Fruit species including oranges have gained more popularity recently. They include high content of nonnutritive, nutritive and bioactive compounds such as flavonoids, phenolics, anthocyanins and phenolic acids, as well as nutritive compounds such as sugars, essential oils, carotenoids, vitamins and minerals. They have distinct flavour and taste, excellent medicinal value and health care functions as well (Gundogdu et al., 2014; Ersoy et al., 2018; Engin and Mert, 2020; Kaskoniene et al., 2020). Citrus fruits are the most commercial fruit crop, intended for regional consumption and export. ‘Washington’ navel orange (
In order to assess the influences of different concentrations of ME on ‘Washington’ navel orange fruits, at maturity stage fruits were harvested from a commercial orchard existing in the Dakahlia Governorate, Egypt (30.04°N, 31.25°E). On the same day of harvesting, 1 h later, 600 fruits were selected to be identical in colour, shape and size as well as without any peel defects. They were divided into two main groups to assess the physical and chemical characteristics; each one contains 300 fruits divided into four treatments. Each treatment consists of 75 fruits with three replicates (25 fruits/replicate). Initially, the fruits were washed by distilled H2O to remove the microbial and dust content loaded from the field. At laboratory temperature, the exogenous ME treatments were applied using four different concentrations (0 mM, 10 mM, 100 mM, and 1,000 mM) by immersing for 20 min (Jannatizadeh, 2019). Treated fruits and control were stored at 4 ± 1°C and 95 ± 1% RH for 4 weeks. All chemical reagents and ME were procured from Sigma, USA.
The CI-symptoms in orange fruits appear as brownish wrinkled areas/spots, where their number and size increase with the duration of cold storage. Based on the orange peel necrosis and the intensity of the browning colour, the CI-index was measured visually in a range of 0 (no CI-symptoms) to 5 (very severe CI-symptoms). CI-index has been determined using the formula following the method of Sayyari et al. (2009); CI (%) = σ (value of scale) × (number of fruit with the corresponding scale number)/(total number of fruit × 4) × 100. The water loss was determined at the end of each storage utilising the equation following the method of Sun et al. (2019) and it was expressed as a percentage based on the initial fruit weight prior to the cold storage at harvest time. Water loss (%) = [(initial weight – final weight)/initial weight] × 100. The orange fruit peel colour (hue angle, h°) was evaluated by overall storage duration intervals following the method mentioned by Schirra (1992).
At laboratory temperature, SSC% of orange juice was determined utilising the refractometer, a hand digital, Model MASTER-PM Cat. No2393 (ATAGO, Japan). For TA% determination, 20 ml of orange juice was used to titrate with 0.1 N NaOH. On this basis, the maturity index was assessed, according to Schirra et al. (1998), by calculating the SSC/TA-ratio.
About five grams of peel tissue was blended with 50 mM phosphate buffer (pH 7.8), 0.2 mM of EDTA and 2% polyvinyl polypyrrolidone (PVPP). After centrifugation 30,000 ×
MDA content was assessed following the procedure depicted by Iturbe-Ormaetxe et al., (1998) using thiobarbituric acid method. About 2.5 g of grinded orange flavedo was blended with a mixture of 500 ml of butylated hydroxytoluene (2%, w/v) as well as 25 ml of metaphosphoric acid (5%, w/v) well in ethyl alcohol. Through determining the 1,1,3,3-tetraethyoxypropane in the range from 0 mM to 2 mM of TBARS that has been equal to MDA in the range from 0 Mm to 1 Mm, the calibration curves have been obtained. Stoichiometrically, tetraethyoxypropane is transformed into MDA through the acid-heating step of the experiment. Regarding the IL% determination; 5 g of orange sample (peel) was cut into discs and washed using demineralised water (three times) and placed in 20 ml of 0.4 M mannitol for 3 h at 24 °C; then the sample was measured as the initial electrical conductivity of the solution (EC1). After that, the sample was cooked at 100 °C for 30 min in a water bath to measure the final leakage after leaving the sample at room temperature (EC2). The IL was calculated based on IL (%) = (EC1/EC2) × 100 (Hakim et al., 1999). AA has been estimated by a titration process employing 6% oxalic acid and 2,6-dichlorophenolindophenol reagent (AOAC, 1995).
