Effect of Prolonged Cold Storage on the Dynamics of the Enzymatic and Non-Enzymatic Antioxidant System in the Mesocarp of Avocado (Persea americana) cv. Hass: Relationship with Oxidative Processes

Abstract

This work evaluated the enzymatic and non-enzymatic antioxidant defense systems of avocados cv. Hass stored at 7 ± 0.5 °C for 10, 20, and 30 d, and at the stage of edible ripeness. The enzymatic antioxidant enzyme system included superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT), and the non-enzymatic antioxidant system was composed of total phenolic compounds (TPC), total carotenoids (TC), α-, β-, and γ-tocopherols, as well as hydrophilic (H-AOX) and lipophilic (L-AOX) antioxidant activities. In addition, polyphenol oxidase (PPO) and lipoxygenase (LOX) activities, TBARS values and pulp browning area were determined. At edible ripeness, low SOD and POD but higher CAT activities were evidenced in response to their joint participation in the oxidative stress developed. In addition, low content of γ-tocopherol and higher contents of TPC and TC were evidenced and suggest their individual or joint participation in defense against oxidation. The other compounds and activities evaluated tended to remain constant. Oxidative damage was evidenced by the increase in PPO activity and TBARS values, while LOX did not play a significant role. Pulp browning area did not surpass 6% in affected fruit. The results indicate that under these conditions oxidative stress was largely restrained by the endogenous fruit antioxidant defense system.

1. Introduction

The avocado is a tropical fruit belonging to the Lauraceae family, native to Mexico and Central America [1]. There are several avocado cultivars, but the most commercial is the Hass cultivar. The pulp is the most consumed fraction of this fruit and consists of up to 21% fat (mainly monounsaturated fats, especially oleic acid), proteins, carbohydrates, minerals, vitamins, and antioxidant compounds (carotenoids, phytosterols, phenolic compounds, among others), which are believed to have protective effects on cells against damage caused by reactive oxygen species (ROS) and other free radicals [2,3,4]. Avocado has recently gained substantial popularity and is often marketed as a “superfood” because of its unique nutritional composition, antioxidant content, and biochemical profile [5].

Refrigeration is one of the most widely used postharvest technologies, being an effective and economical treatment to slow down metabolic processes and reduce pathogen growth, ensuring the commercial quality of fruits and vegetables [6]. Although low temperatures delay senescence, it has been reported that they can induce physiological disorders when the temperature drops below the recommended threshold influenced by both the time of exposure and the cold intensity [7], this effect is known as cold damage and is related to modifications in the integrity of cell membranes caused by a state of oxidative stress due to the high production of ROS [8]. This condition triggers the enzymatic and non-enzymatic antioxidant defense system to maintain a balance between oxidant-antioxidant species; however, if the production of ROS exceeds the antioxidant system, irreversible cell damage is established and—in advanced stages—may lead to cell death [9].

Major antioxidants in fruits and vegetables include the enzymatic (SOD, CAT, guaiacol, ascorbate and glutathione peroxidases, and glutathione reductase) and nonenzymatic antioxidants (vitamins, flavonoids, and carotenoids) [10]. Avocado exposed to 3–5 °C for more than two weeks may present physiological disorders mediated by oxidative stress, manifesting themselves through internal pulp browning (grayish flesh, stained flesh, browning of vascular bundles), problems of irregular ripening and increased susceptibility to attacks by pathogenic microorganisms. The time at which chilling injury begins to develop and the severity with which it occurs depend on the cultivar, region or geographical area of production, and stage of development, among others [11]. For avocados, Woolf et al. [12] recommended storage temperatures between 3 and 8 °C for 2 or 4 weeks.

