Fruit appearance is an important aspect of consumer purchase and acceptability where the green fresh condition of the fruit calyx (button) is a desirable consumer attribute of citrus appearance [1
]. Changes in the physical appearance of the calyx can occur as a result of either natural senescence or external stimuli such as heat stress, causing colour change (from green to yellow and brown), with abscission of the calyx. Retention of the calyx is important not only because it enhances cosmetic appeal, but it also protects against fungal infection in the abscission zone of the fruit peduncle and extends fruit storage life [2
]. Both mandarins and to a lesser extent oranges are susceptible to calyx senescence during storage.
Citrus fruit can be stored for up to 8 weeks under optimal conditions, but storage life depends on pre-harvest conditions, cultivar and storage environment, where lower storage temperatures reduce fruit senescence [3
]. However citrus fruit can be sensitive to chilling injury (CI) when exposed to chilling temperatures (<3 °C to 5 °C) depending on citrus type and cultivar.
Citrus fruit are considered non-climacteric, but are responsive to postharvest application of ethylene [3
], which has been shown to induce a desirable colour change in citrus fruit, and is widely used to commercially degreen early season fruit [4
]. A short-term ethylene treatment (4 days with 2 μL L−1
ethylene) has also been shown to protect fruit from stress related postharvest disorders such as CI and non-chilling peel pitting [6
]. Storage of citrus fruit in an ethylene environment at low temperature (0 °C) has been shown to reduce the development of CI symptoms [7
]. However, ethylene has also been shown to produce undesirable effects related to accelerated fruit senescence including abscission of the calyx, calyx browning and calyx drying [8
]. Brown and Burns [9
] found that fruit exposure to ethylene (55 μL L−1
) during degreening induced calyx senescence in ‘Valencia’ oranges and increased calyx abscission. However, studies by Li et al. [10
] showed that when ethylene levels were decreased from 1 to <0.001 μL L−1
, the incidence of calyx senescence in ‘Afourer’ mandarins diminished. The development of undesirable characteristics in citrus as a consequence of ethylene exposure varies with citrus type and cultivar [11
]. Lafuente et al. [6
] found that while an ethylene concentration of 2 µL L−1
at 2 °C storage reduced calyx abscission in ‘Lane Late’ oranges, the same treatment conditions for ‘Navelate’ oranges resulted in high calyx abscission.
Many studies on the effects of ethylene on citrus fruit quality have been conducted examining the effects of commercial degreening treatments, where high levels of ethylene (up to 4 μL L−1
) are applied for a short period of time (a few days) [12
]. These short-term degreening treatments are not comparable to longer treatment and storage times, where fruit can be exposed to low levels (<1 μL L−1
) of ethylene during storage [10
Ethylene exposure at low concentrations during storage has been linked to compositional changes of citrus [15
]. Li et al. [10
] observed lower ethanol levels in ‘Afourer’ mandarins during prolonged storage (10 weeks) in a low ethylene atmosphere while Mahler et al. [17
] found no effect on total soluble solids (TSS) and titratable acidity (TA) levels in ‘Navelina’ orange stored under 0.5 µL L−1
ethylene at 5 or 20 °C. Accumulation of volatiles such as ethanol is often associated with off-flavour sensory qualities, particularly in citrus juice products [18
] and must therefore be avoided during long-term storage.
There are few reports on assessing the effect of long-term ethylene exposure on calyx condition and internal quality attributes of mandarins and oranges held in long-term storage (cold or ambient temperature) [6
]. The few cited studies conducted have not undertaken an extensive evaluation of the impact of ethylene concentrations and storage temperature on fruit quality parameters, including vitamin C content and antioxidant activity. The objective of this study was to assess the impact of four ethylene concentrations and different storage temperatures on calyx senescence and internal quality parameters of ‘Afourer’ mandarins (also known as ‘Nadorcott’ or ‘W. Murcott’) and Navel oranges during long-term storage.
