Exogenous Application of Melatonin on the Preservation of Physicochemical and Enzymatic Qualities of Pepper Fruit from Chilling Injury

Abstract

This study investigates the impact of melatonin (MT) treatment at varying concentrations (0, 25, 50, 75, 100 µmol L⁻¹) on the post-harvest quality and shelf life of long green pepper fruits stored under low temperature for 28 days. Every 4 days, pepper fruits were examined for the chilling injury (CI) index, weight loss (WL), respiration rates, firmness, electrolyte leakage (EL), reactive oxygen species (ROS), malondialdehyde (MDA) levels, non-enzymatic antioxidant (NEA) content, antioxidant (AO) enzyme activity (superoxide dismutase (SOD), catalase (CAT), and peroxidases (PODs)), and cellular degrading enzymes (polygalacturonase (PG), pectin methylesterase (PME), phospholipase D (PLD), and lipoxygenase (LOX)). MT-treated samples exhibited delayed and reduced CI stress compared to controls, with higher exogenous MT concentrations (>50 µmol L⁻¹) offering significant (p < 0.05) CI reductions. During storage, WL was notably mitigated by MT treatment in the tested samples compared to control samples. This study also demonstrated that MT-treated pepper fruits effectively decelerated respiration rates and consequently preserved pepper firmness. A higher concentration of MT-treated pepper fruits demonstrated a significantly (p < 0.05) lowered level of ROS and MDA while maintaining membrane stability, as evidenced by reduced EL. MT treatment with increasing concentration increased the levels of glutathione (GSH), glutathione disulfide (GSSG), ascorbic acid (AsA), dehydroascorbate (DHA), and total phenolic content (TPC) in the pepper fruits compared to control and thus significantly (p < 0.05) suppressed the ROS production (superoxide anion (O₂^•−) and hydrogen peroxide (H₂O₂) radicals) in the pepper fruits. Furthermore, AO enzymes such as SOD, CAT, and POD were also high in the pepper fruits that were treated with higher concentrations of MT (>50 µmol L⁻¹). Additionally, the activities of cellular degrading enzymes (PG, PME, PLD, and LOX), which are linked to senescence and stress-induced physiological disorders, were also effectively regulated by MT-treated (>75 µmol L⁻¹) pepper fruits. Overall, the application of MT at higher concentrations (>75 µmol L⁻¹) demonstrated substantial benefits in preserving the quality and extending the shelf life of pepper fruits during cold storage.

1. Introduction

Long green pepper fruits belong to the Capsicum annuum L. species, which is part of the Solanaceae family and are recognized for their elongated, cylindrical shape, complete with a pointed and frequently curled tip, spanning a length of approximately 6–9 cm. The fruit’s outer skin is notable for its glossy texture and green color and the seeds of the fruit are flat and pale yellow colored [1]. Green pepper fruits are the most widely cultivated chili pepper species worldwide, producing a variety of peppers ranging from sweet to spicy peppers. The peppers’ green pigments are contributed by chlorophyll, while they transition to yellow-orange due to the accumulation of carotenes, zeaxanthin, lutein, and cryptoxanthin. The vibrant red color characteristic of certain chili peppers is primarily attributed to carotenoid pigments, including capsanthin, capsorubin, and capsanthin 5,6-epoxides [2]. The NEA in green pepper fruits varies according to several factors, including maturity level, genotype, and the timing of the seasonal breeding periods [3]. Studies have found that green pepper fruits contain significant levels of health beneficial chemical composition, including vitamins A, C, and E, as well as polyphenols [1]. These components impart the fruits with functional properties, including antioxidant, antifungal, and antibacterial activity [2]. However, the pepper fruits deteriorate quickly due to the physiological and biochemical changes induced by physical and microbial stimuli; severe alterations are also caused by inadequate and inappropriate post-harvest management, including dehydration, color loss, skin shriveling and texture softening, etc.

The primary cause of pepper fruit softening is its cell wall polysaccharides, and certain enzymes that hydrolyze the cell wall—like PG, PME, pectin lyases (PLs), β-galactosidases (β-Gals), endo-1, and 4-β-D-glucanases (EGases)—become extremely active and negatively alter the texture of the pepper fruits [4]. Furthermore, pepper fruit deterioration due to its weakened stress response system has also been widely reported, as shown by decreased activity of AO enzymes such as SOD, POD, and CAT, which are the robust antioxidant systems in plants that effectively slow down the senescence process [5]. Proper storage is crucial for preserving the freshness of green pepper fruits for extended periods. This involves keeping them at low temperatures combined with high relative humidity (RH) to ensure freshness during transportation and to prolong their post-harvest shelf life. Low-temperature storage could reduce the physiological, biochemical, and microbiological processes that cause quality degradation and preserve the nutritional value [1]. Fresh green pepper fruits may rapidly be perishable at ambient storage, as they lose moisture and induce microbial growth when stored for a long time. The optimum temperature for storing fresh green pepper fruits is 7.5–13 °C with a RH of 95–98%, and they may remain fresh for 2–3 weeks. Storing at temperatures above 13 °C accelerates ripening and stimulates the growth of microorganisms that might cause spoiling. CI can occur when green pepper fruits are stored at temperatures lower than 7.5 °C [6]. Pepper fruit CI symptoms include pericarp color changes, water-soaked areas, seed browning, electrolytic leakage, elevated respiration rate, increased peroxidation and its byproduct (MDA), and increased accumulation of ROS [7].