The present research aimed to evaluate simultaneously the enzymatic antioxidant system: superoxide dismutase, catalase, and peroxidase activities; and the non-enzymatic antioxidant system: total phenolic compounds, tocopherols, and total carotenoids; as well as measurements of hydrophilic and lipophilic antioxidant activity in the mesocarp (pulp) of avocado cv. Hass at harvest, during storage at ~7 °C for 10, 20, and 30 d, and at their respective stages of edible ripeness. In addition, polyphenol oxidase and lipoxygenase activities were analyzed, and thiobarbituric acid reactive substances and the extent of cold damage in the mesocarp (pulp browning area) were determined.

2. Materials and Methods

2.1. Plant Material Storage and Shelf-Life Conditions

Avocados cv. Hass were harvested from the field of the Programa de Investigación en Frutales of the Universidad Nacional Agraria La Molina (La Molina, Lima, Peru) located at south latitude: 12.0817° S, west longitude: 76.9432° W at 243.7 m.a.s.l. A random sampling of one hectare of the field was carried out and 150 fruit were collected. The fruit were harvested at physiological maturity. The avocados were washed and selected and damaged fruit was discarded. The fruit presented weight, dry matter, and fat average values of: 210 g, 25.67 ± 1.29% and 14.49 ± 1.19%, respectively. The fatty acid profile consisted of oleic, palmitic, linoleic, palmitoleic, linolenic, and stearic acids representing 48.4, 21.9, 15.8, 12.0, 0.88, and 0.86%, respectively.

Ten fruit were separated for initial sample (IS) measurements and the rest were stored at 7 ± 0.5 °C and 85% relative humidity (RH) for periods of 10, 20, and 30 days (R10d, R20d, and R30d). For each period, 30 avocados were removed and five of them were subjected to the respective analyses and the remaining 25 were transferred to a shelf-life period (~20 °C, 80% RH) until reaching edible ripeness (4–8 N firmness). Avocados stored for 10, 20, and 30 d reached edible ripeness at ~9.8, 7.6, and 4.6 days, respectively. Five independent fruit were sampled at IS, after 10, 20, and 30 d cold storage and at their respective edible ripeness (SLR10d, SL20d, and SLR30d). The sampled fruit were subjected to peel and pit removal, and the mesocarp of each fruit was chopped and immediately frozen in liquid nitrogen, then ground and stored at −80 °C until analysis. For firmness determination during shelf-life conditions, 25 avocados were used.

2.2. Pulp Browning Area

Mesocarp chilling injury was assessed as pulp browning area. All samples prior to conditioning were sectioned longitudinally and quickly photographed. For the determination of browned area, 25 avocados at edible ripeness were evaluated per condition. The extent (area) of dark spots at the pulp level was determined using ImageJ v.1.8.0 software. The images were obtained from avocados cut lengthwise in half and placed on a white background. The photos were captured with a Samsung Galaxy A10 cell phone, without flash and zoom, with a resolution of 889 × 1317 pixels of contrast and sharpness. In the avocados with evidence of damage, the perimeter of the fruit cut longitudinally was delimited, as well as the browned area, and based on these, the corresponding areas were calculated, establishing the percentage of the browned area with respect to the total area (half) of the fruit.

2.3. Soluble Protein Assay

The soluble protein content of the enzymatic extracts was performed using the Bradford method as recommended by George & Christoffersen [13]. The results were expressed in mg protein/mL.

2.4. Enzymatic Analysis

2.4.1. Catalase Activity (CAT)

For this determination, the method reported by Uarrota et al. [14] with adaptations was used. Briefly, the enzyme extract was obtained by homogenizing 200 mg of the sample with 1 mL of the extraction solution (0.2 M potassium phosphate buffer at pH 7, 5 mM disodium EDTA dihydrate, 0.1% Triton X-100, 20 mM DTT, and 0.18 M PVP) and 40 µL of 125 mM PMSF. The mixture was cold sonicated (~2–4 °C) for 30 s, centrifuged at 2700 g for 15 min at 4 °C, the recovered supernatant was passed through a 0.22 µm filter and stored at 4 °C until analysis. The CAT activity was measured in a mixture composed of 500 µL of extraction buffer, 5 µL of enzyme extract, and 45 µL of 122 mM hydrogen peroxide. The absorbance of the mixture was then monitored in a UV/VIS microplate reader (Biotek, VT, USA) at 240 nm every 15 s for 5 min at 25 °C. The specific CAT activity was expressed in U/mg protein, where U corresponded to mM hydrogen peroxide formed per min.

