Ozonated Water for Enhancing Quality and Antioxidant Activity in Ready-to-Eat Table Grapes During Cold Storage

Abstract

Table grapes are widely cultivated worldwide and are highly appreciated by consumers for their sensory characteristics and high nutritional value. Recently, many researchers have focused on applying specific post-harvest preservation strategies, such as the use of ozone, which represents an environmentally and health-friendly approach to sanitizing and preserving fresh fruits. In addition, ozone acts as a stressor, stimulating the defense mechanism of fruits and the synthesis of polyphenols, important antioxidant compounds. This study aimed to investigate the effect of different concentrations of ozonated water (8 ppm and 12 ppm), cold stored for 12 (T1) and 24 (T2) days, on the weight loss, texture and color parameters, phenolic content, and antioxidant activity of ready-to-eat Italian ‘Regal seedless’ grapes. Weight loss increased during cold storage for all treatments; however, the control exhibited higher values at 12 and 24 days (0.30% and 1.06%, respectively). Cold storage clearly affects the color of grapes, resulting in a loss of brightness and saturation, an increase in the yellow component, and a reduction in hue. At T2, ozonated water showed a significant increase in total phenolic content (TPC), antioxidant activity (DPPH, ORAC), and some phenolic compounds, including gallic acid, kaempferol, and resveratrol, compared to the control. The results indicated that using ozonated water on grape berries could be an effective strategy for enhancing their shelf life and nutraceutical value in post-harvest treatment.

1. Introduction

Table grapes are extensively cultivated worldwide and highly valued by consumers due to their sensory attributes and significant nutritional content. These fruits are classified as non-climacteric, exhibiting minimal physiological activity. They exhibit sensitivity to water loss and are susceptible to various postharvest diseases, which can negatively impact their commercial value and overall quality edibility [1,2,3]. The most common method for controlling the decay of clusters during cold storage is fumigation with sulphur dioxide (SO₂) [4]. However, elevated concentrations of SO₂ can result in bleaching damage to the fruit and may even pose a risk to human health [5]. Nowadays, the use of ozone serves as an alternative technique to SO₂ for extending postharvest life and facilitating transportation to more distant countries. The use of ozone within the agri-food sector represents an environmentally friendly and health-friendly approach for sanitizing and preserving fresh food [6,7,8]. Its application on fresh fruit extends shelf life, mitigates the development of post-harvest diseases, reduces spore production, inhibits ethylene oxidation, and decelerates both the respiration and ripening processes of the fruit [9,10]. Ozone can also oxidize chemicals and remove pesticide residues from vegetable products, making them safe for sale or further processing [11]. In addition to enhancing the quality of fresh products, postharvest treatments with ozone have been shown to stimulate the production of phenolic compounds, thereby eliciting chemical defense responses, such as the synthesis of polyphenols [12].

In recent years, numerous studies have concentrated on the application of ozone in table grapes to enhance their quality and shelf life.

Numerous studies focus on the impact of ozone on phenolic content in fruits, as this is significantly influenced by factors such as the dosage used, the method of application (whether in gaseous form or through ozonated water), and the duration of exposure. Generally, higher ozone concentrations lead to increased oxidative stress, which reduces polyphenol levels [13]. Conversely, lower doses of ozone can promote controlled oxidation, potentially enhancing the biosynthesis of these compounds [14]. Additionally, the application method, whether as gas or dissolved in water, plays a vital role. Ozone in its gaseous form is significantly more stable than ozonated water, proving more effective in inducing oxidation and thus reducing polyphenol content. In contrast, ozonated water, being less oxidative, could increase the accumulation of these bioactive compounds [15]. Furthermore, longer exposure periods lead to a decrease in polyphenols compared to shorter durations [16]. Numerous studies have reported the accumulation of polyphenols in table and wine grapes following ozonation treatments. Three studies highlighted the effects of pre-storage treatments with gaseous ozone at varying concentrations on table grapes, indicating an increase in total polyphenols, particularly in total stilbenes [1,17,18]. Three additional studies have investigated the impact of gaseous ozone applied during multiple days of cold storage on table grapes, revealing a significant enhancement in bioactive compounds within the grapes exposed to ozone [19,20,21]. Furthermore, only two studies on the use of ozonated water as a pre-storage treatment on table grapes were found. Silveira [22] documented the effects of immersion in ozonated water (2, 4, 6, or 8 mg/L of ozone) for four minutes at a temperature of 5 °C on both Thompson seedless and Black seedless varieties. The total phenolic content and antioxidant activity were elevated in grapes treated with ozonated water. Geransayeh [23] reported that ozone, dissolved at a 0.3 ppm concentration with three treatment times (5, 10, and 15 min), in water, was applied to the ‘Bidaneh qermez’ grape cultivar, causing lower decay incidence and longer storability.