Melatonin (MT), chemically known as N-acetyl-5-methoxytryptamine, plays a significant role in controlling CI in various fruits and vegetables, mainly through its effects on membrane integrity, antioxidant activity, and metabolism regulation [8]. MT treatments have been shown to prevent membrane lipid peroxidation and maintain a higher ratio of unsaturated to saturated fatty acids, thus preserving the cell structure and function and, delaying the development of CI in fruits and other produce [9]. MT treatment could enhance the antioxidant defense system in plants by increasing the activity of SOD, POD, CAT, and the AsA-GSH cycle and thus increase the scavenging ability of ROS, thereby protecting the plants from oxidative damage caused by chilling stress [10]. Kong et al. [11] reported that MT treatment up-regulates the transcription level of genes associated with antioxidant enzymes in bell peppers and increases the content of proline, which acts as an antioxidant and thus effectively reduces the CI in pepper fruit. MT treatment up-regulates the expression of genes associated with polyamine, γ-aminobutyric acid (GABA), and proline content in harvested peaches, enhancing their chilling tolerance [8]. MT applied to the surface of green bell pepper fruits slows post-harvest softening by preserving cell wall structural integrity, membrane stability, and antioxidant system homeostasis [7]. The activities of the AO enzymes, especially CAT and POD, are increased by MT treatment at physiological levels; however, the amount of MT needed to achieve the best physiological activity depends on the type of plant and species. MT treatment for controlling the CI in bell peppers is limited, and the available studies are also limited to a single concentration [12]. Furthermore, Ahamad et al. [9] reported that the bell pepper fruits were dipped in various MT concentrations (70, 120, 170, 220 μmol L⁻¹) for 20 min and stored at 10 ± 1 °C for 20 days. They found that the bell pepper treated with 120 μmol L⁻¹ MT significantly suppressed respiration and weight loss, delayed chlorophyll degradation, and prolonged the postharvest shelf-life of the bell pepper by 20 days. Additionally, MT (120 μmol L⁻¹) treatment reduced loss of firmness and titratable acidity, and retained total phenol, flavonoid, and ascorbic acid content. It hindered malondialdehyde accumulation by enhanced DPPH radical scavenging and antioxidant enzyme activity like superoxide dismutase, catalase, and peroxidase. It is not sufficient to understand the exogenous application of MT on the preservation of physicochemical and enzymatic qualities of pepper fruit from CI.

In this study, we examined the efficacy of utilizing different doses of MT (25, 50, 75, and 100 µmol L⁻¹) as an external coating to maintain the physicochemical and enzymatic properties of fresh long green pepper fruits against CI. The primary goal was to determine the effectiveness of this treatment in extending shelf-life, maintaining fresh green pepper fruit quality, and measuring varieties of physicochemical and NEA parameters during internal storage under CI-inducing environmental conditions.

2. Materials and Methods

2.1. Chemicals and Reagents

All the chemicals and reagents (sodium hypochlorite and MT) used in Section 2.2 were of food grade, whereas the chemicals and reagents (potassium phosphate, dihydroethidium, calcium chloride, scopoletin, horseradish peroxidase, trichloroacetic acid, thiobarbituric acid, sulfosalicylic acid, triethanolamine, 2-vinylpyridine, sodium phosphate, ethylenediaminetetraacetic acid, nicotinamide adenine dinucleotide phosphate, 5,5′-Dithio-bis-(2-nitrobenzoic acid), dithiothreitol, N-ethylmaleimide, orthophosphoric acid, iron chloride, 2,2-dipyridyl, ethanol, Folin–Ciocalteu reagent, sodium bicarbonate, methionine, nitroblue tetrazolium, riboflavin, polyvinyl polypyrrolidone, hydrogen peroxide, polyvinyl pyrrolidine, sodium chloride, tris-acetate buffer, polygalacturonate, dinitro salicylate, sodium hydroxide, Tris-HCl buffer, phosphatidylcholine, chloroform, ammonium tetrarhodanatodiammonchromate, Tween 20, and linoleic acid) used in Section 2.3 were of analytical grade. All the chemicals and reagents in the above-mentioned sections were procured from Sigma Aldrich (Thailand) Co., Ltd., Bangkok, Thailand.

2.2. Plant Material and Treatments

Long green pepper fruits (Capsicum annuum L. (Capsicum annuum Linn. var acuminatum Fingerh)) were harvested fresh, 35 days post-anthesis, from a contracted garden in southern Thailand. The peppers were inspected for any apparent and/or microbial damage; damaged fruits were discarded, ensuring only undamaged ones were collected for treatment. Each selected fruit exhibited uniform color and maturity. After that, distilled water was used to thoroughly wash them to get rid of any dust or debris. Following the washing, the fruits underwent a disinfection process in a solution containing 0.5% sodium hypochlorite for 5 min. Subsequently, the fruits were organized into five groups. The control group received no further treatment (control). The treatment groups were exposed to varying MT concentrations—precisely, 25 (MT25), 50 (MT50), 75 (MT75), and 100 (MT100) µmol L⁻¹. The application of MT treatments involved an immersion method where pepper fruits from each MT group were submerged in their designated concentrations for 20 min. The control group underwent a similar submersion process but in distilled water only. After immersion, the fruits were air-dried using a portable electric fan for 30 min. Then, 20 fruits from each group were packed in low-density polyethylene bags (0.025 mm thickness), with each treatment receiving 15 bags per replication. These were stored at a temperature of 4 °C and 95% RH for 28 days. The fruits’ various qualities were assessed at 4-day intervals throughout storage (as detailed in Section 2.3).

2.3. Quality Analysis

2.3.1. Chilling Injury (CI) Index

The CI index was assessed on the surface of the pepper fruits by focusing on surface pitting irregularities [13]. The quantification of CI was accomplished by utilizing a grading system comprising six categories: 0 = absence of pitting; 1 = minimal (0% < damage < 10%); 2 = minor (10% < damage < 20%); 3 = moderate (20% < damage). The following equation was used to determine the CI index in the bell peppers:

$$\mathrm{CI} \mathrm{index} = \mathsf{\Sigma }\frac{ \left(\mathrm{CI} \mathrm{scale} \times \mathrm{number} \mathrm{of} \mathrm{fruit} \mathrm{at} \mathrm{the} \mathrm{CI} \mathrm{scale}\right)}{\mathrm{total} \mathrm{number} \mathrm{of} \mathrm{evaluated} \mathrm{fruit}}$$

(1)

2.3.2. Determination of Weight Loss (WL)

The WL of fresh green pepper fruits was measured in accordance with the method of Njie et al. [14]. WL was quantified with an electronic scale, with readings taken in grams. Every four days, the weight of 20 fruits from each treatment group was recorded and compared to their initial weight, and the results were calculated and expressed using the following formula:

$$\mathrm{WL} \mathrm{rate} (\%)=\frac{\left(\mathrm{Initial} \mathrm{weight}-\mathrm{Weight} \mathrm{at} \mathrm{sampling} \mathrm{day}\right)}{\mathrm{Initial} \mathrm{weight}}\times 100$$