2.4.2. Superoxide Dismutase Activity (SOD)

The methodology reported by Fuentealba et al. [15] was used with very few modifications. Enzyme extraction was performed by homogenizing 200 mg of sample with 1 mL of the extraction solution (100 mM potassium phosphate buffer, pH 7.8, containing EDTA at 0.1 mM concentration). The mixture was sonicated, centrifuged, filtered, and stored at the same conditions as the CAT analysis. For the analysis, 50 µL of the enzyme extract was mixed with 250 µL of a solution composed of 194 mg of 13 mM methionine, 6 mg of 75 µM nitro blue tetrazolium (NBT), and 3.73 mL of 1.3 µM riboflavin, the mixture was illuminated with a fluorescent lamp for 20 min, after which the absorbance was measured at 560 nm. Absorbances of the blank (non-illuminated samples) and control (reaction buffer) were considered in the assay and in the calculations. The specific activity of SOD was expressed as U/mg protein, where U corresponded to the amount of enzyme required to inhibit NBT by 50%.

2.4.3. Peroxidase Activity (POD)

POD activity followed the method described by Woolf et al. [16] with slight modifications. The enzyme extraction was performed by homogenizing 100 mg of the sample with 1.5 mL of the extraction solvent (0.1 M sodium phosphate buffer at pH 7, with 100 mM disodium EDTA dihydrate and 0.1 % Triton X-100), with 15 mg of PVPP and 15 µL of 100 mM PMSF, followed by sonication, centrifugation, filtration, and storage at the same conditions as stated in previous paragraphs. The enzymatic reaction was performed by mixing 160 µL of 20 mM guaiacol and 40 µL of the enzymatic extract followed by incubation for 5 min at 30 °C, and finally, adding 80 µL of 50 mM hydrogen peroxide. The change in absorbance was evaluated at 460 nm for a period of 5 min. The specific activity of the enzyme was calculated from the slope of the linear section of the curve that evaluated the variation of absorbance over time (U) on the protein content, expressing the results as U/mg protein.

2.4.4. Polyphenol Oxidase Activity (PPO)

The procedure described by Fuentealba et al. [15] was followed with slight adaptations. Briefly, 200 mg of sample was mixed and homogenized with 1 mL of 0.1 M citrate-phosphate buffer (pH 6.5) for 30 s followed by centrifugation and filtration, then the enzyme extract was recovered and stored at 4 °C. The reaction was performed with 171 µL of extraction buffer, 43 µL of enzyme extract and 86 µL of 0.2 M catechol. Readings were taken at 420 nm for 10 min. The specific activity of the enzyme was calculated from the slope of the linear section of the curve that evaluated the variation of absorbance over time (U) on the protein content, expressing the results as U/mg protein.

2.4.5. Lypoxigenase Activity (LOX)

The methodology followed by Jacobo-Velásquez & Hernández-Bremen [17] with adaptations was followed. Briefly, enzyme extraction was performed by mixing and sonicating 1 g of sample with 2 mL of 100 mM sodium phosphate buffer (pH 6), followed by centrifugation, the supernatant was recovered, filtered, and stored in the same way as the other enzyme extracts. Next, the enzymatic reaction was produced by mixing 500 µL of phosphate buffer, 30 µL of the enzyme extract, and 40 µL of linoleic acid at 16.64 mM. Readings of the reaction were taken at 234 nm every 30 s for 5 min. The specific activity was calculated from the slope of the linear section of the curve over time and divided by the protein content and expressed as U/mg protein.

