Effects of Ozone Treatment on Reactive Oxygen Species Metabolism and Storage Quality of Flat Jujubes (Ziziphus jujuba Mill. cv. Panzao)

Abstract

Moderate ozone exposure has emerged as a sustainable strategy to enhance postharvest quality in perishable fruits. This study investigated the effects of ozone treatment (2.14–19.27 mg/m³) on flat jujube during 70-day cold storage (0 °C). Results demonstrated that following 70 days of storage, the ideal ozone concentration (10.72 mg/m³, T2) led to a decrease in weight loss of 44.8% and preserved 66.7% firmness when compared to the control check (CK) group. The T2 group suppressed the respiration rate and delayed declines in total soluble solids (TSSs) and titratable acid (TA). Mechanistically, ozone enhances enzymatic activity, with T2 elevating superoxide dismutase (SOD), catalase (CAT), and peroxiredoxin (POD) activities while reducing the accumulation of reactive oxygen species (ROS) and lipid peroxidation. Total phenolics and flavonoids in T2 remained 42% and 52% higher than CK at 56 days, correlating with elevated 2,2-Diphenyl-1-picrylhydrazyl (DPPH) and 2,2′-Azinobis-(3-ethylbenzthiazoline-6-sulphonate) (ABTS) scavenging activities. Browning inhibition (25% lower than the CK group) is linked to suppressed polyphenol oxidase (PPO) activity and phenolic oxidation. Principal component analysis (PCA) confirmed ozone’s efficacy in delaying senescence via ROS homeostasis and antioxidant synergy. These findings establish moderate ozone as a novel, eco-friendly intervention to extend jujubes’ shelf life, emphasizing its dual role in quality preservation and oxidative stress regulation.

1. Introduction

Flat jujube (Ziziphus jujuba Mill. cv. Panzao), named for its resemblance to a flat peach, is characterized by its thin skin, large size, and sweet and sour taste [1]. Jujube fruit is not only a delicious fruit but also a nutritious and healthy food, favored by Chinese consumers for its unique flavor and high content of vitamins, minerals, and other healthy ingredients [2]. Fresh jujubes usually ripen during the summer months and are prone to reddening of the skin, senescence, softening of the fruit, and promotion of internal tissue decay [3]. These changes greatly reduce the visual appeal and nutritional value of the fruit, ultimately leading to a significant decline in market sales and consumer acceptance.

Over the years, a vast amount of research has been implemented to investigate the effects of physical techniques, emerging chemical treatments, and biological control on maintaining the quality and minimizing the postharvest losses of jujube fruit [4]. Chemical preservation technology can effectively extend the shelf life of fruits and vegetables by adding “specific chemical substances” to them to maintain their freshness and nutritional value. For instance, Liu et al. [5] demonstrated that chitosan and potassium sorbate treatments reduced the decay rate of red jujubes by inhibiting the relative abundance of phytopathogenic fungi. Zhang et al. [6] found that 1-methylcyclopropene (1-MCP) treatment was beneficial in limiting the development of the mold diameter in jujubes. Although chemical preservation was effective, its use was restricted because of potential health and environmental risks. The field of fruit storage has paid increasing attention to physical and biological preservation techniques that provide a high degree of safety and security. Reported physical preservation methods include elevated O₂ [7], controlled atmosphere [8], cold plasma [9], and heat shock [10]. Biological approaches include salicylic acid [11], exogenous brassinolides [12], and others. Although the above methods are safer and less harmful than chemical methods, they cannot be used on a large scale due to their limited scope of application, high cost, and complex operation. Therefore, the development of a simple and environmentally friendly preservation method with a low cost and high efficiency has become an urgent need to improve the storage quality of jujubes and extend their freshness period.