(2)

2.3.3. Determination of Respiration Rate

The respiration rate of fresh green pepper fruits, quantified by the release rate of CO₂, was determined by following the methodology outlined by Chae et al. [15] with some modifications. In this process, 20 pepper fruits were initially weighed and then placed in plastic jars after being allowed to stabilize at 20 °C for one hour. These jars were then maintained at the same temperature for an additional hour. Subsequently, 1 mL of gas from each jar was collected using a syringe and analyzed through gas chromatography (GC; Auto Systems XL, Perkin Elmer, Waltham, MA, USA) utilizing an HP19001A-NO1 packed column. The analysis was conducted with the oven and inlet temperatures set at 100 °C, while the back inlet, front detector, and back detector were maintained at temperatures of 375 °C, 250 °C, and 150 °C, respectively. The rate of respiration was calculated and expressed as mg of CO₂ per kg of fresh weight per hour (mg CO₂ kg⁻¹ FW h⁻¹).

2.3.4. Determination of Firmness

Firmness of the pepper fruits was assessed using a TX-XT2i texture analyzer fitted with a 5 kg load cell (Stable Microsystems Ltd., Surrey, UK). Before the measurements, the equatorial diameter of each pepper was determined manually. Firmness testing involved applying a uniform force with a 75 mm flat steel plate to compress the fruit to 5% of its diameter at a speed of 0.5 mm/s. After the test, the probe retracted at a speed of 1 mm/s. Firmness was then calculated as the ratio of force to deformation, measured in Newtons (N), to ensure consistency and reproducibility among the samples.

2.3.5. Determination of Electrolyte Leakage (EL)

EL was determined in accordance with the method of Kong et al. [11] with some modifications. A 9 mm disk of tissues was cut down from pepper fruits using a stainless-steel puncher. After that, the disks were cleaned with deionized water and any residual water was removed with a clean paper towel. Following the initial preparation, the samples were submerged in 20 mL of deionized water and maintained at 20 °C for three hours. A conductometer (DDS-307, Yoke, Shanghai, China) was then used to measure the EL, denoted as L₀. To assess further leakage, the samples within the beakers were heated in a boiling water bath at 100 °C for 30 min. After allowing the samples to cool back to 20 °C, a second measurement of EL (L₁) was taken. The following formula was used to calculate the EL values in the pepper fruits.

$$\mathrm{EL} \mathrm{value} (\%)=\frac{{\mathrm{L}}_0}{{\mathrm{L}}_1}\times 100$$

(3)

2.3.6. Determination of O₂^•− Radical

O₂^•− content was measured using the method described by Zhang et al. [16] with some modifications. Specifically, 1 g of green pepper fruit tissue was homogenized in 3 mL of 20 mM potassium phosphate buffer (pH 6.0) for 30 min. After that, the homogenate was subject to centrifugation at 1900× g for 10 min; then, 1 mL clear supernatant was obtained and incubated with 2 mL of dihydroethidium (10 μM) and calcium chloride (CaCl₂, 100 μM) at a pH of 4.75 in the dark for one hour. The absorbance of the reaction mixture was measured spectrophotometrically at 510 nm using a UV-Vis spectrophotometer (RF-15001, Shimadzu, Kyoto, Japan). The obtained results were calculated and expressed as nmol g⁻¹ FW.

2.3.7. Determination of H₂O₂ Radical

H₂O₂ levels in pepper fruits were measured using the method of Cao et al. [12] with some modifications. Initially, 1 g of the pepper fruit tissue was homogenized in 5 mL of 20 mM potassium phosphate buffer (pH 6). After that, the homogenate was centrifuged at 1900× g for 10 min, and then, the supernatant was collected for analysis. The reaction mixture contained 1 mL of supernatant and 3 mL of the extraction buffer that contained 5 μM scopoletin and 3 mg mL⁻¹ horseradish peroxidase and was incubated in the dark for 30 min. The scopoletin reduction in fluorescence post reaction was spectrophotometrically measured at 346 nm and 455 nm against a reagent blank using a UV-Vis spectrophotometer (RF-15001, Shimadzu, Kyoto, Japan). The absorbance value was then converted into the molar concentration of H₂O₂ using a standard curve with a predetermined concentration of H₂O₂. The obtained results were expressed as nmol g⁻¹ FW.

2.3.8. Determination of Malonaldheide (MDA)

MDA levels were determined in pepper fruits using a method described by Zhang et al. [16] with some modifications. Initially, a 3 g sample was homogenized in 15 mL of 5% (w/v) trichloroacetic acid (TCA). After that, the homogenate was centrifuged at 5000× g for 15 min. Then, 1 mL of the resulting supernatant was collected and combined with 3 mL of 0.5% thiobarbituric acid (TBA), prepared in 10% TCA. This mixture was then heated at 95 °C for 30 min and rapidly cooled down to room temperature. A subsequent centrifugation at 5000× g for 10 min was performed. Then, the absorbance of the final supernatant was measured at 532 nm, with a correction for nonspecific absorbance at 600 nm using a UV-Vis spectrophotometer (RF-15001, Shimadzu, Kyoto, Japan). The concentration of MDA was then calculated and reported in nmol g⁻¹ FW.

