Bio-Stimulants Extend Shelf Life and Maintain Quality of Okra Pods

Abstract

Okra (Abelmoschus esculentus L.), a tropical annual crop, is a highly perishable vegetable. Okra pods deteriorate rapidly after harvesting. The pods undergo physical and physiological changes that diminish storability and quality. The purpose of this study was to investigate the effect of bio-stimulants on the storability and quality of okra pods stored at 4 °C and 25 °C for 12 days. Dipping okra pods for 5 min in a solution of 0.5% ascorbic acid, citric acid, or salicylic acid pre-storage significantly extended the shelf life and preserved the quality of the pods compared to the control condition at 4 °C and 25 °C. Citric acid and ascorbic acid were the most effective in preserving most traits. Citric acid reduced the loss in weight, firmness, appearance, and prevented decay at 4 °C and 25 °C. Ascorbic acid decreased the loss of moisture and the degradation of carbohydrates, vitamin C and lycopene at 4 °C and 25 °C. Salicylic acid decreased the degradation of protein at 25 °C. The low temperature was highly effective in decreasing the loss or degradation of most of the studied traits. Taken together, bio-stimulants and storing at 4 °C played a prominent role in extending the shelf life and preserving the quality of okra pods.

1. Introduction

Okra (Abelmoschus esculentus L.) is a very popular vegetable in Egypt and its immature edible fruits, known as pods, are consumed fresh, frozen, and dried. It is used in many traditional dishes. In fact, it is an indispensable dish in the Egyptian cuisine. Okra is a summer vegetable crop, which belongs to the family Malvaceae and it is grown in both tropical and subtropical regions of Latin America, Asia and Africa [1]. Fresh okra pods are rich in fibers, K, Na, Mg, and Ca. One cup of fresh okra pods has approximately 30% vitamin C, 15% folate, and 5% vitamin A of the daily needs. In addition, Fe, Zn, Mn, and Ni have been found in the pods [2,3,4,5]. Okra is consumed throughout the year as are most vegetables lately, so it is necessary to search for ways to supply the market continuously with fresh or minimally processed okra. Some of these ways are growing okra under greenhouses, freezing and drying fruits [6]. Frozen and dry okra pods are widely used in Egypt to supply the market during winter (thanks to the advancement in food processing technology). Demand for fresh supply, minimally processed or refrigerated fruit and vegetables has increased significantly because of the rapid urbanization in developing countries as well as the awareness of the importance of consuming fresh products.

Okra pods are highly perishable and deteriorate soon after harvesting, so they have a short post-harvest life if they are not stored properly [7]. The pods undergo physical and physiological changes that reduce their quality. These changes include water loss, color fading, and decay, and therefore, the pods become squashy and unsuitable for fresh consumption. Thus, the commercial value is highly diminished or lost [8]. Okra pods’ ridges are easily exposed to mechanical injuries and bruises after post-harvest, so the pods turn black on the ridges and calyx disc resulting in value deterioration and price decline [9]. In addition, the operations of okra fruit processing such as trimming, peeling, coring, and cutting cause unwanted changes including browning, pigmentation and promote microbial growth. Furthermore, the moisture loss and respiration rate of minimally processed vegetables are much higher during refrigerated storage compared to non-processed vegetables [10].

Temperature is the most influential factor in okra shelf life in addition to the relative humidity. A high temperature causes rapid deterioration and reduction of the storage period in okra. Storing okra pods at a low temperature reduces the rate of respiration, evaporation, and ethylene production [11]. Under room temperature conditions, okra pods lose quality within two days due to blackening, shriveling and decay causing significant post-harvest loss. Low temperatures (10–13 °C) and the high humidity (93–100%) reduced weight loss, extended shelf life, and stopped browning and rotting of okra pods [12,13,14].

Antioxidants can be used to stop or delay the oxidation processes that cause undesirable changes in okra flavor, color, aroma, or nutritive value. Ascorbic acid (AsA) is essential for the plant antioxidant systems and human health [15,16,17]. It has beneficial effects in controlling enzymatic browning in fruits and vegetables such as green beans [15], plums [18], mung bean sprouts [19], and fresh-cut artichoke [20].

Salicylic acid (SA) is an endogenous plant growth regulator that has been found to cause a wide range of metabolic and physiological responses in horticultural crops affecting their growth and development. Salicylic acid has a high potential to be used in reducing post-harvest loss in plants because it is a natural and safe phenolic compound [21]. Treating strawberry fruits with 1 and 2 mM/L SA, reduced ethylene production and microbial load and retained overall quality [22]. Furthermore, exogenous application of SA at nontoxic concentrations to susceptible fruits and vegetables could enhance resistance to pathogens and control post-harvest decay [22,23,24,25]. Yossef et al. [26] found that SA application on okra plants increased the content of chlorophyll, protein, antioxidant capacity and phenolic compounds as well as yield attributes.

Citric acid (CA) is an organic acid that plays an important role in the CA cycle in mitochondria, which generates cellular energy through phosphorylative oxidation reactions [27]. Treating okra plants with CA at the concentration of 100 mg/L reduced the loss of total chlorophyll, protein, and phenolic compounds [28]. Therefore, there is an urgent need to develop a suitable method to reduce the post-harvest loss in okra and maintain high quality from harvesting to the entire supply chain under different storage temperatures. The objective of this study was to examine the effect of these safe natural food-grade compounds on the quality parameters and shelf life of okra green pod stored for 12 days at 4 °C and 25 °C.

2. Materials and Methods

2.1. Plant Material and Experimental Design

Okra cv balady, an Egyptian local variety, was grown on the Horticultural Experimental Farm, Faculty of Agriculture, South Valley University, Qena (32°44′25.5″ E, 26° 11′22.2″ N) in the summer of 2021. The soil texture was sandy loam. Three seeds per hill were sown in 10.5 m² plots with 3.5 m long and 0.7 m wide rows. The space between hills was 0.3 m. After establishment, the seedlings were thinned to one plant to reach a plant density of 4.76 per m². All the recommended practices required for agronomic and plant protection purposes were followed.

The pods were harvested 95 days post planting. Immediately after harvesting, the pods were placed in a well-ventilated and shaded place and transported to the laboratory within an hour. Pods, free from any defects, diseases and uniform in diameter and length, were selected for the experiments. The selected pods were washed with tap water, dried with tissues, and then immediately immersed for 5 min in dsH₂O (control), 0.5% CA, 0.5% SA or 0.5% AsA. The treated pods were then left until fully dried at room temperature and packed in polypropylene bags. The pods were stored for 12 days at 4 °C and 25 °C with 80% relative humidity.