Ozone, a naturally occurring gaseous molecule characterized by its distinct fishy odor, has been recognized as a potent oxidant in food preservation [13] since its approval by the U.S. Food and Drug Administration (FDA) in 2001 for food applications [14]. The mechanism underlying ozone’s effects is closely linked to reactive oxygen species (ROS), which include radical molecules like superoxide anions (O₂⁻) and hydroxyl radicals (OH), as well as non-radical species such as hydrogen peroxide (H₂O₂) and hydroxide ions (OH⁻) [15]. These signaling molecules are capable of not only regulating the organs and tissues of plants but also stimulating and influencing their metabolic processes [16,17]. However, the generation and removal of plant ROS should be controlled, because the presence of unfavorable environmental conditions can trigger plant cells to produce large amounts of ROS, and the high concentration of ROS will exacerbate the peroxidation of cell membrane lipids and cause massive cell death [18]. It has been shown that ozone treatment not only increases the content of vitamin C and phenols in fruits and vegetables but also activates the antioxidant enzyme system to improve their antioxidant capacity to cope with oxidative stress [19]. These effects are strongly dependent on treatment parameters (e.g., ozone concentration, duration, and fruit type).

Given the advantages of ozone treatment in improvements in quality, ozone is expected to be a preservation method for flat jujube. However, the specific effects of ozone treatment on the nutritional quality and physiological characteristics of flat jujube, as well as its regulatory mechanisms in ROS metabolism, remain insufficiently understood. This study aims to address these gaps in knowledge by (1) evaluating the impact of ozone treatment at various concentrations on the postharvest physiological properties and nutritional quality of flat jujube and (2) elucidating the mechanisms by which ozone modulates ROS metabolism during storage. This research seeks to strengthen the scientific basis for optimizing ozone-based preservation techniques, ultimately offering practical strategies to extend the shelf life and enhance the commercial value of flat jujube.

2. Materials and Methods

2.1. Plant Sample

The flat jujubes were selected at the white ripening stage and transported by air from Kashgar, Xinjiang, to the Tianjin Academy of Agricultural Sciences. Uniformly sized, undamaged, and fresh fruits were selected for the experiment, which were divided equally into four groups of 20 kg each and stored in cold storage (0 ± 0.5 °C; RH 90 ± 5%).

2.2. Ozone Treatment

The ozone concentration was selected based on previous studies [20]. The experimental design comprised four groups: the control check (CK) group, which involved jujubes stored in cold storage without ozone treatment; the T1 group, 2.14 mg/m³ ozone; the T2 group, 10.72 mg/m³ ozone; and the T3 group, 19.27 mg/m³ ozone. Ozone treatments were administered every two weeks (for one hour each session) over 70 days using a fumigation system designed according to Chen et al. [21], which was operated in a 2 m × 1.5 m × 0.8 m chamber (1200 L capacity) equipped with an ozone generator and two MIC-03 ozone sensors (self-made by the National Agricultural Products Preservation Center) for monitoring of accuracy and installed within the cold storage facility. The duration and frequency of ozone treatments were determined by pre-testing. Ozone treatment was first applied to the designated groups on day 0 immediately after sample grouping. Treated and control fruits were then analyzed at days 0 (immediately post-treatment), 14, 28, 42, 56, and 70 of storage. At each point, samples were collected, pulverized with liquid nitrogen, and stored at −80 °C for subsequent analysis. Each experiment was performed in three replicates.

2.3. Quality Measurements

2.3.1. Color Change and Degree of Browning

Color changes in jujubes were measured using a colorimeter (WR-10, Shanghai BG Instruments Co., Ltd., Shanghai, China), with three randomly selected fruits measured for their L* (Luminosity), a* (Red/Green), and b* (Yellow/Blue) values.

The method of Sun et al. [22] was followed with modifications. Briefly, 1 g of the sample was homogenized in 10 mL of saline at 4 °C. The homogenate was then centrifuged at 4 °C and 10,000× g for 15 min, and the supernatant was collected. Following incubation at 25 °C for 10 min, the absorbance at 410 nm (OD₄₁₀) was measured, and browning was calculated as

$$\mathrm{B}\mathrm{r}\mathrm{o}\mathrm{w}\mathrm{n}\mathrm{i}\mathrm{n}\mathrm{g}={\mathrm{O}\mathrm{D}}_{410}\times 10$$

(1)

