Possible use of organic compounds on shelf life and quality properties of peeled pomegranate

Abstract Pomegranate cultivar (“Ardestani”) peeled and packed in polyethylene containers and treated with different natural products. Two concentrations of Aloe vera gel (10 and 15%), two different levels of saffron petal extracts (10 and 20% V/V) and two concentrations of saffron style extract (0.1 and 1% V/V) and control in one storage condition (7°C and 85% RH) were the treatments that applied by a full factorial randomized method. We examined natural substances for their possible application in extending the shelf life of fresh‐cut horticultural products to find a new approach for packaging and exporting pomegranates. About 13.8% mass loss in the 12th day of storage occurred because of higher enzymatic activity and lower membrane resistance. Our results show that all treatments significantly reduced mass loss, and Aloe vera gel treatments combined with saffron petal extract were the best. Although all treatments decreased ion leakage, Aloe vera gel and saffron petal extract reduced it significantly. Ion leakage incidence of arils at day 12 was lower in Aloe vera gel and saffron petal extract treatment compare to control. Application of both saffron extracts on arils reduced decay incidence and chilling injury from 86.67% to 6.67% and 60% to 26.67%, respectively. Total acidity, soluble solids content, total phenol content, anthocyanin content, and antioxidant capacity of arils changed differently in different treatments, and saffron petal extract significantly was the best one and increased anthocyanin content, total phenol content, and antioxidant capacity in arils. The microbial contamination increased in more extended storage, although both saffron extracts were successfully suppressed mold and bacteria growth below acceptable limits in 14 days at 7°C.

ity of fresh-cut pomegranates are microbial growth and activity of the browning enzyme, which is due to the oxidation of phenolic compounds by polyphenol oxidase (Gil et al., 1996).
Fruits and vegetables are metabolically active and subjected to senescence changes that need to be controlled to maintain long-term quality and shelf life (Mahajan, Caleb, Singh, Watkins, & Geyer, 2014).
The use of postharvest technologies has proven effects on mortality reductions of fresh produce in different parts of the world and can be a strategic procedure for reducing poverty, hunger, and malnutrition (Affognon, Mutungi, Sanginga, & Borgemeister, 2015). The choice of postharvest technology depends on the type of product, climatic conditions on production and transportation, pricing, and usability (Kitinoja & Barrett, 2015). The goal of postharvest technology is to reduce the rate of physiological reactions and senescence, and thus minimizing product decomposition. Some postharvest techniques include the use of chemical and physical methods that are effective in reducing microbial contamination (Tripathi, Sharma, Sharma, & Alam, 2013).
Adding chemicals to keep food products safe and sound is usually based on preventing microbial growth or killing and destroying harmful microorganisms. Regarding the general concerns about the effects of chemical preservatives to human health, consumers tend to buy and use products that have no preservatives or natural preservatives.

| Preparation of plant materials
Mature, uniform (similar in shape and size) "Ardestani" pomegranate fruits were harvested from commercial Orchard, Mahvelat, and Khorasan-Razavi province in autumn 2018 and transported to the Laboratory. Fruits carefully examined in terms of being free of pests and diseases, skin lesions, and signs of sunburn. Fresh, unharmed fruits peeled and seeded.

| Treatments application
Pomegranate arils divided into four groups for treatment application.
This experiment conducted as factorial in a completely randomized design with three replications. Each replication contains ten packages (each one with 100 gr pomegranate aril) as observation, and the data for each replication are mean of them.

| Sampling, measurements, and observations
In this study, arils mass loss, ion leakage, chilling injury and decay percentage, soluble solids content, titratable acidity, anthocyanin content, total phenol, antioxidant capacity, and microbial contamination of minimally processed pomegranate arils measured.

| Mass loss
Weighting each package during storage, the mass loss was calculated and expressed in percentage (Eq. 1).
where W i1 stands for package weight at the beginning of the experiment, and W it stands for package weight during storage period on sampling.