Two experiments were conducted to study the effect of bio-stimulants on the storability and quality of okra pods. The first experiment included storing bio-stimulants treated okra pods at 4 °C and 25 °C for 12 days. The analyses were performed at 3, 6, 9 and 12 days for the morphological and physiological traits: (a) weight loss, (b) appearance, (c) firmness, (d) decay, (e) TSS and (f) pH. The second experiment included storing bio-stimulants treated okra pods at 4 °C and 25 °C for 12 days. The analyses were performed at the end of the storage period at 12 days for biochemical traits: (a) moisture, (b) ash, (c) protein, (d) carbohydrate, (e) vitamin C, (f) total phenolic compounds (TPC), (g) antioxidant activity, (h) carotenoids (β-carotene and lycopene), and (i) chlorophyll. Both experiments were repeated twice with three replicates for each one and the averages were used. Five pods were used in each measurement.

2.2. Preparation of Bio-Stimulants Solutions

Five grams of CA, SA and AsA, (Loba Chemie, Mumbai, India) with 99.5% purity were dissolved in 1 L of dsH₂O to give a concentration of 0.5% (5 g/L). These concentrations were selected based on a preliminary experiment and the previous work by [28,29,30] for CA and AsA, and [23] for SA.

2.3. Measurements

2.3.1. Weight Loss %

The percentage of pods weight loss was measured using an analytical balance scale (Axis balance model BTA2100D, Poland) and the percentage of weight loss was calculated according to the following formula:

$$Weight loss\%=\left(Intial weight-Final weight\right)/\left(Intial weight\right)\times 100$$

2.3.2. Appearance

The following scale was used to determine general appearance: 9 = excellent, 7 = very good, 5 = good, 3 = poor, and 1 = unacceptable. A group of three trained laboratory panelists evaluated the appearance score according to Awad et al. [15].

2.3.3. Firmness kg/cm²

The pod firmness kg/cm² was measured using a hand-held texture analyzer (Axis, FB200, Poland). Firmness values of each pod were determined at three different points. The mean of these values was used to express firmness.

2.3.4. Decay %

The decay percentage was calculated according to the following formula:

$$Decay \%=\left(No. of decayed pods\right)/\left(Total No. of pods\right)\times 100$$

2.3.5. Total Soluble Solids (TSS) and pH

Five pods were ground in an electrical blender for two minutes. The supernatant was used to determine TSS and pH. Total soluble solids was determined using a digital refractometer (Milwaukee, model MA871., USA) at the room temperature. Readings were taken as % of soluble solids content in the pods. The mixture pH was determined using a pH meter (Jenway, 3510 pH meter, Scientific Ltd., UK) at the room temperature.

2.3.6. Proximate Composition

Moisture was determined by oven drying okra pods to constant weight. Crude protein, crude fat, crude fiber, and ash were determined as described in the AOAC methods [31]. While carbohydrates were determined according to Manzi et al. [32].

2.3.7. Ascorbic Acid (V.C)

The amount of ascorbic acid or vitamin C was determined according to the 2, 6-dichlorophenol—indophenols dye method of Kumar et al. [33].

2.3.8. Total Phenolic Compounds (TPC)

The amount of TPC was determined using the Folin–Ciocalteu reagent and gallic acid as a standard, according to Singleton et al. [34].

2.3.9. Antioxidant Activity

Antioxidant activity and bioactive compounds were assessed according to methods previously reported by Barros et al. [35].

2.3.10. Carotenoids and Chlorophyll Pigments

A 150 mg fine dry powder of okra pods was added to 10 mL of acetone–hexane mixture (4:6) and vortexed with for 1 min and then filtered through a Whatman no. 4 filter. The filtrate absorbance was measured at 453, 505, 645 and 663 nm by a spectrophotometer. The contents of beta-carotene, lycopene, chlorophyll a and chlorophyll b were calculated according to the following equations as presented by Nagata and Yamashita [36].

beta-carotene (mg/100 mL) = 0.216 × A663 − 1.220 × A645 − 0.304 × A505 + 0.452 × A453.

Lycopene (mg/100 mL) = −0.0458 × A663+ 0.204 ×A 645 − 0.304 × A505 + 0.452 A 453.

Chlorophyll a (mg/100 mL) = 0.999× A663 − 0.0989 × A645.

Chlorophyll b (mg/100 mL) = −0.328 × A663 + A 1.77 × A645

2.4. Statistical Analysis

The analysis of variance (ANOVA) was conducted using SAS 9.4 (SAS Institute, Inc., 2013) software [37]. In more detail, we performed one-way ANOVA followed by the Dunnett’s test procedure for comparing bio-stimulant treatments vs. control. We also conducted a three-way factorial ANOVA (Bio-stimulants [CA, SA, AsA and C] × Storage Period [3, 6, 9, and 12 days] × Temperature [4 °C and 25 °C] to study the interaction effect of the study factors. Then differences were tested by the least significant difference (LSD) test at 5% probability level (i.e., p ≤ 0.05).

3. Results

3.1. Effects of Bio-Stimulants, Storage Period and Storage Temperature on Weight Loss, Appearance, Firmness, Decay, TSS and pH Traits of Okra Pods

The Supplementary Table S1 includes ANOVA results of morphological and physiological traits of okra pods treated by bio-stimulants (B) and stored at 4 °C and 25 °C (T) for 3, 6, 9, and 12 days (SP). The results reveal that there were significant differences among bio-stimulants, storage temperatures, storage periods and their interaction for all studied traits except the interaction (SP × T) of storage period (SP) and storage temperature (T) of the pH trait.

The statistical analysis of morphological and physiological traits in okra pods treated by bio-stimulants is presented in

Table 1. Analysis of variance of bio-stimulants for the morphological and physiological traits of okra pods stored for 12 days.

Trait	MS	F (3,92)	p Value
Weight Loss %	2.47	0.93	0.43
Appearance %	1.22	5.13	0.003 **
Firmness (Kg/cm)	0.10	0.18	0.91
Decay %	31.61	5.46	0.002 **
TSS %	24.98	112.76	<0.001 ***
pH	0.17	10.67	<0.001 **

** and *** are significant and highly significant at 0.05, 0.01 and 0.001 probability levels, respectively. df = (3,92), MS = mean square, F = F statistic based on the ANOVA test.

