Evaluation of Postharvest Storability of Ponkan Mandarins at Different Storage Temperatures Based on Principal Component Analysis

To reduce postharvest losses of Ponkan mandarins caused by outdated storage facilities and preservation technology, we evaluated the preservation effect of different storage temperatures on Ponkan mandarins (5 ±1, 10 ± 1, 15 ± 1, and 20 ±1 °C), and obtained a comprehensive score using principal component analysis (PCA) to determine its suitable storage temperature. The results indicate that, relative to the other three storage temperatures, storage at 10 °C significantly maintains high total soluble solid content, titratable acid, and vitamin C contents; the accumulation of malondialdehyde (MDA) and hydrogen peroxide (H2O2) content decreased and changes in the relative conductivity (REC) were suppressed; and high activities of superoxide-dismutase (SOD), peroxidase (POD), catalase (CAT), and ascorbate peroxidase (APX), as well as high contents of total phenol and total flavonoid were maintained. The PCA and clustering heat map results show that that the comprehensive score was the highest when stored at 10 °C. The data indicate that the suitable storage temperature of Ponkan mandarins at 10 °C significantly decreased MDA accumulation and reactive oxygen species metabolism, maintains high antioxidant capacity, maintains good fruit quality and achieves good storage and preservation effect, which is the appropriate storage temperature for Ponkan mandarins.


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Ponkan mandarins (Citrus reticulata Blanco, PM) are one of the main broad-skin citrus cultivars 32 and are widely cultivated in Southern China. Jing'an Ponkan mandarin is a famous mandarin 33 variety in Jiangxi Province, characterized by a compact plant type, early fruit bearing, rounded fruit 34 shape, bright color, sweet taste, crisp flesh, storage resistance, and late maturity. These mandarins 35 are a major source of economic income for fruit growers in the Jing'an area [1]. 36 Appropriate postharvest treatment can significantly reduce fruit loss, improve fruit quality, 37 and result in higher profits. However, citrus fruit may exhibit various disorders during harvest that 38 limit the storage period and reduce their commercial value [2]. Many factors affect the postharvest 39 storage of fresh fruit, such as low temperature storage, mechanical damage by harvesting and 40 transporting, and harvest periods [3][4][5]. The most common problem during the postharvest storage 41 of citrus is decay. Many reports indicate that the use of polyethylene film bagging [6], the 42 application of 1-methylcyclopropene [7], and coating with plants extracts or its main antifungal 43 constituents, such as cinnamaldehyde, limonene, and clove essential oil [8], during postharvest 44 treatment can effectively decrease the incidence of decay in citrus fruits during storage. increase antioxidant enzyme activity and maintain higher peroxidase (POD), catalase (CAT), 50 superoxide dismutase (SOD), and ascorbate peroxidase (APX) activities [17]. To some extent, the 51 appropriate storage temperature can inhibit the accumulation of MDA content. A too high or too 52 low storage temperature is harmful to fruits. Therefore, determining the suitable storage 53 temperature for Ponkan mandarins after harvest is crucial. Studies

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The decay rate of Ponkan mandarins due to peel browning and diseases was measured and 88 calculated on the initial fruit number for each lot every 15 days, and is expressed in percentage.

Weight Loss
The weight loss of Ponkan mandarins was measured and calculated on the initial fruit weight 91 basis for each lot every 15 days, and is expressed in percentage.

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The L* and CCI was measured using a MINOLTA CR-400 colorimeter (D65 light source; 94 Konica Minolta Sensing, Inc., Osaka, Japan) for 15 randomly selected fruits, described following the 95 method reported by Chen  108 We mixed 0.5 g of sample powder with 10 mL anhydrous methanol extractant, which was 109 shaken evenly. After 30 min of ultrasonic extraction at 50 °C and centrifuging at 5 000 ×g for 15 min, 110 the supernatant was extracted twice with the same extractant at 10 mL. The residue was washed 111 with a small amount of anhydrous methanol and combined with the supernatant. The supernatant 112 was stored at -40 °C for the TPC and TFC assays.