| Ion leakage (IL)
Ion leakage (IL) was calculated with four grams sample for each packet. It was then stored in a becher containing 20 ml of water for 24 hr, and then, the initial electrical conductivity (EC1) was read by Ec meter. The samples then placed in Ben-Marie at 100°C for one hour and, after cooling down to room temp., the secondary electrical conductivity (EC2) was measured. Finally, ion leakage was determined using equation No.2 (Barranco, Ruiz, & Gómez-del Campo, 2005).

| Decadence/Chilling injury
Decadence/chilling injury (DI/CI) percentage of pomegranate arils was measured by observing and counting the number of packages with decayed/injured arils incidence and calculating relative to the total number of packets according to the following equation (Karabulut, Gabler, Mansour, & Smilanick, 2004).
NP i : number of packets with decay/CI incidence at sampling time (in each specific treatment), and NP t : number of total packets in each treatment. The data are reported cumulatively between observations.
If a treatment has 10 percent of decay in first observation and 10 percent in the second one, 20 percent of decadence in second observation was reported. Also, it should be cleared that one package may show both decay or chilling injury incident and counted in both terms in each time.

| Titrable acidity
Pomegranate arils fresh juice was used to measure titrable acidity (TA) according to the described method by Ayala-Zavala, Wang, Wang, and González-Aguilar (2005); Belay, Caleb, Mahajan, and Opara (2018). The TA content of samples was measured potentiometrically by titration with 0.1 mol/LNaOH, to an end-point of pH 8.2. The TA value was expressed as milligrams per liter of citric acid based on fresh weight.

| Anthocyanin content
Anthocyanin content (AC) of pomegranate juice measured using the pH differential method according to Belay et al., 2018;Lako et al. (2007)  where A abs stands for A520-A700, 449.2 g/mol is the molecular weight of cyanidin-3-glucoside, 9 is the dilution factor, 26.900 is molar extinction coefficient, and L represents path length in centimeters.

| Soluble solids content
Soluble solids content (SSC) of pomegranate juice was measured using a hand refractometer (Atago™ MASTER-53M) and expressed as percent. (1)

| Total phenol content and antioxidant capacity
Total phenol content (TPC) and antioxidant capacity (ACP) were measured according to the method described by Du, Li, Ma, and Liang (2009). Five grams of pomegranate arils was extracted and smashed in liquid nitrogen and then 20 ml ethanol: Acetone (7/3 v/ v) solution was added to the sample. After homogenization, it was placed at room temperature for one hour and then filtered with Watten's No. 4 filter paper. Total phenol and total antioxidant capacity were determined from the extracted solution.
Total phenol content was measured according to Folin-Ciocalteu method using a spectrophotometer (Du et al., 2009). A 5 ml of sample (200 μl of the aril extract plus distilled water) was added to 500 μl of Folin (1:1 with water); then, 1,500 μl of sodium carbonate (20 g/ 100 ml) was added after one minute. After two hours of storage at room temperature in no light condition, the absorbance of the extract was measured at 765 nm. Pure gallic acid was used to obtain the standard curve. A 100 μl of the prepared solution of gallic acid at concentrations of 0-1000 with 0.5 ml of Folin 50% and 1.5 ml of sodium carbonate 20% was mixed and kept in darkness for 2 hr; then, absorbance at 765 nm was read, and then, the standard curve was plotted ( Figure 1). Before measuring the samples, the device calibrated with a blank sample containing 100 μl of extraction solvent, 9.9 ml of water, 0.5 ml of Folin (50%), No.5).
(5) %DPPH sc = A CONT − A SAMP ∕A CONT * 100 F I G U R E 2 Effects of different treatments on mass loss in pomegranate arils during storage. Each error bar is constructed using a 95% confidence interval of the mean according to Tukey HSD multiple range test F I G U R E 3 Effects of saffron petal extract (10 and 20%) and Aloe vera gel (10 and 15%) interaction on mass loss in pomegranate arils during storage. Each error bar is constructed using a 95% confidence interval of the mean according to Tukey HSD multiple range test F I G U R E 4 Effects of saffron style extract (0.1 and 1%) and Aloe vera gel (10 and 15%) interaction on mass loss in pomegranate arils during storage. Each error bar is constructed using a 95% confidence interval of the mean according to Tukey HSD multiple range test where % DPPH sc is the inhibitory percentage of DPPH, A CONT is absorbance of DPPH at 515 nm, and A SAMP is sample absorbance at 515 nm.