. There are significant differences for appearance, decay, TSS, and pH, but not true for weight loss and firmness traits.

Table 2. Mean ± SD and Dunnett’s test results for morphological and physiological traits of okra pods as a function of bio-stimulants.

Traits	Mean ± SD	Compared Group	p Value
Weight loss %	C 3.2 ± 2.0 CA 2.5 ± 1.6 SA 2.7 ± 1.5 AsA 2.6 ± 1.5	AsA vs. C CA vs. C SA vs. C	NS
Appearance	C 4.0 ± 0.71 CA 4.5 ± 0.39 SA 4.4 ± 0.38 AsA 4.5 ± 0.39	AsA vs. C CA vs. C SA vs. C	0.01 **
			0.00 **
			0.03 *
Firmness	C 2.7 ± 0.71 CA 2.8 ± 0.73 SA 2.7 ± 0.74 AsA 2.8 ± 0.70	AsA vs. C CA vs. C SA vs. C	NS
Decay %	C 8.1 ± 2.7 CA 5.4 ± 2.3 SA 6.2 ± 2.2 AsA 6.1 ± 0.55	AsA vs. C CA vs. C SA vs. C	0.02 *
			0.00 ***
			0.02 *
TSS %	C 8.7 ± 0.27 CA 11.0 ± 0.28 SA 9.1 ± 0.66 AsA 9.8 ± 0.55	AsA vs. C CA vs. C SA vs. C	<0.00 ***
			<0.00 ***
			0.01 **
pH	C 3.9 ± 0.09 CA 3.7 ± 0.16 SA 3.8 ± 0.12 AsA 3.8 ± 0.13	AsA vs. C CA vs. C SA vs. C	0.03 *
			<0.001 ***
			0.09

NS, *, ** and *** are non-significant, significant, and highly significant at 0.05, 0.01 and 0.001 probability levels, respectively. SD (standard deviations).

displays the means and SDs of the morphological and physiological traits of okra pods stored for 12 days as a function of the different bio-stimulants.

Table 2 incorporates the results of the post hoc analyses using the Dunnett’s test. For the appearance, decay, pH, and TSS traits, the CA-treated pods significantly outperformed the other conditions, especially the control. For the appearance and decay % traits, the SA-treated pods were significantly better than the control treatment. Pods treated with AsA-treatment showed significant attitude for TSS, appearance, and pH.

3.1.1. Weight Loss %

The differences in weight loss percentage were not significant among bio-stimulant treatments (F_(3,92) = 0.94, p = 0.43) (Table 1). The control treatment (M = 3.2, SD = 1.9) was insignificantly higher than other bio-stimulant treatments especially CA-treated pods (M = 2.5, SD = 1.6), which had less weight loss compared to all other treatments (Table 2). This is in agreement with Taain et al. [28] who dipped okra pods in 100 mg/L CA and had a lower percentage of weight loss compared to the control treatment. The role of CA in reducing weight loss may be due to its potential effects on physiological and metabolic processes, such as cell division and elongation, which increase plant biomass and the photosynthetic process in many plant species [38].

Dipping okra pods in bio-stimulant solutions for five minutes significantly affected the weight loss percentage (Figure 1). The weight loss % increased steadily over the storage period from day 3 to day 12 irrespective of the bio-stimulant or storage temperature. As expected, the percentage of weight loss % was higher when okra pods were stored at 25 °C compared to pods stored at 4 °C. Pods treated with CA, SA or AsA showed less weight loss compared to the control irrespective of storage temperature. Dipping okra pods in CA gave the least weight loss over all storage periods under 4 °C, while dipping okra pods in AsA gave the least weight loss over all storage periods under 25 °C relative to the control treatment. The low temperature storage could be more effective in reducing water loss in the samples after harvesting.

There are many factors causing weight loss. During harvesting and handling, injuries occur in the pods leading to excessive moisture loss. Besides, these injuries accelerate the rate of respiration and transpiration, and as a result, the pod food reserve such as carbohydrates and other constituents are depleted [39,40]. Temperature is also a major factor affecting weight loss. The higher the temperature, the higher the weight loss. Higher temperatures increase cell activities and consequently increase respiration and transpiration. Babarinde and Fabunmi [41] found that okra pods stored at 15 °C lost less weight than okra pods stored at room temperature (28 °C). The weight loss increases gradually when increasing the storage time [42]. Bio-stimulants decreased weight loss in low and high temperatures. These findings are like those found by Eman et al. [43] on guava, Awad et al. [15] on fresh-cut green beans, and Ali et al. [44] on litchi fruit. The effect of reducing weight loss by bio-stimulants could be attributed to the ability of these substances to reduce senescence and metabolic activity, [44], or respiration rate [43].

3.1.2. Appearance

Appearance decline was significantly suppressed in response to bio-stimulants compared to that of the control. The CA-treated condition was significant F_(3,92) = 5.13, p = 0.003, and higher (M = 4.5, SD = 0.39) than the control (M = 4.03, SD = 0.71) (Table 2). These results are consistent with those obtained by Taiane et al. [28] and Saleh et al. [29]. The decrease in appearance during the storage period resulted from shriveling, wilting, and color change in okra pods [45]. This could be due to the inhibitory effects of CA-treatment on chlorophyll degradation, ripening, and senescence [28].

A group of trained panelists rated the appearance of the okra pods every 3 days according to Awad et al. [15]. Figure 2 shows the effects of bio-stimulants, storage temperature and storage period on okra pods’ appearance. The appearance of pods almost remained the same for all bio-stimulant treatments after 3 and 6 days from harvest in both high and low temperatures, except for the control and SA, which slightly decreased from day 3 to day 6. However, a sharp decrease was seen from day 6 to day 12 in all treatments. The AsA-treated pods behaved slightly differently, where the appearance did not change until the day 9 at 4 °C, while at 25 °C after the sharp decrease from day 6 to day 9, the appearance did not change from day 9 to day 12. Bio-stimulants improved the pod appearance over all storage periods compared to the control. Dipping okra pods in CA retained the best appearance, while the control showed the lowest appearance decline at 4 °C and 25 °C. The decrease in appearance in all storage stages for all bio-stimulants slightly increased under 25 °C compared to 4 °C.

