**4. Discussion**

Low-temperature storage is used extensively to keep the quality and prolong the shelf life of horticultural products after harvest. Nevertheless, because of their sensitivity to cold stress, the utilization of low-temperature storage has some limitations for chilling sensitive horticultural products, such as subtropical and tropical crops [21]. Chilling-induced stress changes the balance between the ROS-forming system and defensive mechanisms, resulting in oxidation-induced CI [10].

In the present study, HWT-1 min was effective in alleviating CI and retarded the development of CI symptoms in red sweet pepper fruit as compared to control and the longer treatment times, HWT-3 min and HWT-5 min. CI symptoms presented in the same period of storage (week 2); however, the level of damage differed. Finally, the CI severity in HWT-1 min fruit was slight; however, in the control, HWT-3 min, and HWT-5 min fruit, damage was moderate to severe (Figures 1 and 2). Previous studies also found that hot water immersion modified responses to cold stress, delaying the onset of CI, and thus has been proven to alleviate CI in fresh produce, such as sweet pepper [13,36], mumes [18], plums [37], and cucumbers [16].

Weight loss in untreated red sweet peppers was less than in those treated with hot water, except for HWT-1 min fruit. Hot water immersion increased fruit weight loss in some fresh produce, such as mandarins [38], which lost more weight than the control during cold storage. From this study, short exposure to hot water (HWT-1 min) reduced weight loss (Figure 3), probably as a result of the stimulated recrystallization or molecular orientation of the waxes on the cuticle layer, which plays a significant role in regulating the water loss and preserving fruit firmness [16,39]. However, red sweet pepper fruits exposed to hot water for a longer time (HWT-3 min and HWT-5 min) lost more fresh weight, which might be due to tissue damage (Figure 2). Although hot treatment may be of benefit to hot water-treated horticultural products, excessive heat, such as revelation of products to lethal temperatures or an extended treatment duration, might induce cell damage [40]. Excess heat stress may cause the destruction of cells, the loss of membrane integrity, and the elimination of the wax coating the outer surface of the cuticle, resulting in higher loss of water.

In previous studies, Fallik et al. [41] found serious heat injury in sweet pepper when hot water treatment at 55 ◦C was applied, and even observed severe fruit damage when treated at 52 ◦C for 2 min [4,41]. Thus, they [24] suggested 55 ± 1 ◦C for only 12 s to maintain fruit quality during prolonged storage. In contrast similar with our results (Figure 4), Shehata et al. [42] found that the high-temperature treatment (55 ◦C for 1 min) provided a good appearance without visible injury and decay in pepper fruits during cold storage. The difference of heat tolerance in fruits depends on species, genotype, stage of fruit maturity, type and severity of heat treatment applied, and the preconditioning treatments before heat treatment [43].

Cell membrane integrity is most affected by CI. In the cell membrane, the transition from the flexible liquid crystalline phase to a solid gel structure phase is caused at cold storage temperatures, which increases the malfunction of cell membranes [44]. When horticultural product is exposed to damaging temperatures below a certain threshold temperature, cell membranes break, resulting in the leakage of ion, metabolites, and intracellular water, which can be traced as electrolyte leakage [21,45]. Electrolyte leakage in fruits treated with hot water immersion differed depending on the exposure time. During storage at 10 ◦C, brief hot water immersion (HWT-1 min) showed lower electrolyte leakage because mild heat treatment may reduce chilling stress; however, 5 min (HWT-5 min) was an excessive heating time that triggered cell damage and presented the highest leakage (Figure 5). Similarly, treated cucumbers (55 ◦C, 5 min) had lower electrolyte leakage than untreated fruit, thus hot water mitigated CI by maintaining membrane integrity [16].

Lipid peroxidation is the initial phenomenon caused by CI. Cold stress alters the structure of the plant cell membrane, causing membrane integrity to deteriorate due to lipid peroxidation, which is measured as the MDA level. MDA is the oxidative secondary metabolite of polyunsaturated fatty acid in cell membranes; it is a suitable indicator of oxidative destruction to cell membrane integrity under temperature stress, and its level is a useful indicator of the oxidative stress level [9,11,21]. Our result found that the MDA level in red sweet pepper fruit immersed with HWT-1 min was lower than that with prolonged heat exposure (3 and 5 min) and in the control (Figure 6). The low level of MDA and electrolyte leakage in HWT-1 min fruit reflected the lower degree of CI and the incidence of CI in red sweet peppers during cold storage for 4 weeks.