Texture and color are among the most important quality characteristics of table grapes, playing a crucial role in relation to market requirements and consumer preferences. In table grapes, the hardness that influences the crunchiness of berries and the chewiness related to the enjoyment of chewing are sensory quality traits that consumers highly value [1,24]. Few studies have examined the effect of ozone treatment on the mechanical properties of berries [24,25]. Laureano [24] investigated the effects of continuous exposure to ozone gas (O₃, 30 µL/L, 24 h), showing that the treatment increased the skin hardness of Italia and Muscat hamburg table grapes. Gao [25] reported that gaseous ozone delayed decreases in firmness, which should prolong the storage period and improve the storage quality in Muscat hamburg grapes treated with 14.98 mg m⁻³ and 6.42 mg m⁻³. Admane [1] investigated the effects of pre-treatments with ozone (5, 10, and 20 μL L⁻¹) or CO₂ (50 and 70%) on organic Scarlotta seedless table grapes subsequently stored in MAP (2% O₂ and 5% CO₂), showing an ozone preserving effect on berry firmness.

This study aimed to investigate the effect of ozonated water with different ozone doses (8 ppm and 12 ppm) on the color and textural parameters, phenolic content, and antioxidant activity of ready-to-eat Italian ‘Regal seedless’ grapes. Previously, Caponio [26] also investigated the effects of these treatments on grey mold and the berry microbiome.

2. Materials and Methods

2.1. Plant Material and Growth Conditions

The research was conducted in 2022 on Vitis vinifera L. (cv. Regal seedless) vines that were 15 years old, located in a commercial table grape vineyard in a Mediterranean climate (Casamassima, Southern Italy, longitude 40°55′5″04 N, latitude 16°55′29″ E). The vines were grafted onto 140 Ruggeri (V. berlandieri × V. rupestris) rootstocks, with a spacing of 2.5 m between rows and within rows. They were trained using a “double tendone” trellis system and cane pruned (four canes with 12–15 buds per vine). Plant nutrition, pest management, and disease control were implemented in accordance with local standards. The vines received around 25 L per vine through drip irrigation, approximately every 15 days. In the latter half of August, plastic sheets were employed to protect the vines, with the grapes remaining on the plants until the end of November.

2.2. Ozone Pre-Treatments

Grapes were harvested on 21 November. At harvest, the vine yield (kg vine⁻¹) was determined by averaging the yield from 15 vines (five per replicate). Berries from visibly healthy bunches were collected for the experiments, and the pedicle was cut flush as close as possible to its insertion on the berry. All berries were placed in perforated plastic clamshell boxes, each with a volume of 100 g, to be used for the different treatments. Ozonated water at desired concentrations of 8 ppm and 12 ppm was obtained on-site (at room temperature of 17 °C) using a specific prototype designed for agricultural application (SAIM IMPIANTI s.r.l. Fondi, (LT) Italy) that included an ozone generator capable of producing ozone concentrations ranging from 18 Nm³ to 65 Nm³. The resulting ozonated water was used to fill a 70 L plastic washing tank via a circulation pump and was continuously monitored using an ozone analyzer. The effects of the ozonated water at the two concentrations were tested on eight replicates. A batch of eight boxes was dipped in ozonated water at a concentration of 8 ppm for 5 min. Another batch of eight boxes was immersed in ozonated water at a concentration of 12 ppm. The remaining eight boxes served as a control and were dipped in tap water for 5 min. Subsequently, for each treatment, berries were placed on stainless steel meshes for five minutes to drain excess water. The berries were taken and packed into low-density polyethylene bags (10 × 15 cm). All the bags were then heat-sealed with a micro-perforated plastic film to allow exchange with the outside air and stored in cold conditions at 2 °C and 95% relative humidity (RH) for 12 days (T1) and 24 days (T2) (Figure 1).

2.3. Berry Parameters and Weight Loss

Total Soluble Solid (TSS) was quantified in °Brix using a portable refractometer (Atago PR32, Norfolk, VA, USA). Titratable Acidity (TA), expressed in grams of tartaric acid per liter (g/L⁻¹), was determined via titration with 0.1 N sodium hydroxide and bromothymol blue as an indicator. The pH of the juice was measured with a calibrated pH meter (CRISONBASIC 20, Barcelona, Spain).

The weight of the berries was determined using a precision balance (OHAUS Corporation, Pine Brook, NJ, USA, model NVL2101/2) with an accuracy of 0.01 g. The percentage of weight loss was calculated using the formula % WL_t = (W₀ − W_t) × 100/M₀, where % WL_t represents the percentage of weight loss at time t, W₀ denotes the initial sample weight, and W_t indicates the sample weight at time t. Cumulative weight loss was also documented.

2.4. Berry Color and Texture

To measure color, thirty berries were randomly selected. The color values L*, a*, b*, Chrome, and Hue (CIE, 1986) [27] (Hunter Lab System) were determined using a Minolta CR-400 benchtop reflectance spectrophotometer (Minolta Corp., Osaka, Japan). The standard illuminant D65 and an observation angle of 10° served as references.

The textural analysis was performed on the same thirty berries utilized for color properties by a Texture Analyzer, model Zwick/Roell BT1-FR0.5TND14 (Zwick Roell GmbH & Co. KG, Ulm, Germany), equipped with a compression load cell rated for a nominal force of 500 N. The data were recorded at 500 Hz and processed using the software TestXpert^® II ver 3.1 (ZwickRoell GmbH & Co. KG, Ulm, Germany). The compression of the berry was assessed in the equatorial area of the berry. The texture profile analysis (TPA) was utilized among the various textural tests. The variables measured by TPA were Hardness, Cohesiveness, Springiness, Gumminess, and Chewiness.