The most important quality parameter for consumer acceptance of vegetables and fruits is appearance. Soon after harvesting, the okra pod’s ridges turn black, the color becomes dull, and the fruit shrivels. These defects increase with inappropriate storage, handling, and transportation. Under room temperature conditions, okra pods lose quality by darkening and shriveling within two days, resulting in significant post-harvest losses [30]. Exogenous application of ascorbic acid maintained the quality and acceptability of stored shredded carrots [46] and fresh cut beans [15]. Guava fruits treated with citric had less color change and fewer discarded fruits [43]. The external appearance of fresh cut menthe and sage was preserved by the addition of salicylic acid [47]. The positive effect of ascorbic acid, for example, can be due to its inhibitory activity on chlorophyll degradation, maturation, and metabolic activity [48,49].

3.1.3. Firmness kg/cm²

The prolongation of bio-stimulant treatments resulted in an insignificant reduction in okra pod firmness (F_(3,92) = 0.19, p = 0.91). The CA and AsA treatments retained the firmness of okra pods and had higher firmness (M = 2.8, SD = 0.73 and M = 2.8, SD = 0.70, respectively) than the control condition (M = 2.7, SD = 0.71) (Table 1 and Table 2). The current results on okra pod’s firmness are consistent with those reported by Awad et al. [15] on green beans, and those of Liu et al. [18] on plum, who reported that the firmness of the fruits was preserved by the ascorbic acid treatment. Pepper fruit treated with AsA had higher firmness than its control counterpart due to its antioxidant properties [50]. The higher firmness of okra pod samples treated with CA could be explained by the higher hunting of reactive oxygen species of cells, resulting in a lower respiration rate [51]. Plants, on the other hand, can reduce the damage caused by oxidative stress in cells by activating their antioxidant system [52]. Okra pod softening is caused by the breakdown of insoluble protopectin into soluble pectin or by cellular disintegration, which leads to increased membrane permeability. Cheng et al. [39] found that storage temperature highly affected the pod firmness.

Okra pods that received bio-stimulants and were stored at 4 °C and 25 °C for 12 days showed a gradual decline in firmness over time irrespective of the bio-stimulant or storage temperature (Figure 3). Pods treated with CA, SA, and AsA showed less firmness decrease compared to the control treatment irrespective of the storage temperature. As expected, the firmness decline was higher at 25 °C compared to 4 °C. In general, there were no big differences between the control and the bio-stimulants at the end of the storage period.

3.1.4. Decay %

The CA-treated pods resulted in less surface decay (M = 5.4, SD = 2.3) than the control treatment (M = 8.1, SD = 2.7); however, the difference among bio-stimulant treatments was significant (F_(3,92) = 5.46, p = 0.002) at the end of the storage period (Table 1 and Table 2). Treatments with AsA and SA showed the same pattern of results related to the CA-treated pods (Table 2). These results are in line with those reported by Babalar et al. [22], El-Mogya et al. [23], and Yang et al. [53], who indicated that fruit with 10 g/L CA-treatment had a positive effect on reducing the postharvest decay percentage.

Pathogenic microorganisms cause decay in vegetables and fruits. Stated differently, CA-treated pods retarded or repressed the expression of Ac XE Ts, AcEXPs, and AcPE [54]. Plants use a variety of mechanisms to protect themselves from pathogenic attack. One of these mechanisms is the accumulation of SA [23]. The influence of SA in postharvest deterioration control is that it most likely plays a key role in raising hydrogen peroxide in plants, which acts as a signal molecule to activate plant resistance systems against pathogens attack [52].

The effects of bio-stimulants, storage period, and storage temperature (Figure 4). The decay % increased with time at both storage temperatures. Apparently, the low temperature resulted in less pod decay than the high temperature. The bio-stimulants were more effective in decreasing pod decay than the control. The CA-treatment was the most effective bio-stimulant in reducing pod decay, irrespective of storage temperature.

A high temperature accelerates fruit decay, especially in highly perishable vegetables. The decay rate was higher in pods stored at 25 °C compared to those stored at 3 °C, regardless of storage duration [21].

3.1.5. Total Soluble Solids (TSS)

The TSS were significantly higher than the control treatment (F_(3,92) = 112.76, p < 0.001) (Table 1). The CA-treated pods increased TSS rapidly at the end of the storage period (M = 11.0, SD = 0.3) compared to the control treatment (M = 8.7, SD = 0.3) (Table 2). These results were in agreement with those reported by Yang et al. [53] who indicated that CA-treated peach fruits reduced the peak value of ethylene production in fruits, thereby inhibiting TSS synthesis in the early stages of harvest but then was higher than the control treatment. The CA-treated pods were closely connected with ethylene generation and a minimal rise in TSS under lower storage temperature, which may be attributed to lesser reduction in polysaccharide and acid hydrolysis [53].

The effects of bio-stimulants, storage period, and storage temperature on TSS of okra pods (Figure 5). The TSS slightly increased with time in both storage temperatures for all bio-stimulants, except for samples treated with SA at 4 °C and AsA-treatment at 25 °C. At the beginning of the storage period at day 3, the values of TSS were less at 25 °C than the values of TSS at 4 °C. However, at the end of the experiment on day 12, the results varied where the TSS values of the control treatment and the AsA-treatment at 25 °C were higher than their counterpart at 4 °C. Immersing okra pods in CA solution produced the highest TSS in both storage temperatures, while pods immersed in distilled water (control) and stored at 4 °C, or SA solution and stored at 25 °C, produced the lowest TSS.

The TSS accumulation is mainly a mechanism of starch metabolism, which is slow in okra fruits stored at lower temperatures [30,55]. Samples stored in a cool condition had a lower TSS content than those stored at a room temperature. The low temperature and high humidity in the cool conditions slow down starch conversion into sugars. Similar results have been seen in bottle gourd [56], in carrot [57], in tomato [49,58], and in pepper [59]. The TSS may have increased due to the solubilization of cellulose and hemicellulose in the cell wall or because of water loss [60]. On the other hand, Menzel et al. [61] found that solid soluble TSS in strawberry fruits decreased when night temperature increased. Higher TSS retention in SA-treated samples compared to the control treatment can be attributed to lower evaporation, transpiration, and respiration rates, which result in TSS conservation [58].