2.3.9. Determination of Glutathione (GSH), Glutathione Disulfide (GSSG), and GSH/GSSG Ratio

The GSH content was measured according to the method of Kantakhoo and Imahori [17] with some modifications. The green pepper fruit tissue (2 g) was homogenized using 10 mL of 5% sulfosalicylic acid for 10 min and then filtered through two layers of muslin cloth. Then, the homogenate was centrifuged at 11,000× g for 10 min, and then 7.5 M triethanolamine was used to neutralize the supernatant and bring its pH value down to 7.0. Then, the neutralized supernatant was used to measure GSH and GSSG. A volume of 1 mL of the sample was allocated to assess the total GSH levels (GSH and GSSG). A separate 1 mL portion underwent reaction with 20 µL of 2-vinylpyridine for 60 min at 20 °C, facilitating the derivatization of GSH and thereby enabling the exclusive quantification of GSSG in the analysis that followed. For GSSG analysis, 50 µL of the treated sample were added to a mixture containing 150 µL of 125 mM sodium phosphate buffer (pH 6.5) with 6.3 mM ethylenediaminetetraacetic acid (EDTA), 700 µL of 0.3 mM nicotinamide adenine dinucleotide phosphate (NADPH), 100 µL of 0.6 mM 5,5′-Dithio-bis-(2-nitrobenzoic acid) (DTNB), and 10 µL of GSH reductase (GR) at 50 units mL⁻¹. Absorbance readings were taken at 412 nm using a UV-Vis spectrophotometer (RF-15001, Shimadzu, Kyoto, Japan) over a period of 120 s at 30 °C. The concentrations of total GSH and GSSG were deduced from a standard curve of GSH, which utilized sample volumes ranging from 25 to 100 µL. The results were expressed as µmol g⁻¹ FW. The GSH/GSSG ratio was calculated by dividing the values of both.

2.3.10. Determination of Ascorbic Acid (AsA), Dehydroascorbate (DHA), and AsA/DHA Ratio

The quantification of AsA in pepper fruits was measured by following the method of Wang et al. [18]. To extract AsA, 0.1 g of pepper tissue was pulverized in 6% (v/v) perchloric acid (HClO₄) under cold conditions (4 °C) and centrifuged at 12,000× g for 10 min at 4 °C, and then, the clear supernatant was retrieved for subsequent analysis. The concentration of AsA was determined by comparing the absorbance of the reaction mixture (0.1 mL supernatant and 2.9 mL of 200 mM sodium acetate buffer (pH 5.6) at 265 nm) using a UV-Vis spectrophotometer (RF-15001, Shimadzu, Kyoto, Japan) before and after an incubation period of 15 min with 1.5 units of ascorbate oxidase. The results are expressed in µg g⁻¹ FW.

DHA levels in pepper fruits were determined by following the method of Kantakhoo and Imahori [17] with some modifications. Three grams of each sample were homogenized in 12 mL of 6% TCA, aiming to extract DHA while ensuring protein denaturation and sample stabilization. In contrast to total ascorbate determination, the analysis of DHA content proceeded without the reduction step using dithiothreitol (DTT). Instead, 1 mL of the filtered homogenate was directly mixed with 1 mL of 0.4 M phosphate buffer (pH 7.4), omitting DTT to maintain the focus on DHA. This mixture was incubated at 37 °C for 20 min to ensure DHA stabilization. To halt any further reduction in DHA, 5 mL of N-ethylmaleimide were added to the reaction solution, preserving the DHA content for accurate quantification. For color development, 4 mL of a specific color reagent were introduced to the solution. The absorbance of the developed color complex was determined at 550 nm (UV-Vis spectrophotometer (RF-15001) from Shimadzu, Kyoto, Japan), after an incubation period of 40 min at 37 °C. The DHA concentration in the samples was quantified using a standard curve prepared under identical conditions. The colored reagents are listed below: solution A contained 31% orthophosphoric acid, 4.6% TCA, and 0.6% iron chloride, whereas solution B had 4% 2,2-dipyridyl and 70% ethanol. In order to combine solutions A and B, 2.75 parts (A) to 1 part (B) were used. The computed quantity of DHA was estimated using a standard curve and expressed in µg g⁻¹ FW. The AsA/DHA ratio was calculated by dividing both values.

2.3.11. Determination of Total Phenolic Content (TPC)

TPC levels in samples were tested using the method of Bhardwaj et al. [19], with some modifications. Five grams of sample were homogenized in 80% cold ethanol before being centrifuged at 8000× g for 10 min. The supernatant was then collected, and 0.1 mL was mixed with the freshly produced reagent. It started with 0.5 mL of Folin–Ciocalteu reagent and 2.9 mL of distilled water, and then 2 mL of a 20% sodium bicarbonate solution were added. The reaction mixture was thoroughly mixed and incubated for 45 min, and the absorbance at 760 nm was measured with a UV-Vis spectrophotometer (RF-15001, Shimadzu, Kyoto, Japan). Then, the absorbance was calculated with a gallic acid standard curve and represented as µg gallic acid equivalent (GAE) g⁻¹ FW.

2.3.12. Determination of Superoxide Dismutase (SOD)

The SOD experiment was performed using the method of Njie et al. [14], with some modifications. Two grams of sample were homogenized with 8 mL of cold 50 mM sodium phosphate buffer (pH 7.8) that contained 1% (w/v) polyvinyl polypyrrolidone (PVP). After that, the homogenate was centrifuged at 12,000× g for 30 min at 4 °C. The supernatant was collected and measured for SOD activity. To measure SOD activity, the reaction mixture was composed of 1.7 mL of 50 µM sodium phosphate buffer at pH 7.8, 0.3 mL of 130 µM methionine, 0.3 mL of 750 µM nitroblue tetrazolium (NBT), 0.3 mL of 100 µM EDTA-Na₂, 0.3 mL of 20 µM riboflavin, and 0.1 mL of the enzyme extract. This mixture was illuminated with a 4000 lx fluorescent light for 10 min prior to the assessment of SOD activity, which was monitored at an absorbance of 560 nm. The capacity of the system to inhibit the photochemical reduction of NBT was quantified in units of SOD activity (U) per gram of fresh weight (unit g⁻¹ FW), achieving 50% inhibition.

2.3.13. Determination of Catalase (CAT)

CAT activity was measured in accordance with the method of Tan et al. [20] with some modifications. Two grams of sample were homogenized with 10 mL of 0.1 M phosphate buffer (pH 7.0) that contained 0.1 mM EDTA and 1% PVP. Then, the homogenate was centrifuged at 12,000× g for 20 min at 4 °C. Then, the supernatant was collected and used for CAT analysis. The reaction mixture consisted of 0.1 mL of supernatant and 2.9 mL of 50 mM phosphate buffer (pH 7.0) that contained 15 mM hydrogen peroxide. The depletion of H₂O₂ in the reaction mixture was measured at 240 nm using a UV-visible spectrophotometer (RF-15001, Shimadzu, Kyoto, Japan). One unit of CAT was defined as the amount of enzyme required to reduce 1 µmol of H₂O₂ in 1 min, and the results were expressed in unit g⁻¹ FW.