2.5. Grape Samples and Preparation of Grape Skin Extracts (GSEs)

Grape samples were harvested at maturity, and berries were randomly collected and then frozen at −20 °C. Ten berries from each grape were peeled, separating the skins from the pulp. The phenolic fraction was extracted from the skins according to Di Stefano and Cravero [28] with slight modifications. Briefly, the skins were incubated in 25 mL of a solution of water, ethanol, and 37% hydrogen chloride (70:30:1). After 24 h under dark conditions, the mixture was filtered through a 0.45 μm cellulose syringe filter and was immediately analyzed and stored at −20 °C.

2.6. Total Phenolic Content (TPC)

Total phenolic content was determined using the microscale protocol described by Waterhouse [29]. Briefly, 1 mL of water, 0.02 mL of the extract sample, 0.2 mL of Folin-Ciocalteu reagent, and 0.8 mL of a 10% sodium carbonate solution were mixed and diluted to 4 mL. Absorbance was measured at 760 nm after 90 min at room temperature in the dark with an Agilent 8453 spectrophotometer (Agilent Technologies, Santa Clara, CA, USA). Results were expressed as grams of gallic acid equivalent (GAE) per kilogram of fresh skin, based on a gallic acid calibration curve ranging from 50 to 500 mg with R² = 0.998.

2.7. Antioxidant Activity

The total antioxidant activity (TAC) was evaluated using two different assays: the DPPH (2,2-diphenyl-1-picrylhydrazyl) and ORAC (Oxygen Radical Absorbance Capacity) assays. Calibration curves were prepared using Trolox, with values expressed as mM TE/kg fresh skin.

The DPPH assay was conducted according to the technique established by Brand-Williams [30], with some modifications. A stock solution was prepared by mixing 2.5 mg of DPPH radical with 100 mL of ethanol. The solution’s absorbance was adjusted to 0.7 ± 0.02 at 515 nm using an Agilent 8453 UV–Vis spectrophotometer (Agilent Technologies, Santa Clara, CA, USA). Then, 2 mL of DPPH radicals were mixed with 200 μL of the sample extract or standard (ethanol served as a blank). The decrease in absorbance at 515 nm was measured after 30 min of incubation at 37 °C.

The ORAC assay was conducted as previously described by Milella [31]. In summary, ORAC analysis was performed using a FLUOstar OPTIMA plate reader (BMG Labtech, Ortenberg, Germany), with fluorescein as the probe, an excitation wavelength of 485 nm, and an emission wavelength of 520 nm. Fluorescence was measured every 2 min for 120 min at 37 °C. The final ORAC values were calculated by determining the area differences under the fluorescence decay curve (AUC) between the blank and the sample.

2.8. HPLC Analysis

Before conducting the analyses, the grape skin extracts containing polyphenols were dried using a rotavapor and then reconstituted with 1 mL of HPLC-grade ethanol (Carlo Erba Reagents). Stilbene analyses (resveratrol, piceid, and piceatannol) were performed using a high-performance liquid chromatography (HPLC) Waters E-Alliance 2695 system, coupled with a Waters 2996 photodiode array detector. An aliquot of 20 µL of the extract was injected into a Luna C18 (100 × 2.0 mm, 2.5 μm particle size) column, which was equipped with a Security Guard guard column (3.0 × 4.0 mm) (Phenomenex, Torrance, CA, USA). The separations were performed in triplicate, following Romero [32], at a flow rate of 1 mL min⁻¹, and the run lasted 38 min.

Flavonoids, anthocyanins, catechins, and phenolic acids were analyzed using reversed-phase liquid chromatography (RP-HPLC) coupled with a photodiode array detector. The separations were performed in triplicate, as described by Docimo [33], at a flow rate of 0.70 mL min⁻¹, and the run lasted 50 min. Standards including resveratrol, (+)-catechin, gallic acid, syringic acid, and kaempferol-3-O-glucoside were obtained from Extrasynthese (Genay, France). The quantitative determination of compounds was conducted by comparing dose–response curves based on area data from authentic, distinct, and appropriately diluted standard solutions. The Empower Waters software was utilized to identify and quantify all the compounds.

2.9. Statistical Analysis

Data for each treatment and storage time were analyzed using one-way ANOVA, followed by Tukey’s Honest Significant Difference (HSD) test to establish significant differences at p ≤ 0.05. All results are presented as mean values ± standard error. Different lowercase letters indicate statistically significant differences (p ≤ 0.05) among storage times (Harvest, 12, and 24 days). Instead, different uppercase letters indicate statistically significant differences at the same p-value among ozonated water treatments (Control, 8 ppm, and 12 ppm), for each cold storage time (12 and 24 days). Statistical analyses were performed using OriginPro 2024a software (OriginLab Corporation, Northampton, MA, USA).