Hydrogen peroxide is a strong oxidant, a relatively long-lived molecule, and it is moderately reactive. It is produced by the superoxide dismutase that catalyzes a superoxide radical to H2O2. Environmental stresses can cause the production of H2O2; however, excessive H2O2 can cause oxidative damage in plant cells by disrupting metabolic processes and affecting cell membrane integrity [13,46]. In this study (Figure 7), when comparing different times of heat exposure (55 ◦C for 1, 3, and 5 min), HWT-1 min fruit had lower H2O2 contents; this remained constant until week 3 and then rose significantly by week 4. Prolonged heating times (HWT-3 min and HWT-5 min) tended to overheat red sweet peppers, causing higher oxidative stress, which was demonstrated by a higher accumulation of H2O2 from week 2 until the end of storage (Figure 6). H2O2 responds to oxidative stress as a signal in the cell compartments that it originates from, leading to an applicable response in the cellular protection system [12]. In low levels, H2O2 reacts as a signal molecule involved in acclimatory signaling, triggering tolerance to several stresses; in contrast, in high levels, it acts as a providing factor to stress damages and causes cellular destruction [13,47,48]. HWT-1 min could stimulate H2O2 production to a level that triggered the defense mechanism responses, involving enzymatic and non-enzymatic antioxidative scavenging systems, promoting tolerance of subsequent cold storage. Previous study indicated that temperature stresses reacted to antioxidative scavenging systems in plants. Moderate heat treatment of horticultural produce induces mild oxidative stress, which affects the antioxidant condition and induces tolerance to subsequent stress [18]. The oxidative function of H2O2 is intimately linked to the redox state in plant tissues. The redox balance is associated with the expression of genes that contribute to stress resistance, stress acclimation, and defense systems [12].

Heat treatment of horticultural crops before storage not only increases their resistance to heat stress but also improves their tolerance to other various stresses [15]. Temperature stress altered homeostasis in plant cell and major processes in its physiological functions, resulting in the accumulation of ROS levels. ROS may potentially play a function in signal transduction processes that activate stress-response pathways and trigger defensive systems [49]. Mild heat treatment causes slight stress in fresh products, stimulating antioxidant responses in both enzymatic and non-enzymatic systems [11]. Non-enzymatic systems include antioxidant substances, such AsA and GSH, whereas antioxidant enzymes involve superoxide dismutase (SOD), CAT, and AsA-GSH cycle-related enzymes (APX, DHAR, MDHAR, and GR) [9,11]. The AsA-GSH cycle consists of an antioxidant and detoxifying system against ROS that has a significant impact on the resistance to chill damage during postharvest storage [50]. Both AsA and GSH are the significant antioxidants in plants that play important roles in stress resistance. High AsA and GSH contents may be responsible for improving performance during postharvest cold storage [51]. AsA is known to be the most powerful antioxidant substance and plays a critical role in maintaining APX activities to detoxify H2O2 in the AsA-GSH cycle [48]. In the current study (Figure 8A), the content of AsA in HWT-1 min fruit was gradually decreased but maintained at a higher level than those in HWT-3 min, HWT-5 min, and control fruits during the storage at 10 ◦C. The decreasing trend of DHA contents in HWT-1 min fruit was similar to that of AsA contents (Figure 9B). Prolonged exposure to hot water (3 min and 5 min) decreased the AsA and DHA content. Hot-water dipping has also been observed to increase the AsA level in other fresh products, including tomatoes [9], zucchini [17], and mumes [18]. The elevated redox potential in AsA may correspond with the chill acclimation of hot water-treated fruit. The increased activity of the AsA metabolism system may indicate a key role in lowering H2O2 levels throughout low-temperature storage [18]. In HWT-1 min fruit, the high level of AsA was reflected in the lower content of H2O2 during the initial 3 weeks of storage (Figure 7).

Glutathione has a crucial function in the antioxidative defense mechanism, regenerating AsA from its oxidized form, DHA, throughout the AsA-GSH cycle [48]. In this study (Figure 9), the GSH level in HWT-1 min fruit increased after 1 week of storage and

significantly decreased at week 4; throughout the storage period, the content remained higher than in fruits treated differently. Hot water treatment for 1 min increased chilling tolerance by the increase of the GSH production and the GR activities.

The accumulation of ROS induced by various stresses is mitigated by enzymatic protection systems, such as SOD, APX, GPX, and CAT, and non-enzymatic protection systems of low molecular substances, such as AsA, GSH, α-tocopherol, carotenoids, and flavonoids. ROS disturbs many functions of cellular metabolism by injuring nucleic acids, oxidizing proteins, and producing lipid peroxidation [48]. Many processes in plant metabolism may indicate different optimal temperatures, and their related enzymes may be less heat unstable, so they respond differently to the stresses of temperature [18]. CAT is one of the main enzymes that scavenges ROS by decomposing H2O2 into water and oxygen. The increase in CAT activity is considered to be the adapted characteristic that assists in the disposal of H2O2. Abiotic stresses either increase or decrease CAT activity, depending on the strength, period, and kind of stress [12]. In this result (Figure 10), hot water treatments were less effective at stimulating CAT activities. CAT activity in red sweet peppers treated with immersion of hot water (55 ◦C for 1, 3, and 5 min) and untreated fruit tended to decline during cold storage. Similar trends were indicated in mature green mumes [18], which were treated with immersion of hot water (45 ◦C for 5 min) during low-temperature storage for 4 weeks. Cold stress might cause the downregulation of abundant CAT enzyme protein and the decrease in CAT activity in red sweet peppers during cold storage.