2.3.2. Weight Loss, Respiratory Rate, Firmness, Total Soluble Solid (TSS), Titratable Acid (TA), and Ascorbic Acid (ASA) Content

Fruit weight was determined on days 0, 14, 28, 42, 56, and 70.

$$\mathrm{W}\mathrm{e}\mathrm{i}\mathrm{g}\mathrm{h}\mathrm{t} \mathrm{l}\mathrm{o}\mathrm{s}\mathrm{s}(\%)={\displaystyle \frac{A_1-A_2}{A_1}}\times 100$$

(2)

A₁ = Initial storage weight, g; A₂ = post-storage final weight, g.

The respiratory rate of a specified weight of fruit, sealed in a 700 mL respiratory canister for a duration of 2 h, was measured three times for each treatment using a Checkpoint O₂/CO₂ oximeter (0902-0002, Systech Illinois, Thame, UK). Respiratory rate was recorded in mg·kg⁻¹·h⁻¹ at regular intervals.

The equatorial part of the jujube fruit was selected and measured in a texture analyzer (TA.XT. Plus, Stable Micro Systems, Godalming, UK). The maximum compression force at each peak was analyzed, and results were expressed in g. Puncture tests were conducted using a 2 mm diameter (P/2) probe with a pre-compression speed of 5 mm/s, a compression speed of 5 mm/s, a post-compression rebound speed of 5 mm/s, and a puncture distance of 1 cm. Three jujube fruits were selected for every group, and the average values were calculated.

Following the method outlined by Suo et al. [23], with modifications, the juice was extracted from chopped jujube fruits to assess the TSS and TA content using a handheld digital refractometer (PAL-BX/RI, ATAGO, Tokyo, Japan). The results were averaged from three parallel measurements and expressed as %.

The method outlined by Zhang et al. [24] was utilized to determine the ASA content. To identify the ASA concentration, the change in absorbance was recorded at 525 nm, and the results are expressed in mg/100 g FW.

2.3.3. Total Flavonoid and Total Phenolic Content

Total flavonoid and total phenolic content were determined following the method of Wei et al. [25]. A total of 5 mL of 80% ethanol was added to 1 g of jujube fruit to extract the total phenols and flavonoids. The concentration of total phenols was assessed by measuring the absorbance at a wavelength of 765 nm, and the results were reported as g of gallic acid equivalent per kg (g GAE/kg FW). Similarly, the concentration of total flavonoids was evaluated by measuring the absorbance of the solution at 510 nm, with results expressed as mg of rutin equivalent per g (mg RE/g FW).

2.3.4. 2,2 Diphenyl-1-picrylhydrazyl (DPPH) and 2,2′-Azinobis-(3-ethylbenzthiazoline-6-sulphonate) (ABTS) Scavenging Activities

The methods of Lin et al. [26] were followed with modifications. Briefly, 0.1 g of tissue was homogenized in 20 mL of 80% methanol (v/v) using an ice bath. The homogenate was centrifuged at 4 °C, 10,000× g for 20 min. Subsequently, the supernatant was mixed with the working solution, and the absorbance at 517 nm was measured to calculate radical scavenging activity (expressed as %)

2.3.5. H₂O₂ Content, Superoxide Anions (O₂⁻) Generation Rate, and Malondialdehyde (MDA) Content

Determining the H₂O₂ concentration involved weighing 1 g of sample and extracting H₂O₂ with ice-cold acetone at 4 °C. Following grinding and centrifugation at 3000× g for 30 min at 4 °C, 1 mL of the obtained supernatant was used to measure the absorbance at 412 nm. H₂O₂ content is expressed in μmol/g.

The O₂⁻ generation rate was determined according to the method of Wang et al. [27] with modifications and expressed as μmol/min·g.

MDA content was measured according to the method of Yu et al. [28] with modifications and expressed as μmol/g.