2.3.14. Determination of Peroxidase (POD)

POD activity was measured in accordance with the method of Ngoc et al. [21] with some modifications. Two grams of sample were homogenized with 10 mL of 50 mM phosphate buffer (pH 6.8) that contained 1% PVP, and after that, the homogenate was centrifuged at 12,000× g for 20 min at 4 °C. Then, the supernatant was collected and used for POD assay. The reaction mixture contained 0.1 mL of supernatant and 2.9 mL of 50 mM phosphate buffer (pH 6.8) that contained 20 mM guaiacol and 40 mM hydrogen peroxide. The absorbance of POD activity was measured at 470 nm, and the results were expressed in unit g⁻¹ FW.

2.3.15. Determination of Polygalacturonase (PG) and Pectin Methylesterase (PME)

The PG and PME activity in the pepper fruits was measured in accordance with the method of Priya Sethu et al. [22] with some modifications. For PG activity, after homogenizing 2 g of the sample in 0.2 M tris-acetate buffer (pH 4.5), the homogenate was centrifuged at 12,000× g for 30 min at 4 °C. Then, the supernatant was collected and used to measure PG activity. The assay mixture was composed of 0.2 mL of the enzyme extract in 0.15 M sodium chloride (NaCl), 0.2 mL of 0.2 M tris-acetate buffer (pH 4.5), 0.1 mL of 0.01 M CaCl₂, and 0.5 mL of 1% polygalacturonate. For each test, a control was prepared by boiling the reaction mixtures prior to the introduction of the substrate. The reaction was conducted at 37 °C for 1 h, after which it was terminated by heating at 100 °C for 3 min. Subsequently, 0.5 mL of each solution was subjected to analysis for reducing sugars using the dinitro salicylate (DNS) method. One unit of enzyme activity was defined as the quantity necessary to produce 1 µM of reducing sugar per hour, and the results were expressed in unit g⁻¹ FW.

For PME activity, 2 g of pepper fruit were homogenized at 4 °C with 20 mL of 8.8% (w/v) NaCl solution and 0.5 g of PVP. The resulting mixture was then strained through a muslin cloth, and the filtrate was subsequently centrifuged at 12,000× g for 30 min at 4 °C. The supernatant, which contains the crude enzyme extract, was collected, and its pH was adjusted to 7.0. It was then measured using the titration method. The demethylation rate of citrus pectin was determined at room temperature through titration with 0.025 N sodium hydroxide. A substrate consisting of 50 mL of 1% (w/v) pectin from citrus fruit in 0.1 N NaCl was prepared, and its pH was adjusted to 7.0 prior to adding 1.0 mL of enzyme extract. PME activity was quantified based on the enzyme’s capacity to catalyze the consumption of 1 mM of the base per hour under the specified assay conditions, and the results were expressed in unit g⁻¹ FW.

2.3.16. Determination of Phospholipase D (PLD)

PLD activity was measured according to Yi et al. [23] with some modifications. Two grams of green pepper fruit tissue were homogenized in 5 mL of 0.1 M Tris-HCl buffer (pH 7.0), and then, the homogenate was centrifuged at 13,000× g for 30 min at 4 °C. Subsequently, the supernatant was collected and tested for PLD assay. A rotary evaporator was used to prepare and evaporate a substrate that contained 0.05 g of phosphatidylcholine, 3 mL of chloroform, and 3 mL of water at 40 °C. After the substrate had been rehydrated in 250 mL of 100 mM acetate buffer (pH 5.5, containing 5 mM dithiothreitol and 25 mM CaCl₂), it was combined with 1 mL of supernatant and vigorously stirred for 1 h at room temperature. The aqueous phase was then separated using petroleum ether, and 2 g of ammonium tetrarhodanatodiammonchromate were added to the aqueous phase, resulting in the formation of a precipitate. After centrifuging the precipitate for 15 min at 4 °C at 26,000× g, it was dissolved in 3 mL of acetone. The absorbance was measured at a wavelength of 520 nm using a UV-Vis spectrophotometer (RF-15001, Shimadzu, Kyoto, Japan), and the results were expressed in unit g⁻¹ FW.

2.3.17. Determination of Lipoxygenase (LOX)

The LOX activity was measured according to Kissinger et al. [24] with some modifications. A 1 g sample was homogenized in 5 mL of 100 mM Tris-HCl buffer (pH 7.0), which was centrifuged at 10,000× g for 20 min. The supernatant was collected and used as a crude extract. The assay tube contained 1 mL of standard assay mixture (40 mL of 100 mM sodium phosphate buffer with 200 μL of Tween 20 and 40 μL of linoleic acid) and 0.2 mL of raw extract. The absorbance was measured at a wavelength of 234 nm using a UV-Vis spectrophotometer (RF-15001, Shimadzu, Kyoto, Japan) and the results were expressed in unit g⁻¹ FW.

2.4. Statistical Analysis

All experiments in this study were conducted with at least three replications, and the results were presented as mean ± standard deviation. The significant differences between the mean values were measured using the analysis of variance (ANOVA), complemented by the t-test and Duncan’s multiple range test for post-hoc comparisons. The data were tested for significance at a level of p < 0.05. SPSS (version 6 for Windows) was utilized to perform statistical analysis.

3. Results and Discussion

3.1. CI Index, WL, Respiration Rate, and Firmness

CI index is the critical parameter used to evaluate the impact of the chilling stress on tropical and subtropical plants. Figure 1A represents the CI index of the pepper fruits treated with or without MT and stored under prolonged low-temperature storage. According to the CI index results, the onset of CI stress in the pepper fruits was exhibited on day 4 in the control samples and day 8 in the MT-treated samples; however, the intensity of the CI stress was significantly (p < 0.05) higher in the control samples than the MT-treated samples, and it was prolonged as the storage period extended. Among the MT-treated samples, the low concentration had a positive effect on the increment in the CI index in the pepper fruits compared to the control. However, the higher concentration of MT treatment (>50 µmol L⁻¹) outperformed the other treatments in controlling the intensity of the CI index in pepper fruits during storage. Ge et al. [25] reported that the increase in CI in the pepper fruits during cold storage is mainly due to the transcription factor CaMYB340 gene, which induces inhibition of the fatty acid desaturation process and is followed by the breakdown of cell membranes and peroxidation. The CI index and storage quality parameters in pepper fruit can be effectively controlled by MT treatment [8,26].