3.1.6. pH

In the current experiment, the lowest values of pH were recorded (M = 3.7, SD = 0.12) with CA-treated pods compared to the control (M = 3.9, SD = 0.09) (Table 2). Okra pods treated with bio-stimulants showed lower pH values compared to the control treatment, or, in other words, the values were more acidic than the control. These results are expected as all the bio-stimulants are acids. The pH values were significantly different between the AsA and CA treatments on the one hand and the control on the other (Table 2). Conversely, pH values were not significantly different between SA and the control. These results are consistent with those of Wills et al. [27] and Joyce et al. [13] who reported that the impact of salicylic acid on fruit ripening and senescence was accompanied by changes in several quality aspects, including softening, a decrease in total acidity and an increase in sugar content, color development, aroma production. Furthermore, Eman et al. [43] found that CA changed the acidity of guava fruits. The pH is generally used to establish the maturing stage of fruits and vegetables and estimate fruit taste, which is mainly characterized by the balance of sweetness and acidity [62]. Current results are also in agreement with Shehata et al. [49] on tomato plants.

The pH values of okra pods treated with bio-stimulants and stored at 4 and 25 °C for 12 days (Figure 6). The pH values increased with time of storage irrespective of the bio-stimulant or storage temperature. At the beginning of the storage period, the pH values showed a sharp increase from day 3 to day 6, then, they started to slow down from day 6 until day 12. The values of pH at 25 °C were slightly higher than those at 4 °C. The highest pH values were observed in the control condition, while the lowest values were observed in CA-treated pods in both storage temperatures.

3.2. Effects of Bio-Stimulants and Storage Temperature on Nutritional Value and Biochemical Traits of Okra Pods Stored for 12 Days

Supplementary Tables S2 and S3 include the ANOVA results for the nutritional value and biochemical traits of okra pods treated with bio-stimulants and stored at 4 °C and 25 °C (T) for 12 days. There were significant differences among bio-stimulants, storage temperatures and their interaction for all traits except the interaction of vitamin C content.

The statistical analysis on nutritional value traits in okra pods treated by bio-stimulants (

Table 3. Analysis of variance of bio-stimulants for the nutritional value and biochemical traits of okra pods stored for 12 days.

Trait	MS	F(3,20)	P value
Moisture %	73.26	0.78	0.52
Ash %	0.62	1.60	0.22
Fat %	6.79	3.24	0.044 *
Fiber %	12.73	16.58	<0.001 ***
Protein%	0.91	0.03	0.994
Carbohydrate %	254.80	4.24	0.017 *
Vit. C	35.23	1.38	0.28
TPC	615.82	4.47	0.015 *
Antioxidant	4029.0	4.24	0.018 *
β-Carotene	165.78	0.76	0.53
Lycopene	30.93	4.79	0.01 *
Chlorophyll a	24.24	0.93	0.44
Chlorophyll b	27.74	0.85	0.48
Total Chlorophyll	102.87	0.89	0.46

*, and *** are significant and highly significant at 0.05, 0.01 and 0.001 probability levels, respectively. df = (3,20), MS = mean square, F = F statistic based on the ANOVA test

). There were significant differences for fat, fiber, carbohydrate, TPC and lycopene, but not for other traits.

Table 4. Mean ± SD and Dunnett’s test results for nutritional value and biochemical traits of okra pods as a function of bio-stimulants.

Trait	Mean ± SD	Compared Groups	P Value
Moisture %	C 74.4 ± 12.7 CA 80.8 ± 8.8 SA 79.4 ± 8.7 AsA 82.5 ± 7.8	AsA vs. C	NS
		CA vs. C
		SA vs. C
Ash %	C 8.2 ± 1.0 CA 8.7 ± 0.55 SA 8.8 ± 0.44 AsA 8.9 ± 0.30	AsA vs. C	NS
		CA vs. C
		SA vs. C
Fat	C 3.1 ± 1.9 CA 4.6 ± 1.6 SA 2.9 ± 1.4 AsA 5.0 ± 0.30	AsA vs. C	0.09 *
		CA vs. C	0.22
		SA vs. C	0.97
Fiber	C 20.1 ± 0.52 CA 17.2 ± 1.4 SA 19.6 ± 0.87 AsA 20.4 ± 0.30	AsA vs. C	0.95
		CA vs. C	<0.00 ***
		SA vs. C	0.60
Protein %	C 10.9 ± 6.8 CA 10.9 ± 5.9 SA 11.0 ± 5.2 AsA 10.2 ± 5.1	AsA vs. C	NS
		CA vs. C
		SA vs. C
Carbohydrate	C 35.5 ± 4.6 CA 38.8 ± 4.2 SA 45.9 ± 9.6 AsA 49.8 ± 10.5	AsA vs. C	0.01 *
		CA vs. C	0.80
		SA vs. C	0.08
Vitamin C	C 4.0 ± 3.8 CA 8.6 ± 5.4 SA 5.7 ± 3.3 AsA 9.1 ± 6.9	AsA vs. C	NS
		CA vs. C
		SA vs. C
TP	C 38.0 ± 14.8 CA 37.3 ± 10.1 SA 19.9 ± 8.7 AsA 20.4 ± 12.4	AsA vs. C	0.04 *
		CA vs. C	1.0
		SA vs. C	0.04 *
Antioxidant	C 23.3 ± 18.3 CA 77.8 ± 46.7 SA 69.7 ± 8.3 AsA 76.5 ± 35.0	AsA vs. C	0.02 *
		CA vs. C	0.02 *
		SA vs. C	0.04 *
β-Carotene	C 9.9 ± 9.4 CA 22.7 ± 18.7 SA 16.1 ± 15.3 AsA 17.1 ± 14.1	AsA vs. C	NS
		CA vs. C
		SA vs. C
Lycopene	C 0.80 ± 0.66 CA 2.0 ± 1.9 SA 3.4 ± 2.3 AsA 6.1 ± 4.1	AsA vs. C	0.01 **
		CA vs. C	0.74
		SA vs. C	0.23
Chlorophyll a	C 8.9 ± 4.2 CA 13.6 ± 6.7 SA 11.3 ± 4.5 AsA 12.5 ± 4.7	AsA vs. C	NS
		CA vs. C
		SA vs. C
Chlorophyll b	C 5.2 ± 2.3 CA 10 ± 0.39 SA 8.7 ± 6.2 AsA 8.9 ± 6.2	AsA vs. C	NS
		CA vs. C
		SA vs. C
Total Chlorophyll	C 14.1 ± 0 CA 23.8 ± 13.6 SA 20.0 ± 10.8 AsA 21.4 ± 10.8	AsA vs. C	NS
		CA vs. C
		SA vs. C

NS, *, ** and *** are non-significant, significant, and highly significant at 0.05, 0.01 and 0.001 probability levels, respectively. M (mean), SD (standard deviations).

incorporates the overall findings of the Dunnett’s test for the nutritional value and biochemical traits of okra pods. The CA-treated pods significantly outperformed the control in terms of fiber and antioxidants, whereas the AsA-treated pods significantly outperformed the control in terms of lycopene content, fat percentage, carbohydrates, TPC, and antioxidants.