2.3.6. Enzyme Activity Assay

Ascorbate peroxidase (APX), catalase (CAT), superoxide dismutase (SOD), peroxiredoxin (POD), phenylalanine deaminase (PAL), and polyphenol oxidase (PPO) activities were tested according to the requirements of the enzyme activity kit (Nanjing Jiancheng Institute of Bioengineering, Nanjing, China). Kit names and catalog numbers are listed below: APX(A123-1-1), CAT(A007-1-1), SOD(A001-3), POD(A084-3-1), PAL(A137-1-1), PPO(A136-1-1). APX, CAT, SOD, POD, PAL, and PPO activities units were expressed in U/g.

2.4. Statistical Analyses

Figures were plotted using Origin 2024 (OriginLab Corporation, Northampton, MA, USA). Data were analyzed using IBM SPSS Statistics 22 software (IBM Corp., Armonk, NY, USA) through one-way analysis of variance (ANOVA) and multi-way ANOVA, followed by Duncan’s multiple range test. All experiments were conducted in triplicate, and results are expressed as mean ± standard error (SE). Statistical significance was defined as α = 0.05; if p < α = 0.05, then it represents a significant difference. PCA was performed in Origin 2024 to compare fruit quality parameters across treatment groups. Pairwise correlations between indicators were evaluated using Pearson’s correlation coefficient, with significance thresholds set at α = 0.05 (*), α = 0.01 (**), and α = 0.001 (***). Statistical methods for each analysis are specified in the corresponding figure or table captions within the Section 3.

3. Results and Discussion

3.1. Color Change and Degree of Browning

During the storage of fresh jujubes, the surface of the fruits should maintain a naturally bright yellow color (high L* and b* values), and there should be no obvious browning (lower a* value). The color change in jujube peel during storage is shown in Figure 1A–E.

In the present study, the L* values of the ozone-treated groups were significantly (p < 0.05) higher than that of the CK group during 28–70 days of storage, At 70 days of storage, the L* value of the CK group was 39.59, while the L* values of the T1, T2, and T3 groups were 7.77%, 11.71%, and 8.94% higher than that of CK group, respectively (Figure 1A). Liu et al. [29] demonstrated that the application of intermittent ozone treatment effectively reduced browning on mushroom surfaces, leading to noticeably elevated L* values in the group treated with intermittent ozone compared to the untreated mushrooms. It can be seen from Figure 1B that the a* value of jujubes continues to increase during storage. During the ripening process of jujubes, chlorophyll is gradually decomposed, and the color of the fruit changes from green to yellow and then to red [30]. During storage, the ozone-treated group significantly (p < 0.05) suppressed the increase in a, and at 56 days, a* in the T2 group was only 65.18% of that in the CK group. The T1 group significantly delayed the decrease in b* value during the early storage period. Specifically, on day 28, the b* value of the T1 group was 107.23% that of the CK group (Figure 1C). After the storage period, the browning indices for the T1, T2, and T3 groups were 0.28, 0.27, and 0.30, representing a reduction of 22.25%, 25.12%, and 16.60% compared to the CK group. Notably, the CK group exhibited a 1.4–fold increase relative to the T2 group (Figure 1D). This phenomenon could potentially be attributed to the suppression of PPO activity. As is evident from the phenotypic illustration presented in Figure 1E, during the storage period, the color of the jujubes gradually shifted towards a reddish-brown hue. By 70 days of storage, nearly all of the jujubes had fully transformed into a reddish-brown color. Notably, only the samples under the T2 and T3 treatments still exhibited a minor portion of green coloration.