Changes in fruit weight during storage is a critical aspect that can impact the quality and shelf life of fruits, according to Njie et al. [14], and it can be influenced by various factors such as post-harvest treatments, storage conditions, and fruit characteristics [4]. Pepper fruit size and ripeness stage play a crucial role in the WL of pepper fruits [11]. WL in pepper fruits treated with or without MT is depicted in Figure 1B. An overall increase in WL was observed in all samples throughout the storage period. Konishi et al. [4] reported that an increase in pepper fruit WL was related to the thickness of the cuticular membrane and the content of cutin and polysaccharides, which can influence WL through the pericarp of pepper fruits during storage. The present study showed that the control fruits experienced a slightly higher rate of WL compared to the others. Conversely, MT treatment effectively mitigated WL, with varying MT concentrations significantly (p < 0.05) reducing WL in the pepper fruits. By the end of the storage period, fruits from the control group exhibited the highest WL, reaching 8.37%, in contrast to treated fruits, which showed a reduction in WL between 7.5% and 5.57%. It has been shown that MT improves fruit’s ability to withstand chilling by minimizing oxidative damage, which lowers WL and preserves the fruit’s general quality [14]. Zhang et al. [16] reported that MT treatment can effectively reduce WL in fruits by enhancing antioxidant activity and controlling reactive oxygen-induced stress. Wang et al. [10] tested the different concentrations of MT treatments to control jujube fruit quality, and their findings revealed that increasing the concentration of MT positively enhanced the fruit quality and controlled the WL by limiting the senescence process. This is in accordance with the present study.

On the other hand, the respiration rate of the tested pepper fruits during storage was consistently declined, regardless of the sample group (as shown in Figure 1C). Generally, the respiratory rate of pepper fruits can be influenced by various factors, including ethylene production [15], CI [27], and oxidative stress [28]. In comparison with the MT-treated samples, control samples exhibited a significantly (p < 0.05) higher respiration rate. The higher respiration rate in the untreated pepper fruits under chilling conditions is a result of the physiological responses to chilling stress, oxidative stress, energy availability, and CI, all of which contribute to alterations in the metabolic process and respiratory activity [27,29,30]. Among the MT-treated groups, variations in MT concentration markedly influenced the respiration rate, with higher MT concentrations showing a pronounced effect in reducing the respiration rate. Wang et al. [31] reported that MT treatment regulates the AsA-GSH level in fruits and thus reduces the ROS and oxidative stress, thereby contributing to controlling the respiration rate in fruits during cold storage. This is in accordance with the present study, which demonstrated higher levels of GSH and AsA in the MT-treated pepper fruits.

Firmness serves as a critical indicator of freshness and quality in crops, influencing consumer acceptance, transportation, storage, and overall post-harvest handling [32]. Figure 1D shows the changes in the firmness of pepper fruits during storage under cold conditions. Overall, a downward trend was observed in all the samples. A continuous loss of firmness in the pepper fruits can be attributed to moisture loss and degradation of cell wall compositions [24,30]. In comparison with the control samples, the MT-treated fruits significantly (p < 0.05) controlled the degradation of firmness, and the higher MT concentrations positively influenced the firmness of the pepper fruits. Similar findings were also discovered in the studies by Njie et al. [14], Bhardwaj et al. [19], and Sun et al. [33], who found that the application of MT at higher concentrations significantly (p < 0.05) controlled the fruit firmness loss. The decline in pepper fruit firmness is primarily linked to membrane degradation influenced by hydrolytic enzymes, including PG [22], PME [34], LOX [35], and pectate lyase [36]. Furthermore, lipid degradation and ROS production are associated with membrane degradation, leading to fruit softening during storage [37]. This is in accordance with the present study, where the untreated pepper fruits showed an elevated level of ROS and MDA (see Figure 2). Overall, the MT-treated samples significantly (p < 0.05) controlled this incidence and preserved the fruit firmness.

3.2. EL, ROS, and MDA

Chilling stress in plant crops can lead to the generation of ROS with the disruption of cellular processes, thus leading to damage to the cellular membrane and its components as well as adversely affecting metabolic activity [38,39]. O₂^•− and H₂O₂ are important ROS in plant crops that are generated during exposure to prolonged exogenous chilling stress and accumulate in various plant organelles, particularly chloroplasts, mitochondria, and peroxisomes [40]. Figure 2A shows the EL of pepper fruits during prolonged storage under cold conditions. The results show that continuous storage under chilling conditions significantly (p < 0.05) affected the membrane integrity of the pepper fruit. This is in accordance with the study of Hanaei et al. [13], who explored a continuous increase in ionic leakage in the pepper fruits during storage. Kissinger et al. [24] suggested that increased cell membrane ionic leakage in ripened pepper fruits during cold storage is associated with increased moisture loss. Among the different samples, the fruits that contained no MT coating showed a higher level of ionic leakage in the cell membrane, whereas the fruits treated with MT coating significantly reduced such events. Cao et al. [12] found that MT-treated fruits that were exposed under chilled conditions for a prolonged period significantly enhanced the AsA content and thus controlled the ROS-induced membrane degradations. Xie et al. [41] reported that MT could regulate the ROS metabolism and modulate the antioxidant genes, thereby reducing oxidative stress and maintaining overall fruit quality. These findings are in accordance with the present study, where the application of MT could be able to control the ROS and increase NEA.

