (p < 0.01). This states that ozone treatment successfully inhibits the rise in LOX activity and helps preserve the integrity of cell membrane. These findings are comparable to Fernández et al. [44], who stated methyl jasmonate and ozone reduced LOX enzyme activity in wine grapes.

3.3. Effect of Ozone Treatment on Total Phenol and Flavonoid Content

Total phenols and flavonoids, which are secondary metabolites, hold considerable importance in preserving quality, taste, and color of plants, while also boosting the antioxidant properties during storage after harvest [45]. Figure 3 demonstrates the variation in total phenolic flavonoid levels in kiwifruit during storage. The content of total phenolics and flavonoids for all groups showed an initial rise, which was later succeeded by a decline. This noticeable trend can be accounted for by the gradual buildup of phenolic compounds through the phenylpropanoid pathway as the fruits develop. This upward trend was maintained at the start of storage period, resulting in higher total phenol and flavonoid content. However, as storage time increased, polyphenol oxidase catalyzed the oxidation of polyphenols to quinones, while phenolics acted as low-molecular-weight antioxidants to scavenge free radicals, resulting in their gradual depletion [46].

As illustrated in Figure 3A, a higher mass concentration of ozone treatment delayed the peak emergence of total phenol content. In all groups, the peak occurred at 28 days for T1, while for T2 and T3 groups, it peaked at 56 days. This delay may be attributed to ozone’s role in promoting the metabolism of phenylpropanoids, boosting the function of enzymatic antioxidant system, and reducing the levels of ROS that require scavenging. Consequently, total phenols, which act as non-enzymatic antioxidants, have more time to accumulate, resulting in a postponed peak of total phenol content. Thus, the peak appearance of total phenol content was delayed. Moreover, ozone treatment was found to suppress the function of polyphenol oxidase, which consequently diminished the degradation of total phenolic compounds throughout the prolonged storage duration. By the conclusion of storage, the total phenol content in T2 was 0.22 mg/g, which is 1.53 times greater than CK. The flavonoid content in CK was 0.16 mg/g, while the flavonoid contents in T1 and T2 were 1.23 and 1.38 times than that of CK. The findings suggest ozone treatment can significantly increase the levels of total phenolics and flavonoids. This rise could be linked to the stimulation of mangiferolic acid metabolism and the suppression of enzymes that deplete phenolic compounds due to ozone treatment, which in turn raises the overall levels of phenolics and flavonoids [15].

3.4. Effect of Ozone Treatment on VC and GSH Content

VC, or ASA, is a vital nutrient present in kiwifruit and reducing agent in various metabolic pathways, while also exhibiting antioxidant properties [47]. ASA and GSH are essential in regulating the redox balance within fruit cells. The ASA-GSH cycle is a vital way in plant antioxidant metabolism [48]. Changes in VC and GSH content are shown in Figure 4. The VC content steadily diminished over the course of storage. The significant decline in VC content during initial storage phase could be due to the activation of antioxidant system by ozone, which enhanced the activity of APX, leading to an increase in VC decomposition. In contrast, the relatively minor changes observed in the later storage period could be due to ozone treatment, which elevated the activities of glutathione reductase and succinate dehydrogenase, as well as the level of adenosine triphosphate (ATP). This increase may have promoted the synthesis of VC and GSH. Consequently, the VC content was higher in T1–T3 groups compared to CK [49]. The GSH content in T1–T3 were generally higher than the CK group. Specifically, the GSH content of the T2 group was 1.44 times and 1.33 times higher than CK on the 84th and 112th days of storage, respectively. Overall, ozone-treated kiwifruit exhibited higher levels of VC and GSH than CK. The result indicate that ozone can keep kiwifruit in a more favorable redox state during storage. This finding aligns with Wang, Y.J et al. [50] and Li, C et al. [48], who discovered that ozone inhibited the reduction in VC content both in kiwifruit and red pitaya.

3.5. Effect of Ozone Treatment on O²⁻ and H₂O₂ Content

One of the reasons for the shortened storage period of kiwifruit is oxidative stress following harvest. ROS, which include H₂O₂ and O²⁻, can lead to the peroxidation of unsaturated fatty acids in lipids, resulting in cell toxicity and harm [51]. Figure 5 demonstrates the changes in O²⁻⋅and H₂O₂ content of kiwifruit during storage. It shows that the trends in O²⁻⋅and H₂O₂ content across all groups were relatively consistent, with significant increases noted between days 0 and 84. The levels of O²⁻⋅and H₂O₂ in T1–T3 groups were lower than CK, indicating that ozone delayed the peaks of O²⁻⋅and H₂O₂ accumulation. Additionally, ozone successfully decreased the magnitude of these peaks. At the end, the H₂O₂ levels in T1–T3 groups were 2.18, 1.84, and 2.13 mmol/g, respectively, which were lower than the CK group by 8.02%, 22.36%, and 10.13%, respectively.