3.2. Weight Loss, TA, TSS Content, Respiration Rate, Firmness, ASA Content

In this study, we evaluated weight loss, TSS content, TA content, respiration rate, firmness, and ASA content, which are important storage quality parameters of harvested fruits and vegetables. The rate of weight loss increases with the duration of storage (Figure 2A), but the weight loss rate of the ozone-treated group was significantly lower than that of the CK group at 56–70 days of storage (p < 0.05). At 70 days of storage, weight loss was as high as 3.68% in the CK group compared to 2.75%, 2.03%, and 2.40% in the T1, T2, and T3 groups, respectively, with statistically significant differences (p < 0.05). The TA content of jujubes decreased (Figure 2B), and the ozone-treated group effectively delayed the decline in TA content and maintained a high TA content, peaking at 0.38% at 28 days in the T2 group, which was significantly higher than the other groups (p < 0.05). During the ripening process of fruits, sugars and other soluble substances gradually accumulate, leading to an increase in soluble solids content [31]. However, during storage, respiration can consume some of these sugars, resulting in an initial increase in TSSs followed by a decline [32]. Overall, the changes in the TSS content of jujubes during storage conformed to this pattern (Figure 2C). The TSS content of jujube fruits peaked at 42 days and then declined, with 28.20% in the T2 group, which was significantly higher (p < 0.05) than the 25.90% in the CK group.

The respiratory rate decreased sharply during the first 14 days of low-temperature storage, because it was inhibited by the low temperature. During 28–70 days of storage, the respiration rate of the T2 group decreased faster than that of the other groups. By day 70, the respiration rate of the T2 group had decreased to 56% of the value of the CK group. Our findings indicate that treatment with ozone diminished the respiratory rate of the fruit (Figure 2D). This result is consistent with the research conducted by Liang et al. [33]. Fruits with greater firmness are more suitable for long-term storage and transportation, thereby enhancing their commercial value [34]. The firmness of flat jujube showed a decreasing trend during storage, which may be due to the prolongation of storage time, the dissipation of water in the fruit, and the degradation of cell walls [14]. As can be seen from Figure 2E, by the end of storage, the T2 and T3 groups maintained significantly higher firmness levels, with 67.65% and 65.28% of their initial values, respectively, compared to 58.17% for the CK group (p < 0.05). Ozone treatment was effective in delaying the decrease in firmness of jujubes, which is like the results of Liu et al. [35] for ozonated water treatment of freshly cut apples. Minas et al. [36] found that ozone-treated fruits’ softening and cell wall swelling were significantly reduced, and pectin polysaccharide dissolution was delayed, which may be one of the reasons why the fruit maintains better firmness. Figure 2F illustrates that the ASA content exhibited a pattern of initially rising, followed by a decline. During 42 days of storage, the T2 group recorded a content of 470.61 mg/100 g FW, which was greater than the levels found in the other groups (p < 0.05).

3.3. Total Flavonoid Content, Total Phenolic Content, DPPH, and ABTS Scavenging Activities

The changes in total flavonoid and total phenolic content of jujubes during storage are illustrated in Figure 3A,B. The total flavonoid content of jujube fruit decreased during 0–14 days of storage, and at 14 days, the T2 group had the highest content of 0.55 mg RE/g FW compared to the other groups, which was 1.53 times higher than that of the CK group (p < 0.05). During ripening of fruits, the total phenolic content of fruits usually shows a decreasing trend due to oxidation, in addition to a gradual decrease in flavonoid content over time due to storage-related isomerization or oxidation [37]. Phenolics, as ubiquitous natural secondary metabolites found in higher plants, play a crucial role in plants’ growth, particularly in response to oxidative stress and pathogen attacks [38]. At 28 days of storage, the total phenolic content of the T1 and T3 groups was 24.07% and 25.73% higher than that of the CK group, respectively (p < 0.05). At 56 days, the total phenolic content of the T2 group was 61.57 g GAE/kg FW, which was 42% higher than that of the CK group, and the total flavonoid content was 1.51 times higher than that of the CK group at this time (p < 0.05). It was found that lower concentrations of ozone treatment inhibited such oxidative reactions, thus maintaining a high phenolic content, a phenomenon consistent with the findings of Rodoni et al. [39].

