Effects of Different Carbon Dioxide-Modiﬁed Atmosphere Packaging and Low-Temperature Storage at 13 ◦ C on the Quality and Metabolism in Mango ( Mangifera indica L.)

: Mangoes ( Mangifera indica L.) were stored under four different carbon dioxide-modiﬁed atmosphere packaging (MAP) combinations at 13 ± 1 ◦ C to investigate their effects on moisture distribution and content, physiological metabolism, as well as fruit quality. The mangoes stored under C7 combination (7% CO 2 + 3% O 2 + 90% N 2 ) maintained respiration rate, inhibited the increase in 1-aminocyclocarboxylic acid-1-carboxylic acid synthase (ACS) content, and slowed down the senescence process of the fruit. The mangoes subjected to C7 combination also maintained higher ﬁrmness, protopectin, and free moisture content. The C7 combination suppressed the increase in soluble pectin and malondialdehyde (MDA) content, with the lowest weight loss. The yellowing rate of the mango pulp preserved under the C7 combination condition was signiﬁcantly reduced, and the loss of vitamin C was reduced from the 0th to the 6th day of storage. The treatment with lower carbon dioxide content was not as effective as C7 combination. In conclusion, 7% CO 2 + 3% O 2 + 90% N 2 MAP conditions delayed pulp yellowing and biochemical characteristics and maintained ﬁrmness and free moisture content along with better quality of mango for 30 days at low temperature.


Introduction
The mango (Mangifera indica L.) is one of the most important tropical fruits in the world and is known as the "king of tropical fruits" [1]. This kind of fruit is deeply loved by consumers for its attractive color, deliciousness, and unique flavor. At the same time, the mango contains fiber, vitamins, minerals, and several bioactive compounds that can improve human antioxidant defense ability and promote physical health. However, the mango is easily perishable after harvest, and its life is reduced by fungal attack, external environment factors, and postharvest respiration [2].
Fungicides and chemicals used to extend the storage time of mangoes after harvest are harmful to human health and the environment [3]. Therefore, it is necessary to explore safe and environmentally friendly methods to maintain the quality of mangoes and extend their shelf life. Modified atmospheric packaging (MAP) is an effective way to preserve the quality of fruits and extend their shelf lives by changing the gas ratio in the storage environment [4]. Low O 2 and high CO 2 conditions are formed inside the packages for fruit storage, which can inhibit the growth of microorganisms, reduce the respiration rate of fruits, delay the metabolic rate after harvest, and reduce the loss of nutrients [5]. A host of examples have highlighted that MAP could be used to extend the storage period of agricultural products, such as sweet corn [6], kiwifruit [7], etc. The recommended and commonly used controlled atmosphere to extend the shelf life of mangoes is 3-5% O 2 and 5-10% carbon dioxide [8]. However, different results have been reported in the literature to prove that the variety of mango has a significant impact on the preservation in modified atmosphere conditions. When the stored oxygen condition is less than 2%, it may cause anaerobic respiration in mangoes, the development of "off-flavors", discoloration of the fruit, irregular ripening, etc. [9,10]. Therefore, it is important to determine the gas components in MAP storage and use it for different mango varieties to maintain quality attributes and extend the shelf life of mangoes.
Low-temperature storage is a valuable postharvest strategy to maintain product quality and is widely used to extend the shelf life of fruits [11]. The suitable storage temperature for mangoes is 10-13 • C, which varies for different varieties. Below 10 • C, mangoes are susceptible to chilling damage and gradually develop blackening and pitting of the peel, discoloration of the flesh, and reduced aroma and flavor [12]. As far as we know, there have been few reports on the study of low-temperature and MAP storage on mangoes. This study aims to recommend the effect of different carbon dioxide and oxygen MAP storage conditions on the quality and metabolism of fresh mangoes during storage at 13 • C.

Experimental Materials and Treatment
Mangoes were obtained from Shanghai Daozhi Agricultural Products Co., Ltd., Shanghai, China. Fruit with uniform size, maturity, no mechanical damage, and no diseases were selected to help reduce variation among experimental treatments. The mangoes were pre-cooled for 12 h at 13 • C, and the initial mass was weighed. The fruits from each treatment were then divided equally, and each mango was randomly placed into 25 × 36 cm pouches (0.24 mm) made from polyethylene (PE) and polyamide (PA) packing material. The water vapor transmittance of the pouch is 0.8 g (m 2 24 h) −1 , and the oxygen transmittance is 7 cm 3 (m 2 24 h 0.1 MPa) −1 . A total of 126 mangoes were used in this study. Five experimental batches were prepared; namely, CK combination: 21% O 2 + 79% N 2 ; C3 combination: 3% CO 2 + 7% O 2 + 90% N 2 ; C5 combination: 5% CO 2 + 5% O 2 + 90% N 2 ; and C7 combination: 7% CO 2 + 3% O 2 + 90% N 2 . The bag-type vacuum air-conditioning packaging machine (JY-500, Shanghai Jiyi Machinery Co., Ltd., Shanghai, China) was used for the packaging of different experimental batches, after which these pouches were stored independently at 13 ± 1 • C for 30 days, and 6 samples were taken every 6 days for analysis.