2. Materials and Methods
2.1. Plant Materials and Experimental Procedure
Mature mandarin fruit (Citrus reticulata Blanca cv. ‘Afourer’) were obtained from a commercial packinghouse in Gayndah, Queensland, Australia (25°38′00″ S, 151°35′47″ F). Fruit used for this experiment had their calyx intact, were waxed with commercial carnauba wax and were not treated with the growth regulator 2,4-dichlorophenoxyacetic acid (2,4-D). Fruit for the experiment were uniform in size and free from visible defects. Fruit were randomized and placed into 48 replicate net bags (n = 20 fruit per bag). The bags were then evenly distributed into 6.3 L plastic containers (n = 16) fitted with inlet and outlet airflow ports and stored at either 5, 10 or 20 °C. Individual containers within each temperature environment were then connected to one of four ethylene containing humidified air streams (≤0.001, 0.01, 0.1 and 1 μL L−1) operating at a flow rate of 50 mL min−1. The ≤0.001 µL L−1 stream was considered to be ethylene-free (air) as the ethylene concentration is below the limit of detection by gas chromatography. The experiment was monitored daily. Mandarin fruit stored at 20 °C were assessed as weekly removals for four weeks, while fruit stored at 10 and 5 °C were assessed every two weeks for eight weeks.
In a parallel study, Navel oranges (Citrus sinensis L. Osbeck) were harvested at commercial maturity from a New South Wales Department of Primary Industries (NSW DPI) research farm at Somersby on the NSW Central Coast, Australia (33°36′50″ S, 151°28′50″ F). Fruit were transported to NSW DPI postharvest laboratories (Ourimbah, NSW, Australia) (10 min), where the fruit was sanitised with commercial sodium hydrochlorite solution (50 ppm) before being washed and air dried. The fruit was then sized and sorted as previously described, randomized and placed in net bags as individual treatment units (n = 20 fruit). The treatment units were then placed in 60 L steel drums and stored in two different temperatures (1 and 10 °C). The individual drums within each temperature were connected to one of the four humidified air streams containing ethylene (≤0.001, 0.1 and 1 µL L−1) operating at a flow rate of 400 mL min−1. The drums were inspected daily. There were four replicates (in different drums, i.e., independent replicates) for each treatment with assessments after 1, 5 and 10 weeks at both storage temperatures and again with an additional shelf-life assessment following 5 days storage at 20 °C.
2.2. Physio-Chemical Assessments
2.2.1. Calyx Senescence and Chilling Injury Assessment
Calyx senescence was visually evaluated with changes in colour of the calyxes from green to brown and scored according to Li et al. [10
] using a 5-point scale where 1 = green, 2 = slightly yellow, 3 = moderately yellow, 4 = totally yellow and 5 = brown and the mean score of all fruit in the sample then calculated. Calyx abscission was assessed by the presence or absence of the calyx of the fruit. Calyxes which had abscised during storage were counted on each assessment day and the results expressed as percentage calyx abscission. Chilling injury (CI) was assessed using a 4 point scale previously described by Lafuente and Sala [20
] where 0 = normal (no pitting symptoms), 1 = slight pitting (a few scattered pits), 2 = moderate pitting (up to 30% surface covering), 3 = severe pitting (>30% surface covering), with results expressed as a percentages of the total number of fruit evaluated in the experiment.
2.2.2. Weight Loss
Weight loss of the fruit was assessed using an electronic balance (Model Kern & Sohn GmbH, D-72336, Balingen, Germany), where fruit weight of each treatment unit was recorded each assessment day. Weight change was expressed as a percentage value determined by deducting the initial weights (W1) from the final weights (W2) divided by the initial weights and multiplied by hundred percent (%).