Ascorbate peroxidase has an important role in the decomposition of H2O2 and the control of H2O2 levels in cellular compartments. In the present study (Figure 11A), although HWT-1 min did not significantly increase APX activities, they were higher than those in fruits treated for prolonged times (3 and 5 min) and the control. HWT-1 min might be a modest heat exposure that could induce APX activity to maintain low levels of H2O2. APX is considered to play a role in the defensive mechanism against CI development during cold storage, and to enhance the tolerance to cold storage, as well as having a more important action in the ROS control or being responsible for regulating ROS signaling [13].

Ascorbate is a powerful antioxidant that can directly detoxify free radicals. It comes in two oxidized forms: MDHA and DHA. In living organisms, the AsA recycling metabolic system is significant for preserving AsA homeostasis against exogenous stimuli. DHAR and MDHAR are two major AsA recycling enzymes and are members of the AsA-GSH cycle [52]. DHAR catalyzes the recycle action of DHA to AsA, utilizing GSH as a hydrogen donor prior to the spontaneous hydrolysis of DHA to irreversibly form 2,3-diketogulonic acid. On the other hand, MDHAR uses reduced ferredoxin or NAD(P)H as an electron donor to recycle MDHA prior to the spontaneous oxidation of MDHA to form DHA [44]. The activity of MDHAR in HWT-1 min fruit highly enhanced and about doubled after 2 weeks of storage but reduced subsequently (Figure 11B). DHAR activity in HWT-1 min fruit slightly increased throughout the first 3 weeks of storage and reduced thereafter (Figure 11C). The levels of both DHAR and MDHAR activities in HWT-1 min fruit were higher than those in HWT-3 min, HWT-5 min, and control fruits. The increase of DHAR and MDHAR activities indicates accumulative responses to cold stress. In a previous study, the higher H2O2 level produced by heat treatment promoted DHAR and MDHAR activities [13]. Temporally, an increase in gene expression of DHAR and MDHAR was induced in H2O2 accumulation caused by heat treatment [53]. H2O2 responds as a molecular-signaling secondary messenger of metabolic control, and regulator of gene expression, increasing responses and activation of protection pathways to various stresses. Thus, the higher activities of DHAR and MDHAR enzymes play a significant role in alleviating CI.

Glutathione reductase is a key enzyme in the AsA-GSH cycle and catalyzes the conversion of GSSH to GSH by utilization of NADPH as an electron donor. It also plays a crucial role in the defense against ROS by maintaining the reduction state of GSH and the AsA pools [13]. GR activity in HWT-1 min fruit sharply increased and was higher than those in HWT-3 min, HWT-5 min, and control fruits throughout the storage period (Figure 11D). High GR activity induced a larger GSH pool and maintained the AsA-GSH cycle, and consequently enhanced the AsA pool [54]. Therefore, higher levels of GSH content and GR activity imply an influence on chilling tolerance and cold acclimation. The GR has a significant action in resistance systems for chilling stress.

#### **5. Conclusions**

The findings of our study revealed that pre-treatment with immersion of hot water at 55 ◦C for 1 min alleviated CI in red sweet pepper fruit during the chilling storage duration, which may be due to the increase of the AsA-GSH cycle by enhanced antioxidant contents of AsA and GSH and the activity of antioxidant-related enzymes. During cold storage, chilling resistance was improved, and the onset of CI was delayed and mitigated in HWT-1 min fruit, as illustrated by low H2O2 levels, whereas prolonged exposure to hot water (3 and 5 min) caused cellular damage, as showed by increases in weight loss, the CI index, the level of electrolyte leakage, and the MDA level. The non-damaging heat condition of hot water at 55 ◦C for 1 min can allow cold storage at storage temperatures lower than the optimal temperature. This hot water treatment in the present study is a safe treatment, technologically easier, cheaper, and more feasible for suitable postharvest treatment.

**Supplementary Materials:** The following are available online at https://www.mdpi.com/article/10 .3390/foods10123031/s1, Figure S1: Effect of hot water treatment (55 ◦C for 1, 3, 5 min) on *L\**, *a\**, *b\** color in red sweet pepper fruit during storage at 10 ◦C.

**Author Contributions:** Conceptualization, Y.I. and J.K.; methodology, Y.I.; formal analysis, J.K.; investigation, Y.I.; data curation: Y.I. and J.K.; writing—original draft preparation, J.K.; writing review and editing, Y.I. and J.K.; supervision, Y.I. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research received no external funding.

**Conflicts of Interest:** The authors declare that they have no conflicts of interest for this article.