3. Results

3.1. Berry Parameters and Weight Loss

Table 1. Effects of treatments on TSS, TA, pH, and weight loss (%) of table grapes cv. Regal seedless after 12 and 24 days of cold storage (2 °C and 95% RH).

Parameter	Harvest	12 Days	24 Days
TSS (°Brix)
Control	18.38 ± 0.19 b	18.76 ± 0.08 ab A	19.50 ± 0.06 a A
8 ppm		18.37 ± 0.38 b A	18.03 ± 0.15 b B
12 ppm		18.44 ± 0.09 b A	18.20 ± 0.25 b B
TA (g tartaric acid 100 mL−1)
Control	3.54 ± 0.08 a	3.42 ± 0.02 a B	3.59 ± 0.04 a A
8 ppm		3.57 ± 0.05 a A	3.59 ± 0.01 a A
12 ppm		3.42 ± 0.03 a B	3.58 ± 0.02 a A
pH
Control	4.07 ± 0.04 a	4.19 ± 0.08 a A	4.24 ± 0.06 a A
8 ppm		4.08 ± 0.08 a A	4.00 ± 0.03 a B
12 ppm		4.06 ± 0.04 a A	4.21 ± 0.06 a AB
Weight loss (%)
Control		0.30 ± 0.007 c A	1.06 ± 0.005 a A
8 ppm		0.26 ± 0.009 d B	0.86 ± 0.009 b B
12 ppm		0.13 ± 0.005 e C	0.83 ± 0.005 b B

All data are expressed as average values ± standard error (n = 4). For each parameter, means followed by different letters, with uppercase in column for treatments and lowercase in row for time, are statistically different according to Tukey’s test at p ≤ 0.05.

illustrates the effects of ozonated water pre-treatments on the chemical qualities of table grapes cv. Regal seedless after 24 days of cold storage. The TSS value was significantly higher in control berries compared to those subjected to harvest and ozonated water pre-treatments. No significant variations were detected for the TA parameter. In contrast, pre-treated and untreated samples showed slight variations in pH. Weight loss increased during cold storage for all treatments; however, the control exhibited higher values at 12 and 24 days (0.30% and 1.06%, respectively). The ozonated water treatments led to lower water loss; at 12 days, the weight loss in the 8 ppm and 12 ppm treatments was significantly different. A similar trend was noted at the end of storage (24 days), although the 8 ppm and 12 ppm treatments showed no significant differences.

3.2. Berry Color and Texture Parameters

Cold storage significantly alters the color of grapes over time (

Table 2. Effects of treatments on color parameters for ‘Regal seedless’ after 12 and 24 days of cold storage (2 °C and 95% RH).

Parameter	Harvest	12 Days	24 Days
L*     Control	37.28 ± 0.72 a	31.82 ± 0.45 b A	32.46 ± 0.56 b AB
8 ppm		31.30 ± 0.56 b A	31.37 ± 0.36 b B
12 ppm		32.06 ± 0.39 b A	32.85 ± 0.41 b A
a*     Control	−2.09 ± 0.11 c	−1.70 ± 0.08 ab A	−1.51 ± 0.07 a A
8 ppm		−1.69 ± 0.11 ab A	−1.50 ± 0.08 a A
12 ppm		−1.66 ± 0.08 ab A	−1.87 ± 0.07 bc B
b*     Control	3.77 ± 0.22 b	4.92 ± 0.30 a A	5.09 ± 0.36 a A
8 ppm		4.88 ± 0.26 ab A	5.56 ± 0.37 a A
12 ppm		4.90 ± 0.26 a A	5.31 ± 0.39 a A
C*     Control	7.06 ± 0.31 a	5.18 ± 0.33 b A	5.05 ± 0.36 b A
8 ppm		5.49 ± 0.32 ab A	5.88 ± 0.44 ab A
12 ppm		5.64 ± 0.31 ab A	5.30 ± 0.19 ab A
H (°)  Control	117.50 ± 2.12 a	107.64 ± 1.45 b AB	107.18 ± 1.10 b AB
8 ppm		109.70 ± 1.16 b A	108.79 ± 1.38 b A
12 ppm		105.61 ± 1.13 b B	104.52 ± 0.73 b B

All data are expressed as the average value ± standard error (n = 30). For each parameter, means followed by different letters, with uppercase in column for treatments and lowercase in row for time, are statistically different according to Tukey’s test at p ≤ 0.05.

). Brightness (L*) markedly decreases from harvest (37.28) in the following days. Notably, at 12 days, L* values declined across all treatments without significant differences. Meanwhile, at 24 days of cold storage, the treatment with 12 ppm (32.85) exhibited slightly higher brightness compared to the 8 ppm and control treatments. Regarding the red/green component (a*), the berries become less green during cold storage, resulting in increased a* values compared to harvest (respectively, −2.09 vs. −1.50/−1.51 at 24 days). At 12 days, there are no differences between the treatments. Again, at 24 days, the 12 ppm treatment (−1.87) maintains a lower value than both the control (−1.51) and the 8 ppm (−1.50) treatments, suggesting that the 12 ppm treatment appears to slow down this transition, keeping its value slightly closer to that of the harvest. The yellow/blue component (b*) exhibited an increase in b* values over time, resulting in a greater degree of yellowing in the berries, with significant differences compared to the harvest (3.77 vs. 5.09). No significant differences emerged between the ozone treatments at 12 and 24 days. Berry saturation (C), or Chroma, showed a significant decline from harvest (7.06) to 12 days, after which it remained stable until 24 days (5.00). There are no significant differences between the ozone treatments.