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The TPC was assayed following the Folin-Ciocalteu method reported by Goulas et al. [27] with 114 a few modifications. The extraction solution (0.5 mL) was mixed with FC reagent (0.5 mL) and 115 distilled water (5 mL). After standing for 3 min, 1 mL of sodium carbonate solution (10% m/v) was 116 added, and the absorbance at a wavelength of 725 nm was measured after reaction in light for 1 h. 117 Gallic acid was used as the standard sample to create the standard curve, and TPC is expressed as 118 mg/g.

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The aluminum nitrate colorimetric method by Wang et al [28] was applied to determine the 121 TPC, with certain alterations. About 0.3 mL of sodium nitrite (5% m/v) was added to the extraction 122 solution (2.4 mL). After blending and reacting for 6 min, 0.3 mL of aluminum nitrate (10% m/v) was 123 added. Then, after reacting for another 6 min, 4 mL of 1 M sodium hydroxide was added, and the 124 absorbance at a wavelength of 510 nm was measured after reaction in light for another 15 min. 125 Rutin was used as a standard sample to create the standard curve, and the TFC is expressed as 126 mg/g.

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The RI was measured using a GXH-3051H infrared carbon dioxide fruit and vegetable 129 respiration tester (Jun-Fang-Li-Hua Technology-Research Institute Beijing, China) in units of 130 mg·CO 2 ·kg -1 ·h -1 . We selected randomly 15 fruits for measurement after weighing.

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Malondialdehyde (MDA) MDA content (mmol/g) was measured using the thiobarbituric acid (TBA) method described 133 by Dhindsa et al. [29]. We ground 1.0 g peel to a fine powder and then homogenized it in 10 mL of 134 50 mM phosphate buffer saline (PBS, pH 7.8).The homogenate was centrifuged at 12,000 ×g for 20 135 min at 4 °C and 2 mL of the supernatant was mixed with 2 mL of TBA, heated at 100 °C for 30 min, 136 and then rapidly cooled. The supernatant was collected by centrifugation at 4 °C and 3000 ×g for 20 137 min at 4 °C. Absorbance was measured at 450, 532, and 600 nm. MDA content was calculated as 138 follows: (mmol/g) = (6.45 × (A 532 -A 600 ) -0.559 × A 450 ).

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For relative conductivity, 2.0 g of peel was removed with a 1 cm diameter puncher, rinsed 141 twice in distilled water, and blotted dry with clean filter paper. We then added 20 mL distilled 142 water to the cup and immersed the surface of the peel. After 20 minutes, a conductivity meter 143 (DDS-307A; Rex Shanghai, China) was used to measure the initial conductivity (P 0 ) of the sample, 144 and then the sample was boiled for 10 minutes to completely kill the tissue. Then, the sample was 145 cooled to room temperature, and the final conductivity (P 1 ) was assayed; relative electrical 146 conductivity = P 0 /P 1 × 100%.

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The H 2 O 2 content was assayed using the molybdate colorimetric method described by a H 2 O 2 149 test kit built in Nanjing (Nanjing Jiancheng Bioengineering Institute, Nanjing, China). We mixed 0.5 150 g peel powder after grinding with a sample machine with 9 × volume of saline according to the 151 ratio of weight (g):volume (mL) = 1:9. The supernatant was centrifuged for 10 min at 10,000 ×g. For 152 the remaining steps, we followed the kit instructions. 153 The H 2 O 2 content was calculated as follows: (mmol/g) = (determined OD value -blank OD 154 value)/(standard OD value -blank OD value) × standard concentration (163 mmol/L)/sample 155 weight to be tested.