Figure 3C shows that the overall DPPH scavenging activity of the fruit increased initially and then decreased during storage. Throughout the storage period, the DPPH scavenging activity in the T2 group was consistently higher than in the CK group (p < 0.05). After 42 days of storage, the DPPH radical scavenging rate was only 67% in the CK group, compared with 78.76%, 81.82%, and 75.82% in the T1, T2, and T3 groups, respectively (p < 0.05). As depicted in Figure 3D, the ABTS scavenging activity of jujubes decreased during storage, with the T1 and T2 groups maintaining a higher ABTS scavenging activity compared to the CK group. At 70 days of storage, the ABTS scavenging activity of the T1, T2, and T3 groups was 8.93%, 13.79%, and 3.83% higher than that of the CK group, respectively (p < 0.05). In this study, we demonstrated the effectiveness of ozone treatment in preserving the DPPH and ABTS scavenging activities of flat jujube, a treatment that significantly enhanced the overall antioxidant capacity of the fruits. This discovery is consistent with earlier studies suggesting that ozone significantly enhances the antioxidant properties of papaya [40].

3.4. ROS Metabolism and Defense Enzyme Activity Changes

O₂⁻ and H₂O₂, as components of ROS, play dual roles in plant life activities. They serve as key signaling molecules that are intricately involved in regulating plants’ responses to environmental stresses, encompassing both biological and non-biological processes [41]. MDA can reflect the degree of lipid peroxidation in plant membranes, and changes in its content are closely related to cell membrane damage [42]. Based on this, ROS (H₂O₂, O₂⁻) and MDA were identified in this study as the core indicators for assessing the level of oxidative stress in flat jujube during storage.

Ozone treatment showed an inhibitory effect on the regulation of ROS metabolism during the storage of flat jujube. ROS and MDA levels gradually increased with the extension of storage time (Figure 4A–C). Specifically, the H₂O₂ content (Figure 4A) of CK reached 667.95 μmol/g after 70 days of storage, whereas the H₂O₂ content of the fruits subjected to the T2 group’s regimen was 561.32 μmol/g, which is 15.9% less than that of the CK group, significantly so (p < 0.05). In addition, the rate of O₂⁻ generation in the T2 group was only 1.17 μmol/g in the middle of storage (42 days), which was significantly lower than the rate of 1.59 μmol/g in the CK group (p < 0.05). Interestingly, the levels of rate of O₂⁻ generation in the T3 group were quite similar to that observed in the CK group (Figure 4B), which might be due to the high concentration of ozone-induced oxidative stress response in plant cells [43]. Throughout the duration of storage, the levels of MDA in the group treated with ozone remained steadily lower compared to the CK group (Figure 4C), indicating that ozone might preserve cell membrane integrity by altering the thiol groups of membrane proteins and preventing the chain reaction of lipid peroxidation induced by ROS [44]. For example, at 70 days of storage, MDA accumulation reached 0.42 μmol/g in the CK group, whereas it was only 0.22 μmol/g in the T2 group, which was 48% of that in the CK group (p < 0.05).

The enzymatic antioxidant system serves as a primary means of controlling the production of ROS, with APX, CAT, POD, and SOD being the essential enzymes that manage the scavenging of ROS [45]. APX activity in jujube fruits initially increased and then decreased during storage (Figure 4D). There was no significant difference (p > 0.05) in APX activity between the groups during the early storage period (0–28 days). APX activity peaked at 42 d of storage, when it was 217.27 U/g in the T2 group, which was 1.21 times higher than that of 179.40 U/g in the CK group. By 70 days, APX activity had decreased in all groups relative to the peak, but the activity in the T2 and T3 groups remained significantly higher than that in the CK group (p < 0.05). Enzymes such as CAT and POD function in concert, and they further decompose into harmless aqueous H₂O and O₂, and this cascade of enzymatic reactions greatly mitigates the possible damage caused by ROS to plant cells and ensures a homeostatic balance of intracellular ROS levels [46]. They differ in that CAT directly decomposes H₂O₂, while POD removes H₂O₂ by catalyzing the oxidation of H₂O₂ to other substrates [47]. The T2 group consistently showed higher levels of CAT activity relative to the CK group throughout the storage period (Figure 4E) (p < 0.05). CAT activity was 17.0% (p < 0.05) higher in the T2 group (23.94 U/g) than in the CK group at 42 days. POD activity initially decreased and then increased, with that in the ozone-treated group being significantly (p < 0.05) higher than in the CK group on day 14 (Figure 4F); after 42 days, T3 peaked at 55.15 U/g and then decreased, whereas the T2 group continued to increase and peaked at 60.12 U/g on day 70, which was 17.72% higher than the CK group (p < 0.05). As shown in Figure 4G, the SOD activity of the CK group decreased continuously during the storage period, but the ozone-treated group showed larger fluctuations, probably due to ozone-induced oxidative stress. After 56 days, SOD activities in the CK, T1, T2, and T3 groups were 193.48, 224.24, 359.96, and 309.46 U/g, respectively, with the T2 and T3 groups significantly higher than the CK and T1 groups (p < 0.05). Previous studies by Modesti et al. (2018) found that wine grapes exhibited high SOD, CAT, and APX activities after continuous ozone treatment for 12 h in the airstream condition [48]. Concerning the enhancing influence of ozone on antioxidant enzyme activities such as SOD, CAT, POD, and APX, the findings from the current research align with earlier studies conducted on pears [49] and strawberries [50].

