3.1. Effect of Antioxidant Treatment on Browning Index
The treatments CT, AA, and CA showed BI within the acceptable limit until the 21st day, and no significant differences were observed between them (
Figure 1A). There was an increase in BI of the leaves for the OA and EDTA treatments starting from the 9th day, which both were discarded on the 18th day, because the BI exceeded the commercial limit of browning.
The color deterioration kinetics in our research were in accordance with previous reports, such as MPP lettuce cultivars, which exceeded the visual quality limit in the 12th day of storage at 5 °C, exhibiting symptoms of severe darkening on the surfaces and cut areas of the leaves [
28]. Among several factors that caused these changes in color, the most significant were enzymatic activity and pigment degradation [
5].
The total phenolic compounds (TPC) (
Figure 1B) showed variations during storage days. TPC content in samples ranged from 48 to 117 mg GAE 100 g
−1 FW, similar values to those obtained by Tiveron
et al. [
13] in several leafy vegetables. From the 9th day of storage, the highest values (
P < 0.01) of TPC were observed in EDTA samples, and at the end of the experiment, CT and the treatments AA and CA showed the highest values. EDTA as a complexing and chelating agent may have maintained higher TPC values up to the 9th day.
Variations in TPC of MPP may occur due to many aspects such as prolonged storage, temperature, and processing. After a cut in processing, the reactions of the defense mechanisms of the vegetable tissue caused the consumption of the phenolic compounds as substrates by defense enzymes; free conversions can also occur between phenolic groups and their bonds [
29,
30,
31,
32]. The application of antioxidant products in MPP affects the TPC, that may increase or decrease, depending on the chemical action and its concentration [
10,
33].
There were variations in the PPO activity (
Figure 2A) during storage. Differences (
P < 0.01) were observed among the treatments starting from the 6th day; more specifically, samples submitted to OA and EDTA treatments showed reduced activity of this enzyme. At 12 and 15 d, the lowest PPO activity observed was for the CA and EDTA treatments. On the last day of the experiment, CA showed a lower PPO activity than AA and the control. The activity of the PPO in MPP escarole varied between 0.10 and 0.90 U mg
−1 protein, and can be characterized as very low compared to the values obtained in lettuce cultivars, which may vary between 14 and 28 U mg
−1 protein [
34].
PPO is an oxidoreductase enzyme with copper in its active site; in the presence of oxygen, it catalyzes the hydroxylation of monophenols into
o-diphenols, which are subsequently oxidized into
o-quinones. These quinones can be polymerized or combined with amide compounds and carbonyl-amine or react with amino acids or proteins, thus forming dark colored, low molecular weight compounds [
10,
35]. EDTA as well as CA and OA are chelating agents, and they act on PPO, complexing with copper in its active site. The efficacy and mechanisms of action of various antioxidants depend on the vegetable species and cultivar, since PPO may use different phenolic compounds as substrates [
8].
There were peaks in the POD activity (
Figure 2B) in the escarole leaves during storage. In most of the storage period, the POD activity was higher (
P < 0.01) in CT samples until the 18th day, and this treatment did not differ between AA and CA through the end of the experiment. The POD activity varied from 0.47 to 3.13 µmol guaiacol min
−1 mg
−1 protein, higher than the variation obtained in lettuce of 0.10 to 0.35 µmol guaiacol min
−1 mg
−1 by Altunkaya and Gökmen [
36].
POD in the presence of H
2O
2 oxidizes phenolic compounds that are hydrogen donors [
37,
38]. When oxidized, the phenolic compounds become dark. The ascorbic acid and citric acid control the activity of POD by reducing the intracellular pH. However, this reduction depends on the agent and the pH of the solution [
39]. Some studies have shown that AA has a temporary antibrowning inhibitory effect because it does not act directly on the enzyme; rather, it reduces the
o-quinones into their precursor forms of diphenols, and, in the process, the AA is converted into DHA [
10,
40]. On the other hand, CA reduces pH, has a chelating function, and acts directly on the enzymes [
41]. There are some contradictions in the literature about the POD function in browning of MPP [
7,
39]. It has been hypothesized that the formation of quinones generated by PPO can lead to the accumulation of significant levels of H
2O
2, thus allowing for the oxidation of polyphenols by POD enzyme [
42,
43].
The protective action of AA on TPC can be attributed to reduction reactions and regeneration of polyphenols, while CA reduces their oxidation due to its acidifying and chelating action on enzymes that use these compounds as substrates [
36,
44]. There are controversies regarding the involvement of phenolic compounds and the activity of enzymes in browning of MPP. Several papers have reported that there is no clear relationship between phenolic content, ascorbic acid content, and activities of PPO and POD with browning of the leaves in different cultivars of MPP lettuce [
20,
45,
46]. The browning observed in MPP escarole in our research may have been caused by the degradation of chlorophyll, and microbiological growth and weight loss, in addition to enzymatic activity. This hypothesis was supported by the correlation between the values of the traits (
Table 1).