After 24 days of cold storage, a significant reduction in hardness was observed compared to the harvest time. The control showed a 31.3% reduction compared to the initial value (

Table 3. Effects of treatments on texture for ‘Regal seedless’ after 24 days of cold storage (2 °C and 95% RH).

Treatments	Hardness (N)	Cohesiveness (−)	Gumminess (N)	Elasticity (-)	Chewiness (N)
Harvest	8.30 ± 0.34 a	0.26 ± 0.006 c	2.15 ± 0.09 b	0.62 ± 0.006 a	1.15 ± 0.04 b
Control	5.70 ± 0.13 c	0.26 ± 0.005 c	1.50 ± 0.04 c	0.32 ± 0.009 b	0.96 ± 0.03 c
8 ppm	6.99 ± 0.26 b	0.36 ± 0.007 a	2.52 ± 0.10 a	0.59 ± 0.007 a	1.46 ± 0.04 a
12 ppm	7.01 ± 0.26 b	0.30 ± 0.006 b	2.13 ± 0.01 b	0.60 ± 0.011 a	1.05 ± 0.04 b

All data are expressed as the average value ± standard error (n = 30). Values within a column followed by different letters are significantly different (p ≤ 0.05) according to Tukey’s multiple range test.

). Treatments using ozonated water alleviated firmness losses, with reductions of 15.8% and 15.5% at 8 ppm and 12 ppm, respectively. The strength of the internal bonds between the pulp and the skin, known as cohesiveness, indicated that the 8 ppm treatment had the highest value (0.36), which was significantly greater than both the control (0.26) and the 12 ppm treatment (0.30). Gumminess, referring to the energy required to disintegrate a semi-solid berry until it is ready to be swallowed, was lower in the control (1.50 N) compared to the other treatments and the harvest (2.15 N). The 8 ppm treatment (2.52 N) showed the highest value, suggesting greater resistance to chewing. The 12 ppm treatment (2.13 N) displayed intermediate behavior, which was more similar to the harvest condition. Furthermore, elasticity decreased in the control (0.32) compared to harvest (0.62), indicating a loss of elasticity over time. However, both ozone treatments (8 ppm and 12 ppm) maintained higher values (0.59 and 0.60, respectively). Lastly, chewiness exhibited a trend similar to gumminess. The highest value was observed at 8 ppm (1.46 N), followed by the harvest (1.15 N) and 12 ppm (1.05 N). The lowest value was recorded in the control (0.96 N), confirming that ozone contributed to preserving greater fruit firmness.

3.3. Total Phenolics

Figure 2 illustrates the TPC of Regal seedless berries treated with two distinct concentrations of ozonated water (8 and 12 ppm) after 12 days (T1) and 26 days (T2) of cold storage. At harvest, the TPC was 24.43 g GAE kg⁻¹. After 12 days, treatments with ozonated water showed a slight increase in polyphenol content, with values exceeding 30 g GAE kg⁻¹ compared to the harvest and control, though no significant differences were observed between them. At 24 days, the total phenolic content (TPC) in the control was lower than in the grapes treated with ozonated water (8 ppm and 12 ppm), both of which preserved a high polyphenol content similar to that recorded at 12 days. This suggests that ozone washing helps preserve phenolic compounds during storage, while the control experiences natural degradation over time.

3.4. Antioxidants

Total antioxidant activity (TAC) was assessed using the DPPH and ORAC methods. Figure 3 illustrates the total antioxidant activity expressed as DPPH radical scavenging capacity (mM TE kg⁻¹) in grape berries treated with ozonated water at 8 ppm and 12 ppm during storage for 12 and 24 days, compared to the control.

The initial TAC value was approximately 100 mM TE kg⁻¹ at harvest. After 12 days of storage, the control displayed the same antioxidant capacity as the initial value. In contrast, the samples treated with ozonated water (8 ppm and 12 ppm) exhibited significantly higher values than both the control and the harvest values, with antioxidant activity measuring 168.7 and 182.36 mM TE kg⁻¹, respectively. The two ozone concentrations highlighted no significant differences. At 24 days of storage, the antioxidant capacity of the control remained stable, comparable to that at 12 days and at harvest. The treated berries with ozonated water maintained a higher antioxidant capacity than the control, with no significant differences observed between the two doses at 12 days.

Figure 4 illustrates the antioxidant activity of grape berries measured using the ORAC assay (mMol TE kg⁻¹). At harvest, the antioxidant activity was significantly lower than that of all treated samples on two sampling dates; however, in the absence of ozonated water treatment, the value remained consistent over time. After a 12-day storage period, samples treated with ozonated water at concentrations of 8 ppm and 12 ppm exhibited a notable increase in antioxidant capacity of 96% and 63%, respectively, compared to the control group.