PAL plays a crucial role in the metabolism of phenylpropanoids and is directly engaged in the synthesis of plant phenols and flavonoids [51]. This research demonstrated that the activity of PAL reached its highest point in the T2 group after 42 days of storage, showing an increase of 18.35% (p < 0.05) when compared to the CK group (Figure 4H). This increase contributed to a temporary rise in total phenols, subsequently strengthening antioxidant defense mechanisms. During 70 days of storage, the total phenolic content in the T2 group dropped to 41.94 mg GAE/kg FW, indicating a decrease of 8.62% compared to the CK group. This reduction is likely attributable to the oxidative degradation of phenolics [52]. PPO catalyzes the oxidation of polyphenols to form quinones, which further polymerize to form melanin, leading to browning of tissue [52]. In the present study, it was found that ozone treatment significantly inhibited the increase in PPO activity, which was 27.72% lower than that of CK in the T2 group at 73.35 U/g at 70 days, thus delaying the browning process (Figure 4I). This was further verified by a 25.0% reduction in browning value. In conclusion, the antioxidant defense capacity of flat jujube was notably improved through moderate ozone treatment, which also postponed the senescence process of the fruit by modulating the ROS metabolic defense system.

4. Multi-Way ANOVA Based on Measured Parameters

As demonstrated in

Table 1. Multi-way ANOVA based on the parameters of the jujube (F-value).

Source of Variation	Ozone Treatment (A)	Storage Time (B)	(A) × (B)
L*	1.73	30.07 ***	0.17
a*	44.96 **	209.40 ***	4.50 **
b*	2.60	22.49 ***	0.319
Weight loss	99.16 ***	737.19 ***	14.71 ***
TA content	6.48	13.41 ***	0.83
TSS content	2.70	5.92 ***	0.22
Respiration rate	16.94 ***	99.13 ***	3.66 **
Firmness	8.06 ***	52.52 ***	0.54
ASA content	16.25 ***	146.91 ***	2.69 *
Total flavonoid content	19.78 **	383.94 ***	5.48 ***
Total phenolic content	12.60 ***	118.08 ***	5.66 ***
DPPH scavenging activities	7.86 ***	29.33 ***	1.10
ABTS scavenging activities	4.66	63.65 ***	0.53
H2O2 content	9.00 ***	34.53 ***	1.06
O2− generation rate	29.60 ***	60.23 ***	1.97
MDA content	102.41 ***	728.70 ***	20.84 ***
APX activity	19.93 ***	135.43 ***	3.38 ***
CAT activity	32.53 ***	89.26 ***	16.10 ***
PPO activity	31.38 ***	130.31 ***	3.82 **
SOD activity	17.63 ***	204.70 ***	7.48 ***
PAL activity	5.10 **	159.36 ***	1.77
POD activity	14.354 **	276.32 ***	4.50 ***

Single asterisks (*) represent significant correlations at p < 0.05, double asterisks (**) represent significant correlations at p < 0.01, and triple asterisks (***) represent significant correlations at p < 0.001.