3.2.1. Moisture and Ash %

There were insignificant differences in the moisture percentage and ash content percentage of okra pods treated with bio-stimulants at the end of storage compared to the control condition (F _(3,20) = 0.78 and 1.6, p = 0.52 and 0.22, respectively) (Table 3).

The control treatment was lower (M = 74.4 and 8.2, SD = 12.7and 1.0) than the AsA-treated pods (M = 82.5 and 8.9, SD = 7.8 and 0.30), respectively (in Table 4). Awad et al. [15] reported that AsA-treated fresh-cut green beans had more moisture content compared to non-treated fruit. Eman et al. [43] found that CA alone or combined with honey decreased weight loss and respiration rate of guava fruit.

Figure 7A, presents the effects of bio-stimulants and storage at 4 °C and 25 °C for 12 days on okra pod moisture content. Okra pods that received bio-stimulants had more moisture content than the control at 4 °C and 25 °C at the end of the storage period. However, the difference was bigger under 25 °C. As expected, storing okra pods at low temperatures resulted in more pod moisture content. The highest moisture content was recorded in pods treated with AsA, while the lowest was recorded in the control at 4 °C and 25 °C.

Similar results have been reported in prior research [63,64]. The moisture content of food is an index of its water activity and is used as a measure of stability and susceptibility to microbial contamination [65]. The high moisture content in vegetables makes them vulnerable to microbial attack and spoilage [66]. High moisture content also implies that dehydration would increase the relative concentrations of other food nutrients and, therefore, improve the shelf-life and preservation of the fruits [67]. Under high temperatures, the rates of respiration and transpiration increase, so the plant loses more water. Most of the weight loss in the highly perishable vegetables such as okra is the result of moisture loss.

Figure 7B, presents the effects of bio-stimulants and storage at 4 °C and 25 °C for 12 days on okra pod ash content. Okra pods treated with bio-stimulants had more ash content than the C-treated pods at 4 °C and 25 °C. However, the difference was more notable under 25 °C.

The ash content of okra pods stored at 4 °C was higher than the ash content of pods stored at 25 °C. The highest ash content was recorded in pods treated with CA at 4 °C and with AsA-treated pods at 25 °C, while the lowest was recorded in control at 4 °C and 25 °C.

Gemede et al. [68] studied the chemical composition of eight okra cultivars and found total ash content ranged between 5.37–11.30 g/100 g. Combo et al. [69] also found similar results. Surprisingly, the content under high temperature at the end of the storage period decreased compared to values under low temperature. This phenomenon has been noticed by some researchers. Babarinde and Fabunmi [41] found that the ash content of okra pods stored in a container decreased from 8.9% in the fridge to 7.9% at a room temperature. However, it is not clear why the bio-stimulant treated pods had more ash than the control under high temperatures. This suggests that bio-stimulants can play a role in preventing mineral loss in commodities post-harvest.

3.2.2. Fat and Fiber Content for Dry Weight %

The fat content of okra pods increased significantly in AsA-treated pods (M = 8.9, SD = 0.30) compared to the control (M = 8.2, SD = 1.0). However, no significant differences were noted in fat content between the CA and the control, and the SA and the control (Table 4).

The crude fat content was higher than the values reported by Combo et al. [69] who studied the chemical composition of six okra varieties and found that fat content ranged between (2.78 to 3.94 g/100 g) on a dry weight basis. Furthermore, Gemede et al. [68] found similar results in Ethiopian okra indigenous accessions. Romdhane et al. [70] reported lower values of fat for the Tunisian okra cultivars. Fat % is an energy reserve for plants and can be consumed when needed in cellular activities. The metabolic activities in an organism increase when the ambient temperature increases. The highest values of fat in bio-stimulant treated pods could be explained because the bio-stimulants improve metabolic activities and respiration rate [44].

The effects of bio-stimulants application and storing of okra pods at 4 °C and 25 °C on fat % (Figure 8A). Bio-stimulants effectively prevented the degradation of fat under low and high temperature compared to C-treated pods, except for SA-treated pods at the low temperature. This low temperature was more effective in preserving fat content compared to high temperature at the end of the storage period. At 4 °C, the CA-treated pods produced the highest fat %, while salicylic acid treated pods produced the lowest fat %. At 25 °C, the AsA-treated pods produced the highest fat %, while the control produced the lowest fat %.

A significant decrease between the CA-treated and the C-treated pods; however, no significant differences were found among AsA-treated, SA-treated, and C-treated pods. Treating okra pods with CA could be an effective way to prevent the development of undesirable fiber formation (Table 4).

Current results are like those obtained by Gemede et al. [68] and Combo et al. [69]. There have been reports about increasing fiber content since the fruit set over time or during the storage. Gunawardhana and de Silva [71] reported an increase of fiber content in okra pods from fruit set to 12 days. Additionally, Cachero and Bolenias [72] reported a positive relationship between okra fruit age and fiber content.

The effects of bio-stimulants application and storing of okra pods at 4 °C and 25 °C on fiber % (DW) (Figure 8B). The fiber % (DW) for okra pods at 25 was slightly higher than at 4 °C. The AsA-treated pods produced the highest fiber content, while the CA-treated pods produced the lowest fiber content at high and low temperatures.

It seems that fiber formation is a continuous process that occurs in the okra pods after they have been cut from the plant, especially during high temperatures. Apparently, low temperatures slowed down the formation of fiber content due to low metabolic activity. Another explanation for the higher content of fibers at 25 °C is that carbohydrates, protein, and fat were degraded in higher amounts under high temperatures as shown in this experiment, so the relative proportion of fiber on dry weight basis increases.