2.5. Total Phenolic Compounds and Hydrophilic and Lipophilic Antioxidant Activities

The extracts were obtained with the methodology reported by Huamán-Alvino et al. [18]. Two grams of sample was mixed with 10 mL of 80% (v/v) MeOH, the whole mixture was shaken for 1 h and centrifuged at 2700 g for 15 min at 4 °C, recovering the liquid extract. The process was repeated with the residual cake, joining the two extracts. The final extract was used for the determination of total phenolic compounds (TPC) and hydrophilic antioxidant activity (H-AOX). The resulting final cake was used for the measurement of lipophilic antioxidant activity (L-AOX). Then, 10 mL of dichloromethane was added and shaken for 30 min, followed by centrifugation, and finally the recovery of the liquid extract. All liquid extracts were stored at −20 °C until analysis.

TPC were determined using the Folin-Ciocalteau method described by Singleton and Rosi [19]. Sample absorbance was measured at 755 nm and values were expressed as mg of gallic acid equivalents (GAE)/g of dry weight (DW). The H-AOX and L-AOX were determined by the ABTS assay adapted from Arnao et al. [20]. The sample absorbances were measured at 734 nm, and the antioxidant capacities were calculated in µmol Trolox equivalents (TE)/g DW.

2.6. Tocopherol Content

The samples were prepared following the methodology reported by Huamán-Alvino et al. [18]. Analyses were performed by HPLC (Waters 2695 Separation Module, Waters, Milford, MA, USA) coupled to a fluorescence detector according to Chirinos et al. [21] and expressed as μg/g DW.

2.7. Total Carotenoids

The extracts were prepared following the methodology reported by Chirinos et al. [21]. Absorbance was measured at 450 nm and values were expressed as mg de β-caroteno/100 g DW.

2.8. TBARS Assay

For the determination of thiobarbituric acid reactive substances (TBARS) concentrations, the malonaldehyde assay (MDA) was used according to the method of Bailly and Kranner [22] with some modifications. Briefly, 0.4 g of sample was homogenized in 6 mL of an ethanol solution containing 20% trichloroacetic acid and 0.5% thiobarbituric acid for 20 s. The homogenate was brought to 95 °C for 30 min and immediately cooled in an ice bath. The recovered supernatant was then centrifuged at 2700 g for 15 min at 4 °C and the absorbances at 532 nm and 600 nm were measured. The TBARS content was expressed as mg MDA/Kg DW sample.

2.9. Statistical Analysis

Results were reported as mean ± standard deviation of five independent biological replicates (five independent fruit). Analysis of variance (ANOVA) followed by Tukey’s tests were used to assess statistically significant differences among treatments (p < 0.05). Pearson correlation analysis using all evaluated variables was carried out. All statistical analyses were performed with Statgraphics Centurion XV (Stat Point Technologies, Inc., Warrenton, VA, USA). Principal component analysis (PCA) and hierarchical clustering analysis (using Euclidean distance and the Ward algorithm) were performed on the normalized data using MetaboAnalyst 4.0 (Xia Lab, McGill University, Montréal, QC, Canada).

3. Results and Discussion

3.1. Enzymatic Antioxidant System

The results of the activities of CAT, SOD, and POD enzymes involved in the antioxidant defence system at harvest, during cold storage, and at edible ripeness are presented in Figure 1 and Supplementary Table S1. CAT activity did not show significant differences (p > 0.05) for most of the evaluated samples, however, a tendency to increase was observed at edible ripeness, only being significant (p < 0.05) for R20d and SLR20d samples. A different behaviour was observed for SOD and POD enzymes, as lower values of enzyme activities were observed in both cases in ripe fruits (p < 0.05), with respect to those determined at the exit of cold storage. Balois-Morales et al. [23] point out that it is at edible ripeness where the symptoms of oxidative stress (appearance of physiological disorders) are most evident, due to the accumulation of ROS, such as superoxide anion (O2^•-), and peroxide (H₂O₂), among others. Similarly, antioxidant enzymes such as SOD, CAT, and POD regulate ROS by maintaining O2^•- and H₂O₂ at low levels [10].