| Microbial quality
Microbial quality of pomegranate arils was studied according to methods described by Belay et al., 2018. 10 g of pomegranate arils was mixed with 90 ml peptone buffered the water and homogenized for 2 min with a laboratory blender. Plate count agar (PCA) was used for the aerobic bacterial count, while rose bengal chloramphenicol agar (RBCA) was used to count mold. PCA plates incubated at 30°C for three days and RBCA plates at 25°C for five days. After incubation, colonies were counted on each plate, and the results were expressed as log colony-forming unit per weight (log CFU/ml) (Belay et al., 2018).

| Data collection
Data collection and measurements were done at the 1st, 4th, 8th, and 12th day of the experiment. Then, data were subjected to F I G U R E 5 Effects of different treatments on electrolyte leakage in pomegranate arils on each storage period (Up: effects of Aloe Vera Gel and saffron style extract interaction on ion leakage, Down: effects of Aloe vera Gel and saffron petal extract interaction on Ion leakage. T1: day 1, T2: day 4, T3: day 8, T4: day 12).Each error bar is constructed using a 95% confidence interval of the mean according to Tukey HSD multiple range test analysis of variance. Tukey HSD multiple range test at 95% confident interval with SAS-JMP (ver. 14) was the tool for evaluation of the difference between mean values significance.

| Mass loss
Schematic weight loss of treated arils kept at 7°C was showed in Figure 2. By the rise in the concentration of treatments, reduction in mass loss observed at each sampling time. During each observation, AG 15% had the lowest mass loss, and AG 10% and SPE 20% were in next places (p < .05). Although all treatments were reduced mass loss significantly (p < .05) in comparison with control, differences between SSE 0.1%, SSE 1%, and SPE 10% were not significant. Application of 0.1 and 1% SSE reduced weight loss of control treatment at the 12th day of storage from 13.8% to 10.69% and 10.27%, respectively (p < .05). Different studies reported that more extended storage periods of pomegranate arils cause higher weight loss due to more enzymatic activity and lower cell membrane resistance against water loss (Atilgan et al., 2014;Belay et al., 2018). Combined application of Aloe gel with saffron petal and style extracts had the same trend (Figures 3 and   4), while SPE was better in mass loss reduction. Aloe Gel controls micro atmospheric exchanges of O 2 and Co 2 in treated arils, while the antioxidant activity of saffron extracts may reduce oxidation ratio and enzymatic activity. Results suggest that Aloe Gel's treatment was the most effective weight loss prevention treatment ( Figure 2), which is better to be combined with SPE 20% in the application (Figure 3). Essential oils application on postharvest of fresh-cut apple was showed that as their treatment cannot provide a lipid form, multilayer coating on fresh-cut fruits, they could not be able to reduce mass loss, while chitosan and pectin edible coatings were effectual in papaya and pineapple (Sarengaowa, Hu, Jiang, Xiu, & Feng, 2018). Different reports suggest that coating pomegranate fruit or arils with Aloe vera, starch with or without oil combination (glycerol plus Oleum nigella), and lecithin provides a louver to water exchange between product and atmosphere, which reduces the mass loss (Opara, Atukuri, & Fawole, 2015).

| Ion leakage
An increased rate of electrolyte leakage has been used as an indicator of physical damage to cell membranes during low-temperature storage of horticultural produces. Electrolyte leakage measures the integrity of plant cells and tissues, and an increase in EL indicates deterioration in cellular membrane systems. As it is showed in Figure 5,  In all treatments, it is clear that the storage of arils for longer times will cause higher electrolyte leakage ( Figure 5).