3.6. Effect of Ozone Treatment on Enzyme Activities Related to ROS Metabolism

When fruit is exposed to aging or adverse conditions, the dynamic balance of ROS is disrupted. An overabundance of ROS can result in lipid peroxidation, damage to proteins, and degradation of nucleotides, severely impacting the fruit’s normal physiological and biochemical functions. Enzymatic defense systems, such as SOD, POD, CAT, and APX, help plants eliminate excess ROS and protect their cells from oxidative stress [52]. SOD is an enzyme that facilitates the conversion of O₂ to H₂O₂. It acts as the primary defense mechanism for eliminating ROS from plant cells and regulating ROS levels, thereby protecting cell membrane structures. CAT is a crucial enzyme that helps eliminate reactive oxygen species in plants. It can also work in conjunction with POD and APX to facilitate the breakdown of H₂O₂ into H₂O and O₂ [53].

As shown in Figure 6A, SOD activity increased initially, followed by a decreasing trend. During the initial 28 days, SOD activities of all kiwifruit samples increased rapidly, with the ozone-treated groups exhibiting significantly higher SOD activities compared to CK. Furthermore, the SOD activities of all groups continued to rise from days 28 to 56. SOD activity peaked on the 56th day, with T1, T2, and T3 groups showing SOD activities that were 1.28, 1.37, and 1.30 times higher than CK. This study found that ozone treatment effectively enhanced SOD activities, consistent with the findings of Piechowiak et al. [54], which reported a remarkable increase of 313% in SOD activity in kiwifruit exposed to 10 ppm ozone for 15 min.

As shown in Figure 6C, CAT activity demonstrated an initial rise, which was subsequently followed by a decrease in early stages of storage, a trend that paralleled of SOD. At 56 days of storage, the CAT activities in four groups reached peak levels, while SOD activities in T1–T3 groups were 1.24, 1.64, and 1.42 times higher than the CK group. Additionally, the differences in CAT activities between the three ozone-groups and CK were significant (p < 0.01). This finding indicates that ozone treatment during storage enhances kiwifruit CAT enzyme activity, with varying effects depending on the duration of ozone exposure. Comparable findings were noted by Liu et al. [55], who discovered shiitake mushrooms treated with 3.21 mg/m³ of intermittent ozone exhibited higher CAT activity than the CK group.

As illustrated in Figure 6B, POD activity first rose and then fell. The POD activity in ozone-groups were notably greater than that CK for the entire duration (p < 0.05). The peak values of POD activity in T1–T3 groups were 1.18, 1.26, and 1.11 times higher than CK. At 112 days, POD activity in ozone-groups were considerably greater than CK (p < 0.01). These results suggest that ozone during storage maintained higher POD enzyme activity in kiwifruit. Similar findings were reported by Liang et al. [56], who discovered that tomatoes exposed to 17.14 mg/m³ of ozone exhibited higher POD activities compared to the CK group.

APX facilitates the transformation of ASA into monodehydroascorbate, in addition to promoting the oxidation of reduced glutathione to its oxidized form. This dual function serves to mitigate the production of excessive H₂O₂. As illustrated in Figure 6D, the peak activity of APX coincided with that of POD, both reaching their maximum at 56 days. APX activity was consistently markedly higher in T2 compared to CK (p < 0.01). At 112 days, APX activity was 14.90%, 25.62%, and 8.20% higher in T1–T3 groups than in CK group, respectively. These results indicate that ozone treatment during storage effectively maintained APX enzyme activity in kiwifruit. Similar findings were reported by Piechowiak et al. [57], who investigated the effect of 15 ppm ozone for 3 min every 12 h on APX activity in blueberries.

4. Conclusions

Ozone treatment effectively delayed increases in TSS and decreases in TA. It also slowed weight loss and the reduction in fruit hardness. Furthermore, ozone treatment enhanced the activities of SOD, POD, CAT and APX. It preserved levels of VC, GSH, total phenolic content and flavonoid content. Additionally, ozone reduced the accumulation of O²⁻, H₂O₂, and MDA. It decreased LOX activity and postponed the rise in the permeability of the cellular membrane during storage. Exposure to a mass concentration of 21.4 mg/m³ of ozone for three hours every seven days enhanced the activity of reactive oxygen scavenging enzymes, maintained normal metabolic processes in kiwifruit, and minimized fruit weight loss.