In the different cold storage times evaluated in this study, CAT, SOD, and POD in general, did not show major changes, which would indicate that the enzymes remained inactive or their action was limited by the imposed cold and restored at a higher temperature (shelf-life conditions). In addition, the enzymes could have been activated and/or inhibited by the appearance of ROS, presenting different responses associated with the mechanism and conditions of action of the enzymes studied. In accordance with the results found, it has been reported that SOD activity decreases in response to cold stress, as well as when final ripening is reached as part of the fruit senescence process [24,25]. The effect of chilling on SOD activity is also influenced by the duration of cold exposure and the temperature regime employed [26]. SOD catalyzes the dismutation of two O2^•- molecules into H₂O₂, which is subsequently transformed by the action of other enzymes such as CAT or POD. The low values of SOD activity found at edible ripeness would indicate that they acted as a defence against oxidative stress, transforming O2^•- to peroxides, and probably the accumulation of these, as well as their by-products, could have contributed to the decrease in their activity. High concentrations of H₂O₂ that behaves as strong inhibitor of SOD activity has been reported [27].

As with SOD, low POD activities were observed at edible ripeness; although POD removes peroxides, excessive accumulation of peroxides could also have affected its action. Similarly, Kolodyaznaya et al. [28] reported a decrease in POD during ripening of avocado cv. Fuerte during storage and associated this decrease with the inhibitory effect of peroxide. Likewise, the same authors reported increases in CAT activity in avocado cv. Fuerte during prolonged storage times. Zhang et al. [29] in avocado cv. Booth reported an increase in CAT under shelf storage at 20 °C. Activation of physiological and biochemical processes leads to the changes in the activity of catalase and peroxidase in avocado fruit during storage [25]. Switala and Loewen [30] reported that CAT is more effective in H₂O₂ degradation than POD, but CAT has low affinities for H₂O₂ compared to POD, however, CAT tolerates high peroxide concentrations and shows high activities at these concentrations, the latter would be an indication of why the CAT value would show a tendency to increase in activity at edible ripeness.

3.2. Non-Enzymatic Antioxidant System

As a measure of the action of the non-enzymatic antioxidant system, hydrophilic (TPC and H-AOX) and lipophilic (total carotenoids, tocopherols, and L-AOX) antioxidants were evaluated and the results are presented in Figure 1 and Table S1. TPC did not show differences between recently harvested and cold stored avocados at the different times evaluated, but a tendency to increase in quantity was observed at edible ripeness, especially in prolonged cold stored avocados (30 d, SLR30d). Increases in TPC in Hass cultivar at edible ripeness have been reported previously [31,32,33]. Regarding H-AOX values, as for TPC, there were no significant differences (p > 0.05) among all the conditions evaluated.

With respect to lipophilic non-enzymatic antioxidants, in general, the total carotenoid (TC) content in avocado at harvest and during cold storage did not show significant changes, however, a non-significant (p > 0.05) tendency to increase was observed at edible ripeness, possibly related to the arrival of edible ripeness or due to the requirement of its antioxidant action. Carotenoid development occurs when the chloroplast transforms into chromoplast during ripening, resulting in the synthesis of several new carotenoids that are not present in green fruit [34]. In Hass avocado, it has been found that lutein (predominant carotenoid) showed a slight increase during this stage, the opposite case occurred with β-carotene (minor carotenoid) [4]. Stahl and Sies [35] pointed out that carotenoids are most likely involved in the scavenging of two of the ROS, the singlet molecular oxygen and peroxyl radicals.