2.2.3. Measurement of Fruit Firmness
A texture analyser (Lloyd Instrument LTD, Fareham, UK) was used to determine firmness of ten fruit per replicate after the fruit had conditioned to 20 °C. The maximum force (N) was measured by compressing the fruit in the equatorial zone between two flat surfaces closing together at the rate of 1 mm min−1
to a depth of 2 mm. The average of two reading points from each side of the fruit was recorded [21
2.2.4. Respiration Rate
Respiration rate (mL CO2
) was measured on five fruit from each replicate. Fruit were sealed into an airtight 2 L glass jars fitted with a septum for 3 h to accumulate respiratory gases. Carbon dioxide (CO2
) concentration in the jar was determined by withdrawing a 1 mL gas sample from the headspace and injecting into a gas chromatograph (Gow-Mac, Bridgewater, NJ, USA) fitted with two stainless steel columns (60 cm × 1 mm i.d.) connected in series. Operating temperatures for the detector, injector, and column were 110 °C, 50 °C and 110 °C respectively. The carrier gas used was high purity argon (BOC Gases, Sydney, NSW, Australia) at a flow rate of 25 mL min−1
. Respiration rate was calculated according to the method of Huque et al. [22
2.2.5. Internal Fruit Quality Assessments
Two fruit from each replicate were manually juiced and sieved through two layers of cheesecloth. TSS content of the juice was determined using a digital refractometer (Atago Co., Ltd., Tokyo Atago, Japan) and expressed as °Brix. TA was determined by titrating 5 mL of the juice with 0.1 M NaOH to pH 8.2 with an automatic titrator (Mettler Toledo, Greifensee, Switzerland) and data were expressed as percentage citric acid. For ethanol accumulation, 10 mL of juice was transferred into a 20 mL glass vial with crimp-top caps sealed with silicone septa. The sealed sample was then incubated in a water bath of 30 °C for 10 min before analysis. Ethanol accumulation was determined by headspace analysis using a gas chromatograph (Model 580, Gow-Mac, Bethlehem, PA, USA) with a flame ionization detector and a column (Carbwax, Gow-Mac, Bethleham, PA, USA). The injector was set at 190 °C, the column at 68 °C, the detector (FID) at 190 °C with gas flow rates of 30, 30 and 300 mL min −1 for nitrogen, hydrogen and air respectively. After incubating the samples, 1 mL of the headspace gas sample was drawn from the vials and injected into the GC. Ethanol accumulation was calculated and expressed as g L−1.
2.2.6. Vitamin C Content
Ascorbic acid was determined by the indophenol titration method adapted from the method of Nielsen et al. [23
]. Briefly, 10 mL of fruit juice was extracted from fruit and filtered through two layers of cheesecloth. Five (5) mL of metaphosphoric acid-acetic acid solution was then added to the filtered extract and the samples then titrated with the 2,6-dichloroindophenol dye solution until samples turned light rose-pink colour. The quantity of dye used in the titration was then used to calculate the vitamin C content present.
2.2.7. Antioxidant Activity
The antioxidant activity of the ‘Afourer’ mandarin fruit were determined using ferric reducing antioxidant power (FRAP) assay as described by Thaipong et al. [24
], with some modification. A working FRAP solution was prepared by mixing 300 mM acetate buffer, 10 mM 2,4,6-Tris(2-pyridyl)-s
-triazine (TPTZ) reagent in 40 mM HCl and 20 mM ferric chloride in the ratio of 10:1:1 and warmed to 37 °C in a water bath. 2.85 mL of the FRAP working solution was then added to a 150 mL aliquot of mandarin juice and the resulting solution allowed to react in the dark for 30 min at 20 °C. The solution absorbance (λ = 593 nm) was then recorded using a UV-Vis spectrophotometer (Varian Australia Pty. Ltd., Melbourne, VIC, Australia) and the result referenced against a standard curve and expressed as µmol of Trolox equivalents (TE) per 100 mL fresh mandarin juice.
2.2.8. Statistical Analysis
Data from each of the experiments were subjected to two-way analysis of variance (ANOVA) using SPSS Microsoft version 24.0 software package (SPSS, Chicago, IL, USA). Tests for significance difference of treatment means were at p ≤ 0.05. The method used to discriminate among the means (Multiple Range Test) was Fisher’s Least Significance Difference (LSD) procedure at 95% confidence level.