Figure 2B,C show the continuous increment in ROS in the pepper fruits during chilling storage. Among the different samples tested, the control samples showed the highest accumulation of ROS, and it tended to be sustained as storage increased. A similar trend was also observed in the MT-treated pepper fruits; however, the severity of the ROS accumulation in those samples was substantially lower, and the intensity of the MT-treated fruits in controlling the ROS accumulation was dose-dependent. Zeng et al. [42] reported that MT treatment in fruits could indirectly influence stress response pathways, thereby mitigating oxidative stress and enhancing stress tolerance in fruits. Ma et al. [43] found that the application of MT on cassava root could eliminate the accumulation of ROS and maintain cellular homeostasis. Several studies have suggested that the application of MT could primarily activate the gene responses that are linked to the production of AO enzymes, thereby scavenging the production and reducing the accumulation of ROS in chilled injured fruits [44,45,46]. MDA is a byproduct of the oxidation of phospholipids that is induced by higher accumulation of ROS in plant crops [47]. The present study exhibited a continuous accumulation of MDA in the pepper fruits in all the samples (Figure 2D). Valenzuela et al. [27] found that the formation of MDA content in plant crops that stayed under prolonged chilling conditions could adversely affect the cellular membrane phospholipids by triggering lipid peroxidation and, consequently, increased MDA content. The different concentrations of MT treatment significantly (p < 0.05) controlled the MDA accumulation in the pepper fruits, and mainly the fruits treated with above 75 µmol MT/L showed a better controlling effect on MDA. Increased concentration of MT treatment in fruits could effectively control the membrane-degrading enzymes, mainly PLD and LOX activity, and contribute to decreased MDA content [48]. This is in accordance with the present study, where the increased concentration of MT significantly reduced the PLD and LOX activity in the pepper fruits.

Figure 2. EL (A), O₂^•− radical (B), H₂O₂ radical (C), and MDA (D) levels in pepper fruits with or without MT treatments and stored under prolonged low-temperature storage (n = 3).

Figure 2. EL (A), O₂^•− radical (B), H₂O₂ radical (C), and MDA (D) levels in pepper fruits with or without MT treatments and stored under prolonged low-temperature storage (n = 3).

3.3. Non-Enzymatic Antioxidants (NEAs)

NEAs serve as vital components in plant defense mechanisms against chilling stress by enhancing AO enzyme activity, promoting redox balance, and supporting essential physiological processes [40,49,50]. GSH, GSSG, AsA, and DHA are the primary NEAs that exhibit antioxidant activities in pepper fruits [17]. Figure 3 shows the changes in NEA content in the pepper fruits during storage under prolonged chilling conditions. GSH levels in the pepper fruits were gradually increased during storage (Figure 3A). However, there was a slight fluctuation in the GSH levels in samples during the initial days of storage, and afterwards, a steady uptrend was observed, which was maintained throughout the storage. The elevated level of GSH in the pepper fruits was mainly attributed to the AsA level, as it plays a major role in the ascorbate–GSH cycle, influencing the redox state and potentially contributing to the elevation of GSH levels [17]. Among the samples, the MT-treated fruits retained higher GSH levels during prolonged cold storage. Ahammed and Li [51] reported that increasing levels of GSH in plants by MT were mainly attributed to the upregulation of GSH metabolism and metal ion transport genes. Zhou et al. [52] found that MT treatment could increase enzymes such as glutathione S-transferase (GST), which plays a crucial role in GSH metabolism. The present study observed that treated pepper fruits with different concentrations of MT had significantly increased GSH levels during storage. GSSG plays a key role in plants during cold storage by regulating H₂O₂ concentrations and the redox state of cells [53].

The level of GSSG changes in the pepper fruits during storage under cold conditions is shown in Figure 3B. The results showed a continuous declining trend despite the sample variations. This is in accordance with the study by Tan et al. [20], who found that Kulai pepper fruit stored under prolonged low-temperature storage adversely affected the GSSG levels. Rahman et al. [54] found that decreased levels of GSSG in plants could be related to the alleviation of H₂O₂ and converted back to its reduced form (GSH) by the activity of NADPH-dependent glutathione reductase (GR). The higher concentration of MT-treated samples showed a high level of GSSG content in pepper fruits. On the other hand, the lower concentration of MT-treated (<50 µmol L⁻¹) pepper fruits did not significantly affect the GSSG content compared with the control. Ni et al. [55] reported that the application of MT in plants might influence the balance between reduced and oxidized forms of GSH, thus potentially leading to an increase in GSSG levels. The GSH/GSSG ratio is commonly used as a quantifying measurement to assess the scavenging potency of ROS [56]. Figure 3C shows the ratio of GSH/GSSG content in peppers that were treated with MT at ranging concentrations and stored in cold conditions. Overall, the results show a continuous trend of GSH/GSSG accumulation and ratio in the tested samples during storage. The highest level of GSH/GSSG ratio was found at the end of storage, and it was noticed in the 100 µmol L⁻¹-treated samples.

AsA levels in the pepper fruits that were treated with or without MT coating and stored under cold conditions are shown in Figure 4A. Overall, the trend of AsA was found to increase continuously in samples all over the storage area. An increase in AsA levels during prolonged cold storage can be attributed to the accumulation of dry matter content and depolymerization of pepper polysaccharides [30]. Among the sample variants, the control fruits had the lowest level of AsA content. The highest level of AsA was noticed in MT-treated samples. Furthermore, the different concentrations of MT played a significant (p < 0.05) role in the pepper fruits’ AsA levels. Wang et al. [3] reported that exogenous application of MT on pepper fruits enhances the ascorbate-glutathione cycle and promoted the regeneration of AsA and as well as GSH. Shafi et al. [57] found that MT treatment in pepper fruits could enhance the plant resistance against cold stress by enhancing scavenging efficiency against ROS molecules and promoting the antioxidant enzymes activity and metabolite content by regulating stress transcription genes, thus effectively preserving the AsA level. This is in accordance with the studies by Fan et al. [58], Cao et al. [12], and Wei et al. [59]. DHA plays a crucial role in the antioxidant defense mechanisms and is an oxidized form of AsA that can be converted back to AsA through interaction with reduced GSH [60,61]. The present study results show that continuous cold exposure to pepper fruits adversely decreased the DHA levels in all the tested variables (Figure 4B). Among them, the fruits treated with MT retained a slightly low DHA level compared to control samples. Valenzuela et al. [27] also found that fruit stored under prolonged cold stress could adversely affect the DHA contents. Furthermore, the decrease in DHA levels in the pepper fruits could be directly correlated with the increased levels of AsA. This is in accordance with the findings of Sharova et al. [62]. On the other hand, the AsA/DHA ratio of the tested samples showed a continuously increasing trend during storage (Figure 4C). Among the samples, the MT-treated samples (>75 µmol L⁻¹) exhibited a higher AsA/DHA ratio compared to control samples. Overall, the results exhibit that MT-treatment samples performed significantly (p < 0.05) better than the control samples.