SSE(0) SSE(0.1) SSE(1) SSE(0) SSE(0.1) SSE(1)
In papaya, ethylene suppressors could lower electrolyte leakage of fresh-cut fruits by lowering the respiration rate (Muharrem, Donald, Jiwon, & Jerry, 2006). The alginate-based coating containing 0.05% ε-PL significantly reduced electrolyte leakage of kiwi fruits (Li et al., 2017). Modified atmosphere packaging was also recognized as a useful tool in IL reduction of different fresh-cut produces during cold storage. It was also beneficial to use MAP in the storage of intact pomegranate fruits for long-term storage (Valdenegro et al., 2018).

| Decadence/Chilling injury
Since pomegranate is a subtropical fruit, chilling injury and decay of arils may be a significant problem during produce marketing, especially when they were kept in relatively low temperatures.
Using a more environment-friendly technique, which has no remaining residue, may be more favorable. Here, the results of this TA B L E 2 Effect of storage duration and different concentrations of Aloe Gel, saffron petal, and style extracts on TA (mg/l) and SSC (%) of pomegranate arils

| Biochemical composition
Total acidity, SSC, total phenol content, anthocyanin content, and antioxidant capacity of treated pomegranate arils are demonstrated in Table 2 and Table 3 Higher Aloe Gel percentage reduced TA and increased SSC in each storage period. The reduction was significant between control and AG 10%, nor for the AG 10 and 15%. Saffron petal and style extracts increased TA significantly in comparison with control (Table 2).
Anthocyanin content and antioxidant capacity of pomegranate arils decreased, and total phenol content increased significantly in longer storage. Aloe vera Gel did not affect AC (ug.L −1 ) and ACP (%) significantly in each storage period, while TPC (mg.L −1 ) decreased significantly in the higher percentage of Aloe Gel treatments.
Applying different concentrations of saffron petal extract, which contains anthocyanin, on pomegranate arils, increased AC, TPC, and ACP significantly (p ≤ 5%) (Table 3), but saffron style extract was not effective in changing those chemical characteristics of arils significantly (p ≤ 5%). In the application of saffron petal extracts on freshcut watermelon, similar findings in increasing AC were reported (Kaveh, 2017).

| Microbial contamination analysis
Initial microbial count of bacteria and mold was 2.097 and 2.163 log CFU m.L −1 , respectively. Through storage time, In different studies on pomegranate arils, packaging with different films changed Co 2 concentration and intercellular pH, which was successfully decreased microbial contamination (Ayhan & Eştürk, 2009;Banda, Caleb, Jacobs, & Opara, 2015;Belay et al., 2018). In suggest their application in food and pharmaceutical formulations (Muzaffar, Rather, & Khan, 2016). In an unpublished self-research, we had similar results of lower microbial contamination after application saffron petal and style extracts on "Jonagold " apple cubes.

| CON CLUS ION
There are so many different treatments used in pomegranate for more extended storage in favorable conditions and minimal defects of fruit quality. While the fresh-cut industry has pros and cons, in pomegranate, it will provide a possible use of fruit peel in food, health, and cosmetic products. Application of controlled or modified storage successfully increased arils quality in combination with the organic and inorganic compound. Using natural antioxidants and antimicrobials like saffron derivatives (Safranal) will lower preservative application in minimal fruit processing and bring more healthy food to the community. Besides their antimicrobial effects, saffron extracts, especially from unused parts of the flower, may also act like nutritional additives for each red or purple fresh-cut horticultural produce and can increase their nourishment.

ACK N OWLED G EM ENTS
We hereby thank University of Torbat Heydarieh for providing laboratory equipment and other facilities for conducting this research project.

CO N FLI C T O F I NTE R E S T S
Here, we declare that authors do not have any competing interests.

AUTH O R S ' CO NTR I B UTI O N S
H, K., S, V., contributed substantially to the conception and design of the study, the acquisition of data, the analysis, and interpretation.
Both authors have read and approved the manuscript.

E TH I C A L S TATEM ENTS
This study does not involve any human or animal testings.