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Determination of Antioxidant Enzyme Activities

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The POD activity was assayed using the guaiacol method reported by Jin et al. [30]. We 159 extracted 0.5 g of frozen peel with 5 mL of extraction buffer (containing 1% Triton X-100, 1 mmol 160 polyethylene glycol (PEG), 4% polyvinylpyrrolidone) and then centrifuged at 12,000 ×g for 30 min 161 at 4 °C. The supernatant obtained by centrifugation is the crude extract of the enzyme. We mixed 50 162 μL of the extraction with 200 μL of 0.5 mM H 2 O 2 and 3.0 mL, 25 mM of guaiacol . The OD value of 163 the reaction system was recorded at 470 nm every 10 seconds. More than 6 data records were 164 continuously measured.

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The SOD activity was measured using the Nanjing Superoxide Dismutase Test Kit (Nanjing 167 Jiancheng Bioengineering Institute, Nanjing, China). We extracted 0.5 g of frozen peel with 5 mL of 168 extraction buffer and then centrifuged at 12,000 ×g for 20 min at 4 °C. The supernatant obtained by 169 centrifugation is the crude extract of the enzyme. Other steps were conducted according to the 170 manufacturer's instructions. When the inhibition rate of SOD in 1 mL of tissue reaches 50%, the 171 corresponding SOD amount is one unit of SOD activity (U/g).

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The CAT activity was determined following the method reported by Havir et al.
[31] and 174 Robert et al. [32]. We extracted 0.5 g of peel with 5 mL extraction buffer (containing 5 mM 175 dithiothreitol and 5% polyvinylpyrrolid) and then the extraction was centrifuged at 4 °C at 12,000 176 ×g for 15 minutes. The supernatant obtained by centrifugation is the crude extract of the enzyme. 177 The reaction system for measuring CAT activity consisted of 80 μL of supernatant and 2.9 mL of 20 mM H 2 O 2 . The absorbance value of the reaction system was recorded at 240 nm every 30 seconds. 179 More than 6 data points were continuously measured.

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The APX activity was measured by spectrophotometry. We mixed 0.5 g of peel with 5 mL of 182 extraction buffer (containing 0.1 mM ethylene diamine tetraacetic acid, 0.5 mM ascorbic acid, and 183 2% polyvinylpyrrolidone) and then the extraction was centrifuged at 12,000 ×g for 30 min at 4 °C. 184 The supernatant obtained by centrifugation is the crude extract of the enzyme. We collected 080 μL 185 of the supernatant and mixed it with 0.3 mL of 2 mM H 2 O 2 and 2.6 mL of reaction buffer (containing 186 0.5 mM ascorbic acid and 0.1 mM EDTA). 187 The enzyme activity of each sample was determined using three replicates, and one unit of 188 activity is defined as 0.01ΔOD 470 ·g −1 ·min −1 , 0.01ΔOD 240 ·g −1 ·min −1 , and 0.01ΔOD 290 ·g −1 ·min −1 , for 189 POD, CAT, and APX, respectively. 196

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The decay rate and weight loss rate are important indexes for evaluating the storability of citrus 199 fruit [33]. Figure 1A illustrates the decay rate in each group was almost zero during the first 30 days 200 of storage. The rotten fruit appeared in 10, 15, and 20, and 5 °C groups at 45 and 75 days, 201 respectively. The decay rate of fruits stored at low temperature was 3.75-fold lower at the end of 202 storage than that of fruits stored at 20 °C. We found significant differences in each treatment group 203 (p < 0.01). Numerous previous studies reported that low temperature storage slows the decay of 204 citrus [3,34]. Low temperature storage may reduce the occurrence of fruit diseases and insect pests 205 [12]. In line with our findings, low temperature storage reduced the decay rate of Ponkan 206 mandarins.