A similar trend is observed after a 24-day storage period. Samples treated with ozonated water display significantly elevated levels of antioxidant activity compared to the control, with no significant differences detected between the two ozone concentrations.

3.5. Analysis of Phenolic Compounds by HPLC

This study focused on several phenolic compounds in white table grapes, including phenolic acids (gallic acid and syringic acid), flavanols (catechin), flavonols (kaempferol), and stilbenes (resveratrol). At harvest, the gallic acid content was approximately 0.001 mg kg^-1 (Figure 5). After 12 days of storage, the gallic acid content was significantly higher in the ozonated water treatments compared to both the control and the harvest. Even after 24 days of storage, the gallic acid content remained elevated in the ozone-treated samples compared to the control. The treatments at 8 ppm and 12 ppm yielded similar values at 12 and 24 days, with no statistically significant differences. Meanwhile, the control at 24 days showed a significant reduction compared to the harvest.

Regarding catechins, after 12 days, the control did not significantly differ from the value at harvest. Although the treatments at 8 ppm and 12 ppm showed a slight increase in values compared to the control, they did not demonstrate any significant difference. At 24 days, the control exhibited a significant reduction in catechins compared to both the treatments and the value at 12 days (Figure 6).

Syringic acid has the highest concentration at harvest, at 200 mg/kg, with a decreasing trend observed after 12 and 24 days of storage. After 12 days, the control exhibited a lower level of syringic acid compared to the initial harvest; however, this difference was not significant. The treatment with 8 ppm ozone appeared to maintain relatively high levels, similar to the initial value. The treatment with 12 ppm shows a slight decrease compared to 8 ppm, but without a significant difference when compared to the control (Figure 7).

Figure 8 illustrates the evolution of kaempferol content in grapes exposed to different ozone treatments (8 and 12 ppm) and analyzed after 12 and 24 days of storage. At harvest, the initial kaempferol content was the lowest among all conditions, with a value of 17.3 mg kg⁻¹. After 12 days, kaempferol increased, particularly in ozone-treated grapes. The 8 ppm ozone treatment resulted in the highest accumulation of kaempferol (30.3 mg kg⁻¹), followed closely by the 12 ppm treatment (27.6 mg kg⁻¹). The control grapes also exhibited an increase compared to harvest, although with significantly lower values than the ozone treatments (22.4 mg kg⁻¹). Collectively, the control, 8 ppm, and 12 ppm samples were not significantly different from each other (p = 0.07). After 24 days, the kaempferol levels decreased compared to the levels recorded at 12 days, particularly in the control sample, which had reverted to values similar to those observed at harvest (approximately 17.6 mg kg⁻¹). In contrast, the grapes with ozonation demonstrated sustained higher levels of kaempferol compared to the control, with the 12 ppm concentration identified as the most effective treatment in the long term (25.7 mg kg⁻¹), while the 8 ppm treatment maintained intermediate levels (21.5 mg kg⁻¹).

At harvest, the initial total resveratrol content in the grapes was very low, at 0.25 mg kg⁻¹, showing no significant differences compared to what was observed in the samples analyzed after 12 days (Figure 9). In fact, both the control and the samples treated with ozone (8 and 12 ppm) exhibited extremely low resveratrol levels after 12 days, at approximately 0.2–0.3 mg kg⁻¹, with no significant differences between them or in comparison to the harvest. After 24 days, a clear increase in resveratrol levels is evident in the samples treated with ozone, unlike the control. The control at 24 days displayed a content of 0.9 mg kg⁻¹, which remained relatively low and comparable to that of the harvest. In contrast, grapes treated with ozone showed a significant increase: those with 8 ppm reached 4.7 mg kg⁻¹, while those with 12 ppm reached up to 6.3 mg kg⁻¹, with no significant difference between them (p = 0.12). These values are significantly higher than those of the harvest and the 24 days, indicating that ozone strongly induces the synthesis or accumulation of resveratrol over time.

4. Discussion

Ozonated water typically shows better effectiveness than its gaseous form, decomposing more rapidly and leaving no harmful residues [34]. Premjit [34] summarizes recent scientific studies on the properties, chemistry, and production of aqueous ozone, emphasizing the factors that affect the process’s efficiency. However, in the field of viticulture, research on the application of ozone in postharvest treatments for table grapes remains limited. To the best of our knowledge, three studies report the effects of pretreating table grapes with gaseous ozone [6,18,19], while only two document the use of ozonated water [23,24].

In contrast with Campayo [35], who found a slight increase in TSS in ozonated water treatment on Bobal grapevines, our study showed that the total soluble solids (TSSs) levels recorded at 24 days were significantly higher in the control berries compared to those subjected to ozonated water treatments. This observation may be attributed to the greater weight loss observed in the control samples compared to those that underwent ozone pre-treatment. Also, concerning the pH, we found contrasting results, as the control at the end of cold storage showed a higher value compared to the 8 ppm pre-treatment. This suggests that this dose of ozone provides some protection against the naturally occurring pH increase during grape storage senescence. Finally, regarding TA, no significant differences were observed, according to Campayo [35].