The H2O2 content was determined following the procedure depicted by Xu et al. (2012). One gram of peel tissue has been added to 5 ml acetone. After centrifugation 6,000 ×
The average data of two growth seasons (2019–2020) for the present study has been examined statistically. The Co-Stat software package, Ver. 6.303 (789 lighthouse Ave PMB 320, Monterey, CA, 93940, USA) was used. Data have been analysed as a completely randomised design (CRD) with three replications. The means of all examined treatments results have been contrasted utilising Duncan's Multiple Range Test at
All ME treatments showed a higher keeping of fruit peel colour (h°) and a significant decrease in the CI-index and water loss% compared to the untreated fruits (control) during cold storage for 4 weeks. Results of CI-index, water loss% and orange peel colour (h°) as physical properties of the ‘Washington’ navel orange fruits at cold storage intervals up to 4 weeks are shown in Figure 1. The current results indicated that the 1,000 mM ME treatment caused the lowest value of CI-index compared to the other treatments. Such treatment recorded (1.02) at the 3rd week, and then developed slightly to (1.03) at the end of storage time, while the control treatment presented the highest value (4.09) at the end of storage period. With respect to the water loss% and the orange peel colour (h°), the same treatment resulted in a significant decrease of the water loss% (3.84%) and a slight decrease in the peel colour of the fruits (h° = 69.67) compared to the control, which recorded the highest value of water loss% (10.97%) and a sharp decrease in the peel colour (h° = 50.75) at the 4th week of the cold storage period. The variation in physical parameters is due to the effect of low temperature stress, which generates ROS during long-term cold storage (Lo’ay and Doaa, 2020). The most ROS produced is the hydroxyl radical (OH•), which reacts with lipids and proteins of the plasma cell membrane to produce the MDA (Hodges et al., 2004) and protein carbonyl groups (Wang, 2006). After that, the cell membrane loses its structure and functions (Lo’ay et al., 2019), and then the cell dies (Foyer et al., 2017). More generation and accumulation of ROS is associated with more activity of membrane-degrading enzymes such as phospholipase (Aghdam et al., 2018) and LOX (Jannatizadeh, 2019). Consequently, the CI incidence symptoms appear (Lo’ay and Doaa, 2020), which depend on the equilibrium between ROS formation and antioxidant system performance under minimum temperature (Hodges et al., 2004). ME is more effective for membrane safety because it directly scavenges the ROS and indirectly improves the activity of the scavenging systems, which leads to a reduction of ROS accumulation (García et al., 2014).
The effect of ME treatment at four different concentrations on (CI-index), water loss% and fruit peel colour (h°) of ‘Washington’ navel orange fruits during cold storage at (4 ± 1 °C and 95 ± 1% RH) for (4 weeks). The vertical bars represent standard error (±SE of
The differences in chemical quality characteristics of ‘Washington’ navel orange fruits affected by ME treatments and cold storage period up to 4 weeks are represented in Figure 2. It was observed that the untreated fruits (control) gave the highest values of SSC% and SSC/TA-ratio, and the lowest value of TA% during 4 weeks of the cold storage compared to the ME treatments. Also, the 1,000 μM ME treatment provided the lowest changes in SSC% and SSC/TA-ratio over the length of chilly storage compared to other treatments. It recorded 13.42% for the SSC% and 16.53 for the SSC/TA-ratio, while maintaining the stability of TA% at 0.812% after 4 weeks of the cold storage. The variances in SSC% and TA% measurements of stressed orange fruits through cold storage may occur because of the conversion of organic acid into sugar (Baldwin et al., 1995). Also, an increase in SSC/TA-ratio during the storage period may occur because of decreasing AA amount and acidity (Wang et al., 2014) and increasing starch enzyme activity (Brizzolara et al., 2020) under prolong storage duration. The same results have already been observed in lime (Verma and Dashora, 2000) when the fruits are stored at chilled temperatures.