, storage time had an extremely significant impact on all indicators (p < 0.001), indicating that it was the primary factor contributing to a decline in quality. With prolonged storage time, there was an intensification of fruits’ weight loss, softening, ROS accumulation, and degradation of antioxidant substances. Analysis of the interaction effects between ozone treatment and storage time revealed that the weight loss, total phenolic content, MDA, APX, CAT, SOD, and POD exhibited extremely significant interaction effects. This suggests that as storage time increased, the efficacy of ozone treatment on these indicators became more pronounced. For instance, the inhibitory effect of ozone on weight loss was particularly significant during the later storage period of 56–70 days (p < 0.001), and the protective effect of ozone on fruit membrane lipids also increased over time. At 70 days, the MDA level in the T2 group was only 48% of that observed in the CK group. The interaction effects indicate that this advantage became increasingly significant in the later storage period (p < 0.001).

5. Principal Component Analysis and Correlation Analysis

5.1. Principal Component Analysis

The first two principal components, PC1 and PC2, are extracted by normalizing the data for the underlying indicators and usually explain most of the variance in the data. PC1 and PC2 are assumed to represent the main information if their cumulative contribution exceeds 70% [12].

PC1 and PC2 together explain 89.8% of the variance in the original data, as can be seen in Figure 5A. Figure 5B shows that L*, degree of browning, weight loss, and firmness have higher loadings on PC1, indicating that PC1 mainly captures the changes in these indicators directly related to jujubes’ ripening and quality. PC2 loadings mainly included nutritional indicators such as TSSs, TA, and ASA. The scores of the samples on PC1 gradually decreased with the extension of storage time, reflecting the gradual deterioration in jujubes’ quality. The ozone treatment group showed a smaller offset on the PC1 axis, especially in the late storage period (56–70 days), when samples scored significantly higher on PC1 than CK, suggesting that the ozone treatment slowed down the deterioration process. T2 may have performed the best, as it was closer to the region of high TA and ASA values on the PC2 axis.

5.2. Correlation Analysis

The Pearson correlation network system was utilized to evaluate the connection between quality and physiological metabolism (Figure 5C). The findings from the research indicated a significant negative correlation between L* and the degree of browning (r = −0.949, p < 0.01), demonstrating that the reduction in brightness was directly associated with the rise in browning [53]. Phenols are the core antioxidant components, and total phenols were positively correlated with the antioxidant activities of DPPH (r = 0.864, p < 0.05) and ABTS (r = 0.962, p < 0.001), and a* was significantly negatively correlated with total phenols (r = −0.832, p < 0.01), which may be due to the increase in red coloration caused by oxidation of the phenols [51]. CAT was weakly correlated with H₂O₂ (p = 0.034), and the metabolism of hydrogen peroxide required the synergistic scavenging of free radicals by other enzymes [46]. Therefore, from the perspective of correlation, it can be argued that ozone treatment can enhance the storage quality of flat jujube by impacting its antioxidant components and by activating the defense system of flat jujube in response to oxidative stress, thereby addressing physiological decline.

6. Conclusions

The present study elucidated that moderate ozone exposure (T2: 10.72 mg/m³) was effective in alleviating postharvest senescence of flat jujubes by coordinating ROS metabolism and strengthening antioxidant defenses. The T2 group minimized weight loss, maintained firmness, and stabilized TSS/TA content. Ozone increased the activities of SOD, CAT, APX, and POD and inhibited the accumulation of ROS (H₂O₂ and O₂⁻) and MDA, thereby protecting membranes’ integrity. Increased retention of phenolics and flavonoids enhanced DPPH/ABTS scavenging activities and improved antioxidant capacity. Reduced PPO activity and phenolic oxidation retarded browning, as confirmed by higher L* and b* values. It is noteworthy that a higher ozone concentration (19.27 mg/m³, T3) induced a higher stress response. The integration of enzymatic and non-enzymatic antioxidant systems emphasizes the role of ozone in ROS homeostasis, providing a scalable solution for flat jujube storage. Subsequent research should investigate transcriptomic and metabolomic interactions to gain insights into the regulatory functions of ozone and enhance its utilization in various fruit types.