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The weight loss of each group increased with storage time, as shown in Figure 1B, which was 208 mainly caused by water loss due to transpiration. Low temperature storage was shown to 209 significantly reduce the weight loss of Ponkan mandarins (p < 0.01). The lowest weight loss was 3% 210 at 5 °C, which is 3.67-fold lower than that of at 20 °C. As some studies reported, low temperature 211 storage can reduce fruit weight loss rate [9,25], which may be due to the slow water transpiration of 212 fruits caused by low temperature, creating a relatively high humidity environment and maintaining 213 the moisture content of fruits. gradually with the increase in storage time (Figure 2A). However, under low temperature storage, 220 the rate of CCI increase was significantly slower (p < 0.05) than at 15 or 20 °C. The value of L * 221 decreased slightly with the prolongation of storage time ( Figure 2B). During the whole refrigeration 222 period, L * was lower and significant (p < 0.05) at 15 and 20 °C than at 5 and 10 °C under low 223 temperature storage. In this experiment, Ponkan mandarins maintained a good appearance and 224 luster after long storage at a suitable storage temperature. These results are consistent with those reported in other studies [36,37]. Temperature affects the accumulation of carotenoids in citrus 226 fruits, thus significantly affecting its coloration. At the optimum temperature, the more the yellow 227 carotenoids accumulate in the pericarp, the better the color change, the higher the color index, and 228 the brighter the appearance. 229 As one of the organic acids involved in plant respiration, TA content is regarded as an 230 important index for evaluating the respiration rate of horticultural crops [25]. In our study, after 231 storage for 120 days, the TA content was significantly lower at 10 °C than at 15 and 20 °C (p < 0.01) 232 ( Figure 2A) of each treated fruit decreased rapidly ( Figure 2B). The TSS content of fruits at 10 °C was 240 significantly higher (p < 0.05) than other treatments ( Figure 2B) during the middle and late storage 241 periods (60-120 days). Similar results were reported by Alhassan et al. [15] for Afourer mandarins 242 and Navel oranges.

Effects of Different Storage Temperatures on Antioxidant Contents
244 VC is one of the key factors used to evaluate the quality of mandarin fruit. As shown in Figure  245 3A, the VC content of fruits stored at different temperatures first increased and then decreased 246 during the storage period.  The contents of total phenols and flavonoids in Ponkan mandarins increased at first and then 255 decreased at different storage temperatures ( Figures 3B, C). Total phenol content reached a peak at 256 45 days and then decreased ( Figure 3A). The decrease at 10 °C was slower and the rate was 257 significantly lower than that of the other three treatments (p < 0.05). The changes in flavonoids 258 content were similar to that of total phenol content, reaching the peak at 60 days, then decreasing. 259 At 10 °C, the TFC was 1.49 times higher than that at the beginning of storage ( Figure 3B), and was 260 significantly (p < 0.05) different from that of the other three treatments. In this study, high TPC and 261 TFC were maintained at 10 °C ( Figures 3B, C), consistent with previous reports that low 262 temperature storage can maintain high levels of TPC and TFC [40]. In this study, the results 263 indicated that the appropriate storage temperature could improve the disease resistance of fruits by 264 increasing the secondary metabolites with defensive ability in fruit tissues.

Effects of Different Storage Temperatures on Respiratory Intensity, MDA, REC, and H 2 O 2 266
The respiratory intensity of Ponkan mandarins increased under different temperature 267 treatments ( Figure 4A). Low temperature storage significantly slowed the increase in the 268 respiratory rate of Ponkan mandarins, and a significant difference (p < 0.05) at 5 and 10 °C was 269 found between the earlier and later stage of storage and at 15 and 20 °C (p < 0.05). Similar results 270 were reported by Cheng et al. [41] for Annona chinensis storage; the respiratory rate of A. chinensis 271 storage at 4 °C was significantly lower than that at 8 °C. This is analogous to low temperature 272 storage, which can reduce the respiration of fruits during storage. 273 Active oxygen metabolism increased during storage, reactive oxygen species (ROS) 274 accumulation destroyed cell membrane structure, and MDA content subsequently increased [42,43], which then accelerated fruit senescence. The MDA content at the beginning of fruit storage was 1.3 276 mmol g -1 . At the time of storage, the MDA content of the treated group increased, and that of the 10 277°C group increased 3.15-fold, which was lower than the other three treatment groups, and was 278 significant (p < 0.05) ( Figure 4B). MDA is the final product of membrane lipid peroxidation, which is 279 closely related to aging and is one of the direct indicators of membrane oxidative damage. These 280 results are similar to those reported by Wang et al. [16], where low temperature storage at 0 and 10 281°C inhibited the increase in MDA content in cherry fruits compared with high temperature storage 282 at 20 and 30 °C. Therefore, the inhibition of MDA content may be related to the senescence and high 283 VC content of fruits stored at low temperature. To further understand the reason for this change, in 284 future analysis, we should determine the protective enzyme activity that delays lipid peroxidation 285 and cell aging.