The effect of ME treatment at four different concentrations on (SSC%), (TA%), and SSC/TA-ratio of ‘Washington’ navel orange fruits during cold storage at (4 ± 1°C and 95 ± 1% RH) for (4 weeks). The vertical bars represent standard error (±SE of
The change in AEAs in all treatments increased slightly at the 1st week, compared to the initial values, and then became differential from the 2nd week up to the 4th week of cold storage period based on ME concentrations. The activities of antioxidant enzymes, i.e. APX, CAT, SOD and POD in ‘Washington’ navel orange fruits affected by ME treatments and cold storage period for 4 weeks at
The effect of ME treatment at four different concentrations on AEAs of ‘Washington’ navel orange during cold storage at (4 ± 1°C and 95 ± 1% RH) for (4 weeks). The vertical bars represent standard error (±SE of
The level of MDA and IL% increased significantly at
The effect of ME treatment at four different concentrations on the MDA accumulation (μM · g−1 FW), (IL%), AA content (mg · 100 g−1 FW) of ‘Washington’ navel orange during cold storage at (4 ± 1°C and 95 ± 1% RH) for (4 weeks). The vertical bars represent the standard error (±SE of
The changes in generation rates of H2O2 and O2•− as well as antioxidant capacity (using DPPH%) affected by ME treatments and the cold storage period up to 4 weeks are shown in Figure 5. The generation rates of H2O2 and O2•− increased perceptibly in all treatments except 1,000 μM ME treatment that increased slightly generation rates of H2O2 and O2•− starting from the 2nd week until the end of the storage period, while recording the lowest rates of H2O2 (0.11 mmol · min−1 · g−1 FW) and O2•− (0.31 mmol · min−1 · g−1 FW) in the 4th week compared to other ME treatments and control. The antioxidant capacity of ‘Washington’ navel orange fruits affected by all treatments and storage period (4 weeks) was registered as the free radical scavenging impact using DPPH reduction% method. Observing the antioxidant capacity, the treated fruits exhibited varying and strong degrees of antioxidant profile compared to untreated fruit (control), which showed the lowest activity with inhibition% at 32.44% in the 4th week. The highest antioxidant activity was revealed by 1,000 μM ME treatment, which exhibited inhibition% at 42.86% in the 4th week and was more effective than the other treatments. The current results are in agreement with Ma et al. (2016) and Rapisarda et al. (2008), who reported that ME may be able to quench the generation of H2O2 and O2•− directly, or through enhancing the activities of antioxidant enzymes during the cold storage. Additionally, the relationship between CAT and APX alongside other antioxidants could be significantly linked to quenching of H2O2 and O2•− (Foyer et al., 2017 and Yang et al., 2011). On the other hand, Aghdam and Fard (2017) stated that strawberry fruits affected by ME treatments demonstrate significantly high DPPH scavenging activity during cold storage.
Shows DPPH reduction%, O2•− (mmol · min−1 · g−1 FW), and H2O2 (mmol · min−1 · g−1 FW) of ‘Washington’ navel orange during cold storage at (4 ± 1 °C and 95 ± 1% RH) for (4 weeks). The vertical bars represent the standard error (±SE of
The use of 1,000 μmol ME treatment exhibited remarkable tolerance against the cold storage stress, that is represented in minimising both CI-index and the water loss and preserving the orange peel colour (h°) during cold storage duration. Furthermore, the same treatment enhanced the activities of antioxidant enzymes, recorded the lowest values of MDA and IL%, minimised the H2O2 and O2•− production and presented higher antioxidant capacity. Finally, applying ME treatment at 1,000 μmol could be recommended as the best treatment to improve the quality and storability of ‘Washington’ navel orange fruits during cold storage.