The percentage of weight loss increased during cold storage for all treatments; specifically, the control sample showed higher values at 12 and 24 days (0.30% and 1.06%). These results align with those reported by Admane [1], which indicated significant differences in weight loss between grapes treated with 20 µL L⁻¹ O₃ and the control for Scarlotta seedless berries stored for 45 days at 5 °C and 90% RH. Furthermore, Geransayeh [23] reported that ozone treatment resulted in a 0.06% decrease in weight loss compared to the control. In our study, we observed the same behavior, but only at 24 days of cold storage, likely due to the different variety (Regal seedless in our case), maturity index (Regal seedless 18.4 °Brix vs. Scarlotta seedless 15.34 °Brix), and the method of O₃ application (gaseous form vs. ozonated water).

Cold storage clearly affected the color of grapes, resulting in a loss of brightness and saturation, an increase in the yellow component, and a reduction in hue. Specifically, the 12 ppm treatment maintained greater brightness and slowed the loss of the green component (a*), preserving the color while accelerating the change in hue, demonstrating a shift towards redder tones. The 12 ppm ozonated water treatment appeared to have a moderate effect on maintaining the freshness of the berries’ color, but did not prevent their yellowing and loss of saturation over time. Chroma and the b* component did not differ significantly between the ozone treatments. The most likely cause of the reduction in color saturation of the berries may be attributed to the degradation of the green pigments over time, rather than the influence of the ozone factor in this process [20]. Ultimately, the hue angle (H°) shifted towards less yellow–green tones, as the values significantly decreased from harvest (117.50°) to 12 and 24 days, indicating a change in hue. These results are consistent with Geransayeh [23], who reported that the hue angle decreased in the ozone treatment over the cold storage duration. Regarding the textural parameters, the findings indicated that treatment using ozonated water demonstrated greater efficacy in preserving the texture of Regal seedless table grapes compared to the untreated control group. Specifically, the treatment at 8 ppm improved the cohesion of pulp, thereby improving deformation resistance. The reduction in hardness was significantly less pronounced in the ozone-treated grapes compared to the control, indicating that ozonated water contributed to the preservation of berry firmness. Furthermore, other mechanical properties, such as cohesiveness, gumminess, elasticity, and chewiness, remained to a considerably greater extent in the ozonated samples. These results are in agreement with Karaca [36] and Horvitz [37], who found that ozone-treated table grapes can be easily handled postharvest due to their greater skin hardness.

TPC was significantly affected by ozonated water treatment during cold storage. At 12 days, a slight increase was observed, although there was no significant difference between the control and pre-treated samples and those at harvest. At 24 days, berries washed with ozonated water exhibited a significant increase in total phenolic content (TPC) compared to the control, with no significant differences observed between the two treatment doses (8 ppm and 12 ppm). These results agree with Silveira [22], who reported that the TPC of Thompson Seedless and Black grapes, sanitized with ozonated water (2, 4, 6, 8 ppm O₃) and stored for 21 days at 5 °C, increased compared to the control sample. Even in this study, no significant differences among ozonated water treatments were observed. Our findings align with the study by Admane [1], which evaluated the effects of three concentrations of ozone gas (5, 10, 20 μL L⁻¹) as pre-treatments on the quality and shelf life of Scarlotta table grapes over long-term storage. After 45 days of cold storage, the TPC in the skin of berries remained stable in both untreated samples and those pre-treated with O₃ at 5 μL L⁻¹. In contrast, the TPC increased in berries treated with O₃ at concentrations of 10 and 20 μL L⁻¹.

The evaluation of TAC using the DPPH and ORAC assays indicated that the berries treated with ozonated water (8 ppm and 12 ppm) displayed a higher antioxidant capacity than the control at 12 and 24 days. Nevertheless, the response was not dose-dependent, as no significant differences were observed between the two treatments, despite a 50% increase in ozone concentration. The beneficial effect of O₃ on TAC is often associated with an increase in TPC. Silveira [22] reported that Thompson seedless and Black grapes, sanitized with 2, 4, 6, and 8 ppm of ozone and stored for 21 days at 5 °C, exhibited higher antioxidant capacity (measured by the FRAP and DPPH methods) compared to the control. Our findings on the increase in TAC after treatment with ozonated water differ from those reported by Admane [1], whose research showed no changes in the TAC of samples treated with gaseous ozone.

Numerous studies indicate that the effect of ozone on phenolic compounds depends on the balance between two mechanisms: (1) ozone facilitates the release of phenolic components through the partial breakdown of cellular structures, thereby enhancing extraction efficiency and liberating phenolic compounds that adhere to the cell wall [38]; (2) ozone, acting as a stressor, activates pre-existing enzymes involved in defense mechanisms, leading to rapid phenolic accumulation [39], as found by Gao [25] in Muscat hamburg grapes. Consequently, considering these findings, it can be argued that our results are reasonably associated with the two mechanisms previously discussed.

