Reduction of chilling injury of ‘Washington’ navel orange fruits by melatonin treatments during cold storage

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 × g for 20 min at 4 °C, supernatant was recovered and utilised for the activities of antioxidant enzymes as described by Aghdam and Fard (2017). For determination of catalase (CAT), 3 ml of the reaction mixture (12.5 mM H₂O₂, 0.2 ml of enzyme extract and 50 mM (pH 7.0) phosphate buffer) was used. One unit of CAT enzyme activity was identified based on a decreasing of absorbance at 240 nm per min, where the decomposition of H₂O₂ has been occurring. For determination of ascorbate peroxidase (APX), 3 ml of the reaction mixture (0.1 ml of enzyme extract, 9 mM AA, 50 mM phosphate buffer (pH 7.0) and 12.5 mM H₂O₂) has been used. Following the decrease in absorbance at 290 nm due to AA consumption, the APX activity has been assayed. Superoxide dismutase (SOD) activity has been specified based on its capability to prevent the nitro blue tetrazolium (NBT) photochemical reduction. For SOD determination, 3 ml of the reaction mixture (14 mM methionine, 50 mM phosphate buffer (pH 7.8), 1 mM (NBT), 3 mM of EDTA, 0.1 ml of enzyme extract and 60 mM riboflavin) was used. Through observing the absorbance at 560 nm, the blue formazan formation was detected. One unit of superoxide dismutase activity was identified as the enzyme amount which inhibits the reduction of NBT to the extent of 50%. For peroxidase (POD) activity, 3 ml of the reaction mixture, including of phosphate buffer (pH 6.0), enzyme extract, 0.1% guaiacol and 2% H₂O₂, was used after incubation for 5 min. The absorbance of mixture was recorded at 460 nm, where one unit was displaying an increase of absorbance per minute (Tian et al., 2005). Determination was calculated as Unit · g⁻¹ protein based on the total soluble protein in samples by the method of Bradford (1976).

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 × g for 15 min at 4 °C, the clear extraction has been packed. We added 1 ml of the latter clear extraction to 0.2 ml ammonia and 0.1 ml titanium sulphate (5%), and then centrifuged 6,000 × g for 10 min at 4 °C. The pellets (titanium-peroxide complex) that formed have been dissolved in 3 ml of sulphuric acid 10% (v/v) and centrifuged 5,000 × g for 10 min at 4 °C. The absorbance of the resulting supernatant was determined at 410 nm. Using H₂O₂ as a standard curve, the H₂O₂ content was calculated and then expressed as mmol · min⁻¹ · g⁻¹ fresh weight (FW). O₂^•− production rate was determined through the formation of nitrite from NH₂OH in the presence of O₂ according to the procedure depicted by Yang et al. (2011). At 530 nm, the absorbance has been recorded. To determine the production rate of O₂^•− from the reaction equation of NH₂OH with O₂, a standard curve with NO₂ was used. The production rate of O₂^•− has been identified as mmol · min⁻¹ · g⁻¹ FW. Regarding the antioxidant capacity, 3 g peel tissue was added to 30 ml methyl alcohol and then centrifuged at 10,000 × g for 15 min. The resulting clear extraction (1 ml) was added to 3 ml of 0.1 mM 2,2-DPPH that dissolved in methyl alcohol. At room temperature, the reaction mixture was incubated in dark for 20 min. Using a spectrophotometer, the absorbance has been determined at 517 nm and consequently the DPPH radical scavenging impact has been recorded. The DPPH% of the sample has been determined (Cao et al., 2018) as follows: [(Abs control − Abs sample)/Abs control] × 100.

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.

Figure 2

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.

Figure 5