The profile of tocopherols in order of concentration for all samples corresponded to: α-tocopherol > β-tocopherol > γ-tocopherol. In this study, α- and β-tocopherols remained at almost constant concentrations, with no significant changes (p > 0.05) in all the samples evaluated. The γ-tocopherol content was noteworthy as its concentration decreased in the 30 d cold stored samples and then remained constant until edible ripeness. With respect 10 and 20 d cold stored samples, it was observed that as fruit ripen or reached edible ripeness, their values tended to decrease compared to their cold stored counterparts. The decrease in γ-tocopherol could be related to its action as an antioxidant against the undergoing oxidative stress. Tocopherols are recognized as potent lipophilic antioxidant compounds. It has been stated that antioxidant activity of tocopherols in lipids follows this order: γ > δ > α > β [36]. Campos et al. [33] state that changes in tocopherol content are associated with the antioxidant defense system and the presence of phenolic compounds, through the regeneration of partially oxidized polyphenols, under oxidative stress conditions. No significant changes (p > 0.05) were observed in L-AOX during cold storage and at edible ripeness.

Finally, during the ripening process, avocado undergoes many physiological and biochemical changes, including the biosynthesis and accumulation of pigments, lipids, vitamins, and antioxidants, both hydrophilic and lipophilic [37]; therefore, the presence of these pigments and lipids as a whole and according to their nature and mechanism of action would be the response to the L-AOX or H-AOX values found.

3.3. Other Markers of Oxidative Stress

The determination of PPO and LOX activities and the measurement of TBARS were considered to evaluate other responses originating from the occurrence of the oxidation process in the samples. The results are shown in Figure 2 (the total values can be observed in Supplementary Table S2).

The performance of the PPO and LOX oxidative enzymes in avocado products is considered negative since they turn the fruit quite unstable. [38]. In this study, PPO activity increased significantly (p < 0.05) at day 30 of cold storage, and for all three periods evaluated, high values of PPO activity were obtained at edible ripeness. Because of oxidative stress, tissues are damaged and phenolic compounds are exposed and oxidized by PPOs, resulting in the appearance of dark coloration (presence of melanin, pigment of high molecular mass, and dark color) [39]. LOX instead did not show significant changes under the conditions evaluated. This group of enzymes catalyze the oxidation of polyunsaturated fatty acids (PUFA, such as linoleic and α-linolenic acids) promoting the initiation and propagation of the free radical chain reaction [40], with the consequent formation of by-products, among them, hydroperoxides, originating aldehydes, and alcohols responsible for off-flavors, producing an organoleptic degradation. The role of LOXs in plant senescence has been suggested. Rosahl [41] pointed out that loss of membrane integrity, an inherent feature of senescence, is thought to occur via lipid peroxidation, a process that is envisaged to be initiated or promoted by free radicals or enzymatically by LOXs [42]. The constant LOX activity could be due to the low substrate content, i.e., PUFA; since the most important lipid fraction of avocado is mainly composed of MUFA and SFA [33]. Another indicator of the development of oxidative stress via lipid peroxidation corresponds to the determination of TBARS. As can be observed, this value showed significant increases (p < 0.05) at edible ripeness, reaching the highest value in ripe avocados that were cold stored for 30 d, suggesting that lipid peroxidation occurred because of fruit ripening and senescence, as well as the effects of cold storage. Bill et al. [43] mentioned that avocado storage at low temperatures generates cell damage, altering membrane fluidity, inducing peroxidation and decomposition of membrane lipids, caused by the accumulation of ROS.

Finally, the pulp browning area (%) was evaluated in the avocados that presented this physiological disorder (Figure 3, Table S2), and it should be noted that not all ripe avocados showed browning. For the cold storage period evaluated (10, 20, and 30 days) and later ripening at 20 °C, 8.0, 12.0, and 20.0% of fruits, respectively, showed very slight browning (with an area of 0.76, 4.13, and 5.11%, respectively). The browning found in the mesocarp could be the result of the exposure of the fruit to cold, especially at longer times, affecting the integrity of the membranes and thus favoring the contact between PPO and polyphenols, promoting browning reactions. Chilling injury symptoms can occur after 4 weeks of cold storage at about 6 °C, depending on different factors such as maturity stage and growing conditions [44], such damage is closely related to the establishment of oxidative stress. In the present study, temperatures and storage times that could limit the occurrence of this damage were imposed, although it did occur, albeit to a low degree, thanks to the timely action of the antioxidant defense system. Among the external pre-harvest factors that could contribute to the appearance of stress are the degree of physiological maturity of the fruit, the origin, harvest time, fruit size, fruit composition, and crop nutrition. For instance, low concentration of calcium in avocado tissues affects the stability of their cell membranes, making them more sensitive to low temperatures, producing an increase in the incidence of physiological disorders (e.g., chilling injury) [45].