3.2. The Conservation of Endogenous Ascorbic Acid Prevents Pigments Loss
Ascorbic acid content (
Figure 3A) decreased in all treatments during storage. The AA and CA treatments showed the highest retention (
P < 0.01) of endogenous ascorbic acid content during storage. The OA and EDTA treatments showed values similar to the control. The ascorbic acid values in this study varied from 23.2 to 13.6 mg 100 g
−1 FW during storage. Escarole has high ascorbic acid content if compared with lettuce cultivars, which may vary from 1.5 to 12.3 mg 100 g
−1 FW [
11]. Both AA and CA may have acted synergistically in the preservation and increasing of ascorbic acid content. In several studies, it has been reported that the increase and better retention of endogenous ascorbic acid for MPP occurred when treated with AA solutions [
4,
17].
Studies on the biochemical mechanism of browning in MPP leafy vegetables have reported the possible involvement of a high endogenous content of ascorbic acid as a natural antibrowning agent [
39,
46,
47]. The degradation of ascorbic acid may be related to non-enzymatic browning, due to its oxidation into DHA. The DHA can subsequently react with aldol or amine groups, which are responsible for condensation of the quinone and conversion into dark compounds [
48]. DHA levels have been positively correlated with browning in lettuce [
49]. Vegetables with high endogenous ascorbic acid content are able to control the accumulation of reactive oxygen species (ROS) effectively. AA is a more efficient agent against the action of ROS than phenolic compounds, which controls only part of the ROS formed [
50].
CA was the most effective treatment for the retention of chlorophyll content (
Figure 3B) and carotenoids (
Figure 3C), showing the highest values (
P < 0.01) of these pigments during storage. The total chlorophyll values in the MPP escarole were similar to those found in rocket and baby spinach (40–50 mg 100 g
−1 FW) [
9]. The carotenoid levels obtained were lower than those observed in lettuce, equivalent to 2.0 mg 100 g
−1 FW [
51].
There was a positive correlation between the AA content of samples and chlorophyll and carotenoid content with
r = 0.872 and
r = 0.866, respectively (
Table 1). CA and AA have exhibited similar mechanisms against pigment deterioration, reducing chemical and biochemical reactions that oxidize chloroplast structures [
52]. The application of exogenous CA acts synergistically with the endogenous AA content of tissues. Considering that citric acid controls PPO, there is less demand for the consumption of ascorbic acid, which ends up being used for other purposes, as in its competitive action in the enzymatic reactions that result in brown compounds, thus preventing the loss of color and pigments [
33,
53].
3.3. Influence of Treatments on Escarole Metabolism
There were differences in O
2 (
Figure 4A) and CO
2 (
Figure 4B) concentrations inside the package between treatments during storage. The treatments OA and EDTA had significantly lower CO
2 concentrations (
P < 0.01) starting from the 12th day and significantly higher concentrations of O
2 (
P < 0.05) throughout the entire storage period than the other treatments, reaching values close to that of air (20.9%). This might have accelerated the metabolic processes of these samples, increasing the deterioration rate and thus the BI, explaining in part why they were discarded earlier than the others.
The reduction of the respiratory rate by the application of OA is associated with the decrease in metabolic activity reported in MP rocket and spinach [
9]. The effect of EDTA on the respiratory rate has not been fully elucidated, but this treatment could have reduced MPP escarole respiration through its chelating action on lipids, inhibiting oxidation [
54,
55].
The samples treated with AA had the lowest (
P < 0.05) O
2 concentration (17.6%) until the 21st day, during which no significant differences were observed. AA and CA treatments maintained the highest CO
2 values (
P < 0.01) until the 21th day, when they differed from the CA. The samples immersed in CA showed similar CO
2 reduction inside packages to the OA and EDTA treatments. CA application may cause reactions in the cytosol, reducing phosphofructokinase activity, thus also reducing glycolysis and respiration rate. The decrease in glycolysis may also be associated with the reduction of intracellular pH caused by CA [
56].
The weight loss (WL) was higher (
P < 0.01) for both OA and EDTA treatments, which reached percentages of 0.66 and 0.59%, respectively, at the 15th day (
Figure 4C); this might be explained due to the higher concentration of O
2 contained inside the headspace of these samples, which could have accelerated metabolism and senescence processes. There were no differences among the other treatments during storage. The values of WL in our research were lower than previous studies of application of organic acid treatments in MPP leafy vegetables, which have varied from 0.5 to 4.2% [
2].
We observed a relationship between WL and the other parameters (
Table 1). WL has a high correlation with several quality factors in fruits and vegetables, such as appearance, texture, and nutritional value. There was a positive correlation between WL and BI; when WL increased, there was a reduction in firmness and increase of wilting and darkening of tissues [
57]. A negative correlation with ascorbic acid content, chlorophyll, and carotenoids was also verified. At a very high degree, the loss of water could rupture the cell wall, which would cause the decompartmentalization and consequently the contact of the enzyme ascorbate oxidase with the ascorbic acid, thus decreasing the content of this acid [
58].