TPC levels in the pepper fruits that were treated with or without MT coating are shown in Figure 5. Overall, the trend of TPC in all samples was found to have increased during storage. Among the samples, the MT-treated pepper fruits with higher concentrations showed a significantly (p < 0.05) higher level of TPC, followed by the lowest MT levels. On the other hand, the control fruits exhibited the lowest amount of TPC. The increase in TPC levels in the fruits is majorly influenced by temperature stress-induced enzymatic activity, particularly phenylalanine ammonia-lyase (PAL) [63]. Naghizadeh et al. [64] reported that MT treatment could significantly enhance PAL activity in plants. This increase in PAL activity is associated with the accumulation of polyphenol compounds, flavonoids, and anthocyanins in plants under various biotic and abiotic stresses.

3.4. Enzyme Activities

The activity of AO enzymes, particularly SOD, CAT, and POD, in pepper fruits treated with or without MT coating and stored under prolonged cold conditions is depicted in Figure 6A–C. Overall, AO enzymes in the pepper fruits demonstrated a consistent upward trend throughout the storage period. Among the tested AO enzymes in the pepper fruits, CAT activity was predominant, followed by SOD and POD activity. The prolonged exposure to low temperatures might activate the enzymatic AO system in pepper fruits to respond to physiological stress by increasing the activities of SOD, CAT, and POD [10]. Rodríguez-Ruiz et al. [5] reported that CAT activity in pepper fruit is higher than that of SOD and POD due to the modulation of the CAT gene and its activities during the fruit ripening and senescence process. Among the tested samples, the MT-treated samples displayed slightly higher AO enzyme activity. Compared to pepper fruits treated with varying MT concentrations, those treated with higher concentrations exhibited increased activities of AO enzymes. The increase in AO enzyme activity in MT-treated pepper fruits may be attributed to the upregulation of specific genes (CaSOD, CaCAT, CaPOD, and CaAPX) and proteins involved in antioxidant defense mechanisms [11]. Cellular degrading enzymes (CDEs), such as PG, PME, PLD, and LOX, play crucial roles in plant crops, particularly during the senescence process or under stress-induced physiological disorders. The activity of these enzymes in pepper fruits treated with or without MT coating was examined, revealing distinct patterns of enzyme behavior during storage (Figure 7). PG activity in the pepper fruits, as depicted in Figure 7A, increased continuously throughout the storage period in all samples. Notably, fruits treated with higher concentrations of MT (>75 µmol L⁻¹) exhibited significantly (p < 0.05) lower PG activities. Furthermore, other concentrations of MT also effectively reduced PG activity compared to the control. Similarly, the PME activities also showed an increasing trend in all pepper fruits during the storage period (Figure 7B). Despite this general trend, PME activities were comparatively lower than PG activities. Control samples displayed the highest PME activity, while MT treatment significantly (p < 0.05) reduced the intensity of PME activity. However, it was not sufficient to halt the increasing trend of PME activity during storage. Fruits treated with higher MT concentrations significantly (p < 0.05) suppressed PME activities. Sun et al. [33] reported that the exogenous MT treatment of jujube fruits stored under cold conditions led to significant control of CDEs, particularly PG and PME activity, and it was mainly attributed to the controlled effect of MT on these enzymes’ gene expressions. PLD is a crucial enzyme involved in phospholipid degradation during plant response to environmental stress, playing a key role in maintaining cell membrane integrity and function [65]. PLD activity in the pepper fruits slightly fluctuated (Figure 7C). Despite these fluctuations, the overall PLD activity was higher compared to the initial day. MT-treated fruits exhibited slightly lower PLD activity than the control, with higher MT concentrations resulting in marginally lower PLD activity among the treated samples. The LOX enzyme is involved in the oxidation of polyunsaturated fatty acids, leading to the production of fatty acid hydroperoxides, which adversely affect the plant cell membrane integrity [66]. LOX activity was generally lower compared to other tested CDE activity across all sample variants (Figure 7D). Control samples demonstrated significantly (p < 0.05) higher LOX activity compared to MT-treated ones. High MT concentrations (>50 µmol L⁻¹) more effectively controlled LOX activity than lower concentrations. Studies have reported that MT treatment could adversely affect the production of MDA and H₂O₂ by enhancing the AO enzyme activities and thus effectively controlling the PLD and LOX activities in the plants [21,41]. Kong et al. [11] found that the application of MT on the pepper fruit significantly controlled the production of MDA and suppressed the PLD and LOX activity by inhibiting their specific gene expressions.

4. Conclusions

The present study demonstrates the beneficial effects of melatonin treatment on preserving the quality and extending the shelf life of long green pepper fruits during cold storage. The application of melatonin at various concentrations notably reduced weight loss, decelerated respiration rates, and maintained firmness in pepper fruits, indicating a significant protective effect against the physiological stresses associated with low-temperature storage. These effects are attributed to melatonin’s ability to enhance antioxidant activity, thereby mitigating oxidative stress, as evidenced by the lower levels of reactive oxygen species and malondialdehyde, and regulating cellular degrading enzyme activity, such as polygalacturonase, pectin methylesterase, phospholipase D, and lipoxygenase. Furthermore, melatonin treatment improved the non-enzymatic antioxidant profile of pepper fruits, increasing levels of beneficial compounds such as glutathione, ascorbate, and total phenolic content, which are essential for antioxidant defense mechanisms. The study highlights the potential of MT, especially at higher concentrations (100 µmol L⁻¹), as an effective post-harvest treatment to enhance pepper fruit quality and defense system against chilling stress and to reduce physiochemical loss, offering promising applications for the agri-food industry in managing perishability and promoting sustainability. The use of a plastic polymer other than polyethylene could also be studied. It would be interesting to study which is the most suitable plastic polymer in conjunction with the use of melatonin.