3.2.3. Crude Protein and Carbohydrates % (DW)

The SA-treated pods had higher protein content (M = 11.0, SD = 5.2) than the control (M = 10.9, SD = 6.8) (Table 4). Shi et al. [73] found that spraying cucumber plants subjected to heat stress with SA decreased ion leakage and H₂O_s and improved photosynthesis. The bio-stimulants may improve okra metabolism and decrease protein degradation. This result suggests that bio-stimulants can protect the protein from degradation under high temperatures. Bio-stimulants improve plant metabolism under normal and stress conditions. The respiration rate of fresh cut menthe and sage dried for 15 days were improved by the application of SA and CA [47]. Additionally, no significant differences were observed among CA, SA, AsA, and C-treated pods.

The effects of bio-stimulants application and storage temperature on okra pods protein content % stored for 12 days (Figure 9A). Storing okra pods at 4 °C highly decreased the degradation of protein content % compared to storing pods at 25 °C. Bio-stimulants treated pods had more protein content % under high temperatures, although they have less protein content under low temperatures compared to the control. The SA-treated pods produced the highest protein content % at 25 °C, while the control treated pods produced the highest content at 4 °C.

It seems that the degradation of protein content % in okra pods stored at 25 °C is a natural process that occurs in the fruit in the field. Cachero and Bolenias [72] found a negative correlation between fruit age and protein content. The highest protein content was found in 3-day fruits and the lowest was in 24-day fruits. Protein is one of the energy reserves in plant tissues alongside carbohydrates and fats. Therefore, it is normal to be influenced by temperature. The high temperature increases metabolic activities, respiration, and transpiration. Cheng et al. [39] found that okra pods stored at 25 °C lost more protein than okra pods stored at 4 °C.

The AsA-treatment increased carbohydrates content by 28% compared to the C-treated pods (Table 4). Carbohydrates are the plant’s main source of energy and they change continuously during plant growth. Once the fruit has been cut, the reserve of carbohydrates depletes gradually. There was also a significant increase between AsA-treated pods and the C-treated pods, and the SA treatment and control. However, no significant differences were found between the CA treatment and the control condition.

The effects of bio-stimulants application and storage temperature on the okra pod’s carbohydrate content % stored for 12 days (Figure 9A). Bio-stimulants treated pods had higher carbohydrate content than the control condition regardless of storage temperature. The AsA-treated pods yielded the highest carbohydrate content, while the control yielded the lowest carbohydrate content % (DW) at low and high temperatures. Storing okra pods at low temperatures moderately decreased the degradation of carbohydrates content %.

In some fruits such as okra, carbohydrates content % decreases with aging. Cachero and Bolenias [72] found a negative correlation between fruit age and carbohydrate content. Current results showed that bio-stimulants prevented carbohydrates degradation at low and high temperatures.

3.2.4. Ascorbic Acid (Vitamin C) (mg/100 g for Fresh Weight FW)

In more detail, no significant differences were found among CA, SA, and AsA treatments, and the C-treated pods. The AsA-treated pods gave the highest amount of ascorbic acid (mg/100 g FW) (M = 9.1, SD = 6.9) compared to the control condition (M = 4.0, SD = 3.8) (Table 4). The reason for fruits treated with AsA retaining the highest amount of vitamin C compared to the control treatment could be due to the role of ascorbic acid in reducing vital processes occurring inside fruit cells and thus reducing vitamin demolition, as well as the role of AsA in reducing some vital processes, such as oxidation found by Taain et al. [28].

Figure 10 shows the effects of bio-stimulants and storage temperature on the okra pod’s vitamin C content. Storing okra pods at 4 °C highly prevented the degradation of vitamin C content. Relatedly, bio-stimulants clearly prevented the degradation of vitamin C at low and high temperatures. However, the effect was bigger at the high temperature. The AsA and CA-treated pods had the highest vitamin C values compared to the control condition at low and high temperatures.

I The low temperature was effective in preventing vitamin C degradation. Similar results have been seen by Cheng et al. [39] and Babarinde and Fabunmi [41]. Using citric acid or citric acid and honey together decreased the degradation of ascorbic acid in guava slices and fruits [43]. In addition, CA and SA treatments decreased the degradation of vitamin C in menthe and sage at cold and hot temperatures [47]. Vitamin C is a sensitive substance and can be degraded by many factors pre- and post-harvest such as light, temperature, N fertilization, irrigation frequency, drought, and storage period. The most influential factor in vitamin C post-harvest is storing fruits and vegetables at a high temperature for a long time. Storing commodities at a low temperature, low oxygen and CO₂ decreases the loss of vitamin C [74]. Kader and Morris [75] found that vitamin C content decreased by 5 and 12% when tomato plants were left 24 h at 30 and 40 °C, respectively.

3.2.5. Total Phenolic Compounds TPC (mg GAE/100 g DW) and Antioxidants Activity (IC50/100 g DW)

The SA and AsA-treated pods had lower values (M = 19.9, 20.4, SD = 8.7, 12.4, respectively) than the control (M = 38.0, SD = 14.8) (Table 4). The effects of bio-stimulants on TPC in okra pods significantly decreased (p = 0.02) compared to the control treatment (Table 4). In addition, there were significant differences among the CA, SA, AsA, and the control treatments. These results are in agreement with those obtained by Saleh et al. [29]. Such results may be due to the drop in TPC to polyphenoloxidase oxidation, which produces colored quinones compared to the control treatment [39]. Taain et al. [28] reported that CA reduced the loss of TPC in okra. Abdel-Hamid [47] found that total phenols decreased with time at low and high temperatures in menthe and sage and the application of CA, SA and chitosan decreased the degradation of the TPC. Similar results on okra from Benin were reported by Adetuyi et al. [64].

Figure 11A, shows the effects of bio-stimulants and storage temperature at 4 °C and 25 °C on TPC (mg GAE/100 g DW) in okra pods stored for 12 days. The low temperature was effective in preventing TPC degradation. Surprisingly, bio-stimulants treated pods had less TPC content than the control treatment irrespective of storage temperature, except the CA-treated pods at the high temperature.

The effect of bio-stimulants on antioxidant activity (IC50/100 g DW) in okra pods significantly increased compared to the control treatment. The CA-treated pods had an increase in the amount of antioxidant activity by 57% over the control. There were also significant differences among the CA, SA, AsA, and the control condition (Table 3 and Table 4). These results may be due to the CA and AsA, which play roles in some signal transduction pathways that lead to the activation of secondary metabolism routes in plants and fruits and enhanced antioxidant activity [76].