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The relative electrical conductivity of Ponkan mandarins increased at different storage 287 temperatures ( Figure 4C) because the gradual senescence of fruits during storage may lead to 288 increased permeability of the pericarp cell membranes. After storage for 120 days, relative 289 conductivity was 30.1% at 10 °C, and 32.6%, 36.2%, and 39.2% for 5, 15, and 20 °C, respectively. The 290 relative conductivity was significantly lower (p < 0.05) at 10 °C than for the other three treatments. 291 Hydrogen peroxide is one of the important representatives of ROS, and its accumulation will 292 cause fruit senescence. During the whole storage process, the H 2 O 2 content of Ponkan mandarins 293 increased first and then decreased, but increased overall ( Figure 4D). At 10 °C, the increase in H 2 O 2 294 content was lower compared with the initial storage, by only 1.19 times, significantly lower than the 295 other three treatment groups (p < 0.05). In this study, low temperature storage delayed fruit 296 senescence, resulting in a reduction in the accumulation of hydrogen peroxide.

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The activities of POD, CAT, SOD, and APX are closely related to antioxidation and anti-aging 299 in plant tissues. Those enzyme activities in Ponkan mandarins increased at first and then decreased 300 at different storage temperatures ( Figure 5).

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The POD activity peaked at 30 days and then decreased rapidly ( Figure 5A). The rate of 302 decline at 10 °C was significant (p < 0.05), which was slower than the other three treatments. The 303 CAT activity was significantly higher (p < 0.05; Figure 5B) at the later stage of storage at 10 °C, and 304 the decline rate was slower than that of the other three storage treatments. The SOD activity was 305 1.29 times higher than that of the initial storage ( Figure 5C), and significantly higher than that of the 306 other three treatments (p < 0.05). APX activity decreased significantly during storage at 15 and 20 ° 307 C compared with lower temperatures (p < 0.05; Figure 5D).

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The excessive production and accumulation of ROS caused by fruit senescence destroys the CAT usually changes with the change in ROS level. APX is the key enzyme for removing hydrogen 316 peroxide in chloroplasts and the main enzyme for vitamin C metabolism. In our study, the results 317 indicated that low temperature storage enhanced the activities of POD, CAT, SOD, and APX, and 318 lessened the accumulation of MDA (Figures 4 and 5 for SOD, CAT, and APX activities ( Figure 6).

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After 60 days of storage, the score dropped sharply (Figure 7), indicating that the optimal 335 storage time of Ponkan mandarins is 60 days. When the storage time is prolonged, the antioxidant 336 and anti-aging ability of the fruit gradually decreases. The comprehensive score of 10 °C storage 337 fruits was always the highest among the four storage temperatures, which maintained good fruit 338 quality. Therefore, the optimum storage temperature of Ponkan mandarins is 10 °C.

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We found a significantly positive correlation between H 2 O 2 content and SOD activity (r = 0.364,

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In conclusion, the storage temperature of 10 °C can effectively maintain the quality of the fruit 360 by improving the storability of Ponkan mandarins, reducing the accumulation of MDA and H 2 O 2 , 361 reducing the level of ROS, maintaining high levels of defense enzyme activities, improving disease 362 resistance, delaying fruit aging, and prolonging shelf life. We did not delve into the molecular 363 mechanism of how low temperature maintains fruit quality, which requires further exploration.  370

Conflicts of Interest:
The authors declare no conflict of interest.