3.4. Multivariate and Correlation Analysis of Enzymatic and Non-Enzymatic Systems and Oxidative Stress Markers

Principal component analysis (PCA) for the initial sample (at harvest) and the 10, 20, and 30 d cold stored (R10d, R20d, and R30d) and at edible ripeness samples (SL10d, SL20d, and SL30d) is presented in Figure 4. The score plot (Figure 4a) explained 52.9% of the total variance in two principal components. Two well-marked clusters can be observed, cluster 1 encompasses the initial (IS) and cold stored samples for 10 and 20 d (R10d and R20d), while cluster 2 grouped all the samples at edible ripeness. The heat map based on cluster analysis (Figure 4b) revealed differences in the parameters evaluated for the different samples. Thus, IS, R10d, and R20d samples presented higher contents of L-AOX, γ-tocopherol and SOD and POD activities, while the SL10d, SL20d, and SL30d samples displayed higher CAT and PPO activities and TC and TBARS contents, and they presented higher incidences of pulp browning. These results show that the oxidative stress generated between cold storage exit and edible ripeness, SOD and POD would have participated and could have seen inhibited their participation as antioxidants at high concentrations of peroxides generated. The actions of these enzymes would have been supported by the non-enzymatic defense system mainly γ-tocopherol, also present in low amounts, supporting its consumption and important participation. For the other tocopherols (α- and β-tocopherols) this trend was not observed. The higher TC contents might be a consequence of their synthesis as a requirement to support the greater amounts of oxidative species. Under this same condition, a higher activity of CAT was also found potentially acting as an antioxidant enzyme due to the limited activity of SOD and POD, trying to stop the imbalance presented before the generation and accumulation of oxidation products, which is evidenced by high values of PPO and TBARS. The greater presence of TPCs, especially in SRL20 and SRL30d, would be associated with their prior synthesis to act as antioxidants in the advanced stages of oxidative stress, to which could be added their regeneration by the action of tocopherols, especially γ-tocopherol.

Pearson correlation analysis (r) (Figure 5 and Supplementary Table S3) revealed moderate and positive correlations between SOD and POD, TC and α-tocopherol, TPC and H-AOX, PPO and TBARS, browning and TPC, and browning and TBARS (ranged from 0.5587 and 0.7825) as well as moderate and negative correlations between SOD and PPO, SOD and TBARS, CAT and POD, POD and PPO, browning and SOD, and browning and POD (ranged from −0.5246 and −0.6275).

4. Conclusions

The enzymatic and non-enzymatic antioxidant defense systems evaluated in Hass avocado stored at 7 ± 0.5 °C for up to 30 days revealed participation in restraining the oxidative stress manifested between the time the fruit was removed from cold storage and reached edible ripeness. As lines of defense against the oxidative stress presented, the participation of SOD and POD is deduced, but it seems that their activities were limited in the presence of ROS generated by the action of the enzymes, with CAT acting under this situation. In addition, our results revealed that within the non-enzymatic antioxidant system, γ-tocopherol would have been very active, as it was found in low amounts due to its consumption when facing oxidative molecules or supporting the stabilization of TPCs. The higher TC contents would account for its synthesis to help slow down oxidation. Lipid peroxidation was also present and was detected by increases in TBARS values. The physiological disorder was observed with the increase in PPO activity and with the appearance of small brown areas at the pulp level, thus accounting for the joint and important action of the antioxidant defense of avocado to prevent further damage to the fruit. It is not excluded that other pre-harvest factors, independent of the chilling condition to which Hass avocado was subjected may have also contributed to the oxidative stress developed.