The effects of bio-stimulants and the storage temperature at 4 °C and 25 °C for 12 days on the antioxidant activity (IC50/100 g DW) of okra pods (Figure 11B). The lower the antioxidant activity value, the higher its effectivity. As observed, high temperatures increased the antioxidant activity compared to the control treatment. Surprisingly, compounds extracted from the control condition had higher antioxidant activity than compounds extracted from bio-stimulant treated samples. The CA-treated pods had the highest antioxidant activity at 25 °C and the SA-treated pods had the highest antioxidant activity at 4 °C among the bio-stimulants treated pods.

Figure 11B, presents the effects of bio-stimulants application and storage at 4 °C and 25 °C for 12 days on the okra pod’s antioxidant activity (IC50/100 g DW). Sreeramulu et al. [77] determined the antioxidant activity and TPC of nineteen vegetables commonly consumed in India, and okra fruits had the highest levels of TPC (167.70 mg gallic acid/100 g), which is ranked third behind purple cabbage and broad beans.

3.2.6. Carotenoids and Chlorophyll Pigments (mg/100 g DW)

The CA-treated pods recorded high mean values of beta-carotene than the control condition (Table 4). This result could be attributed to the role of citric acid for reducing respiration and maturity process [27,57] leading to slowing down fruit ripening and lycopene biosynthesis. The effect of CA, SA, and AsA-treatment on beta-carotene in okra pods had no significant differences between the control and the other bio-stimulant treatments.

Beta-carotene content (mg/100g DW) of okra pods treated with bio-stimulants and stored at 4 °C and 25 °C for 12 days (Figure 12A). The low temperature was highly effective in preventing the degradation of beta-carotene compared to the high temperature. Bio-stimulants were also effective in decreasing the degradation of beta-carotene irrespective of storage temperature. The CA-treated pods produced the highest beta-carotene content, while the control produced the lowest beta-carotene content at 4 °C and 25 °C.

The effects of bio-stimulants on lycopene (mg/100 g DW) in okra pods significantly increased lycopene content (F_(3,20) = 4.79, p = 0.01). The AsA-treated pods were significantly different compared to the control treatment; however, no significant differences were obtained between the CA and SA treatments, and the control treatment (Table 3 and Table 4). This result could be ascribed to the role of ascorbic acid in reducing vital processes occurring inside fruit cells and thus reducing vitamin demolition, as well as the role of AsA in reducing some vital processes, such as oxidation as reported by Taain et al. [28].

Lycopene content (mg/100 g DW) of okra pods treated with bio-stimulants and stored at 4 °C and 25 °C for 12 days (Figure 12B). Okra pods stored at a low temperature showed very high lycopene values compared to those stored at a high temperature. The low temperature was very effective in preventing lycopene loss. Bio-stimulants treated pods produced higher lycopene content compared to the control treatment regardless of the storage temperature. The AsA-treated pods produced the highest lycopene content, while the control produced the lowest lycopene content at 4 °C and 25 °C.

The effects of bio-stimulants on chlorophyll a, b, and total chlorophyll (mg/100 g DW) in okra pods was insignificant (p = 0.45, 0.48, and 0.48, respectively). There were no significant effects between the AsA, CA, and SA treatments, and the control treatment. The highest accumulation amount of photosynthesis pigments in okra pods was obtained with CA followed by AsA treatment. This result could be due to bio-stimulants such as CA and AsA, which decreased the degradation of chlorophyll in plants [48].

The content of chlorophyll a, chlorophyll b and total chlorophyll, respectively, of okra pods treated with bio-stimulants and stored at low and high temperatures for 12 days (Figure 13A). The low temperatures effectively decreased the degradation of chlorophyll a, chlorophyll b, and total chlorophyll. Bio-stimulants decreased the degradation of chlorophyll irrespective of the storage temperature. Bio-stimulants treated pods produced higher chlorophyll values compared to the control condition. The CA-treated pods produced the highest chlorophyll a, b and total values at 4 °C and chlorophyll b at 25 °C, while the AsA-treated pods produced the highest chlorophyll a and total content at 25 °C. The control-treated pods produced the lowest values at all storage temperatures except the SA-treated pods, which produced the lowest chlorophyll b at 25 °C.

Beta-carotene, lycopene, and chlorophyll had almost similar patterns under low and high temperatures. However, the difference in beta-carotene and lycopene between pods stored at low and high temperature was higher than those for chlorophyll. Chlorophyll content increases over time during okra fruit growth [73]. Carotenoids and chlorophyll degrade gradually in postharvest commodities. Treating menthe and sage with CA, SA, and chitosan decreased the degradation of chlorophyll [48]. Values of beta-carotene in this experiment are higher than those reported by Gemede et al. [68] who found that beta carotene in Ethiopian okra pods was (185 μg 100 g⁻¹ fw). Balasubramanian et al. [78] measured the values of carotene and chlorophyll content in okra which were 10 and 60 mg 100 g⁻¹ fw, respectively, and attributed the variations to differences in genotype and/or pod size at the harvest stage. Martinez et al. [79] reported a decrease in the chlorophyll content during French beans pod development. It is obvious that bio-stimulants play a role in protecting okra pod pigments from degradation, especially during high temperature.

4. Conclusions

Our study clearly proved that the quality and appearance of okra pods stored at 4 °C and treated with AsA, CA, and SA are well preserved compared to the control treatment or storing at 25 °C. Low temperatures were highly effective in maintaining all the quality traits, so okra pods can be stored up to 12 days with high quality. Bio-stimulants were effective in reducing the decline in weight loss, firmness, appearance, moisture, ash, fat protein, carbohydrates, vitamin C, carotenoids, and chlorophyll. Furthermore, they prevented the increase of decay, TSS, pH, and fibers. Bio-stimulants were more effective at high temperatures.

Overall, the CA-treated okra pods outperformed other bio-stimulants in terms of weight loss, firmness, pod color deterioration, and pod decline. Furthermore, TSS increases, and the pH becomes more acidic. As a result, it is the most effective treatment for carotenoids and chlorophyll. The appearance and firmness of the AsA-treated okra pods improved. Furthermore, they demonstrated good performance in terms of moisture, ash, fat, fiber, carbohydrate, vitamin C, and lycopene content. The SA treatment improved protein and TPC levels. Therefore, bio-stimulants used in this experiment can be used alone or in combination with low temperatures as safe natural food-grade substances to preserve the quality and appearance of okra pods.