Interestingly, the TPC and TAC analyzed between 8 and 12 ppm concentrations of ozonated water did not differ. This suggests that a concentration of 8 ppm is sufficient to achieve the desired beneficial effects, making the treatment more economical and environmentally friendly. In particular, regarding the economic aspect, it has been highlighted that the lower 8 ppm concentration requested during the experiment results in a lower oxygen flux (−40%) and requires a lower concentration compared to that of 12 ppm.

To better understand the results obtained, specific phenolic compounds were identified, including gallic acid, catechin, syringic acid, kaempferol, and resveratrol. In particular, the behavior of gallic acid, kaempferol, and resveratrol was similar. The effect of ozone on gallic acid is evident after just 12 days, showing a significant increase compared to the control and harvest, and it remains stable until 24 days, maintaining values similar to those observed at 12 days. Both doses of ozone induce comparable levels of 0.0013 and 0.0014 mg kg⁻¹, respectively, while the control demonstrates a progressive decrease over time. This suggests that ozonated water has a distinct stimulating effect on the production of this phenolic compound, helping to maintain its concentration during storage. The kaempferol content increases after just 12 days in samples treated with ozone, showing a more pronounced effect at a dose of 8 ppm (30.3 mg kg⁻¹), followed by 12 ppm (27.6 mg kg⁻¹). These values are significantly higher than those of the harvest and control. After 24 days, kaempferol levels decrease in the samples treated at 8 ppm, while they remain elevated in the samples treated at 12 ppm, suggesting that the higher dosage is more effective in sustaining this flavonol over time. According to Carbone [40], no literature is available regarding the effect of ozone on kaempferol content in grapes. However, our results could be in agreement with the trend found by Foy [41], who reported a positive association between ozone tolerance and kaempferol content in soybean. Unlike kaempferol and gallic acid, resveratrol exhibits a significant increase only after 24 days in samples treated with ozone. Treatment with ozonated water has no immediate effects on resveratrol production, as demonstrated by the unchanged levels at 12 days. However, with prolonged storage (24 days), a marked positive effect is observed, particularly with the 12 ppm dose, which shows the highest level of resveratrol, although without statistically significant differences compared to the 8 ppm dose. This result aligns with the findings of Sarig [17], in which five distinct varieties of table grapes were subjected to exposure to ozone gas for varying durations (up to 30 min), demonstrating that the phytoalexin resveratrol was elicited by the ozone treatments. Furthermore, González-Barrio [18] demonstrated that the postharvest treatment of seedless white table grapes, Superior, using different ozone gas concentrations (3.88 and 1.67 g/h) for 1, 3, and 5 h induced an increase in stilbenoid biosynthesis, including trans-resveratrol, piceatannol, and vinifera, during storage at 22 °C.

These data suggest that ozone may act as an effective inducer of phenolic compound synthesis, such as kaempferol, gallic acid, and resveratrol, potentially enhancing the nutraceutical properties of grapes during storage. We found that the catechin content in ozonated treatments did not increase compared to the harvest content. This contrasts with Carbone [40], who found an increase in catechin content in grapes due to short-term postharvest treatment of white wine grapes, and Hernandez [20], who found an increase in total flavan-3-ol content in an enriched ozone atmosphere with cold-stored table grapes. However, looking at the 24-day sampling, it is possible to highlight that ozone preserved catechin degradation, which is evident in the control on the same date. On the other hand, a similar preservation effect was found for syringic acid, which, in particular, was twofold that of the control.

The results of our study on phenolic compounds indicate that ozonated water pretreatment can have two distinct effects. Firstly, ozone preserved the catechin and syringic acid content in the harvest from natural degradation over time. The second and more significant effect was observed in the cases of gallic acid and kaempferol, which were generally enhanced by the ozone treatment, particularly at 12 days. Overall, our findings, as confirmed by Modesti [42], support the hypothesis that ozone can be used to enhance the compositional and nutraceutical quality of berries.

5. Conclusions

This study showed preliminary results of the effects of ozonated water pretreatment on table grapes in post-harvest. The results demonstrated a significant increase in total phenolic content (TPC), antioxidant activity (DPPH, ORAC), and some phenolic compounds, including gallic acid, kaempferol, and resveratrol, compared to the control. This evidence suggests that using ozonated water for the post-harvest treatment of grape berries could represent an effective strategy to improve the shelf life and nutraceutical value. Furthermore, these data may support the use of ozone as a viable alternative to the conventional application of sulfur dioxide (SO₂) for the long-term preservation of table grapes. The benefits derived from these findings may also suggest alternative applications for ozone. Indeed, a short pretreatment is clearly more cost-effective than the extended use of low-concentration ozone gas during the cold storage period. Moreover, this case study highlights the sustainability of ozone use, especially in the medium term, as well as its effectiveness in sanitizing and preserving fruit during storage. However, since there are still few studies on the effects of ozone on table grapes, more research should be directed towards optimizing the application conditions (concentration and treatment duration) to maximize the benefits and minimize any negative effects on the grape’s organoleptic quality.

The current experiment focuses on the specific application of ozonated water only for the Regal seedless variety. In this context, subsequent studies should investigate other similar use cases to expand the study sample.