Respiration Rate
The respiration rate was estimated following the method of Deng et al. [13] with slight modifications. A single mango was sealed in a container filled with 30 mL 0.2 M NaOH (Sinopharm Chemical Reagent Co., Ltd., Shanghai, China; all sources except indicated) and placed at room temperature for 0.5 h. Then, the collected solution was added with 30 mL saturated BaCl 2 and 3 drops of phenolphthalein, and the solution was titrated with 0.1 M oxalic acid to the end point. For each experiment, four mangoes in each packaging combination were used to measure the respiration rate, and expressed as CO 2 mg kg −1 h −1 .

ACS Content
The ACS content was measured by the method of Nair et al. [14] with some modifications. One gram of mango pulp was homogenized by 9 mL phosphate buffer (Sangon Biotech (Shanghai) Co., Ltd., Shanghai, China) and centrifuged at 5000× g for 30 min. The 2 mL supernatant was placed in a test tube containing 1 mL 500 µM S-adenosylmethionine (SAM), and incubated for 30 min at 30 • C. Then, 0.1 mL 50 mM HgCl 2 and 0.3 mL of 5% NaOCl and NaOH (2:1, v/v) was added to the test tube (placed on ice). It was incubated in ice for 25 min, and injected with 1 mL of gas for gas chromatography (Agilent 6890N-5973B, Agilent Technologies Inc., Palo Alto, CA, USA). The ACS content was expressed as ng L −1 .

Magnetic Resonance Imaging (MRI) and Free Moisture Content (FMC)
During storage, fresh mango pulp was wrapped in preservative film and vertically placed into a low-field NMR analyzer (MesoMR23-060H-I, Suzhou Niumag Analytical Instrument Corporation, Suzhou, Jiangsu, China) for testing. The main parameters of the instrument were adjusted according to the method of Kirtil et al. [15]. The transverse relaxation time T2 was calculated using the low-field NMR analyzer through the CPMG pulse sequence. T2 inversion software (V1.0, Niumag nuclear magnetic resonance image processing software, Suzhou, Jiangsu, China) was used to measure the inverse spectrum of T2 and discovered the distribution of the free moisture content in the pulp. The main parameters of the instrument were as follows: the repeat sampling waiting time (TW), 5000 ms; the echo time (TE), 0.45 ms; the number of echoes (NECH), 18,000; and the number of scans (NS), 8.

Mass Loss (ML)
The mass loss of the fruit was evaluated using a digital balance. Fruits with four replicates in each treatment were individually massed at the beginning of storage and during each storage time. Mass loss was expressed as a percentage with respect to the initial mass by Lo'ay et al. [16].

Fruit Firmness
The mango firmness (g) was determined using the texture analyzer (TA-XT Plus C, Stable Micro Systems Co., Ltd., Surrey, UK). The firmness of each fruit was measured at six opposite points perpendicular to the equatorial area by using a SMS-P/2 cylinder. The speed of penetration and depth of penetration was set at 2 mm s −1 and 10 mm, respectively. Four replicates were used for each treatment.

Protopectin and Soluble Pectin Content
The preparation of the protopectin for content analysis was according to the method of Ren et al. [17] with some modifications. Soluble pectin content was assayed as described by Jongsri et al. [18]. The above two results were expressed as nmol g −1 . One gram of mango pulp was used for the analysis of protopectin and soluble pectin content, respectively. After the pulp was washed alternately with 80% ethanol and acetone (Sangon Biotech (Shanghai) Co., Ltd., Shanghai, China), it was placed in dimethyl sulfoxide solution for 15 h, and the supernatant was discarded by centrifugation at 4000× g for 10 min. The extraction was measured by the carbazole colorimetric method to read the absorbance at 530 nm (N4S UV-visible spectrophotometer, INESA Analytical Instrument Co., Ltd., Shanghai, China), and the content of protopectin and soluble pectin was calculated using the D-galacturonic acid standard curve.

Vitamin C (VC) Content
Vitamin C content of mango pulp tissue was determined as described by Chen et al. [20], and expressed as g 100 g −1 FW. We dispersed 10 g of mango pulp in 20 g L −1 oxalic acid solution and diluted it to 100 mL. After being shaken to homogenate, 10 mL of filtrate was accurately measured and titrated with 2,6-dichloroindophenol solution to the end point.

Malondialdehyde (MDA) Content
The MDA content of mango pulp tissue was assayed according to Zheng et al. [21], and expressed as nmol g −1 based on fresh weight. Dispersed 1 g of mango pulp evenly in 15 mL of 10% TCA solution, centrifuged at 10,000× g for 20 min. Two milliliters of sample supernatant were aspirated and reacted with an equal volume of 2-thiobarbituric acid. At last, we determined the values at 450, 532, and 600 nm absorbance (N4S UV-visible spectrophotometer, INESA Analytical Instrument Co., Ltd., Shanghai, China). The formula for calculating MDA content is as follows:

Statistical Analysis
Data were subjected to analysis of variance (ANOVA) using IBM SPSS Statistics 25.0 for Windows (Version 25.0, IBM SPSS Statistics, New York, NY, USA), and significance analysis was detected with Tukey's multiple range tests at a level of 5%. All the results were shown as means ± standard error of the mean.

Respiration Rate and ACS Content
The respiration rate of mangoes stored at 13 • C for 30 days under different carbon dioxide-MAPs is shown in Figure 1A. Under the treatment conditions, the respiration rate of mangoes increased with storage time from 0 to 18 days, and there were no significant differences in the change pattern. In many previous studies, the respiratory rate of climacteric fruits increased rapidly at the end of the storage period, such as dragon fruit [22] and longan fruit [23]. For the experimental groups, CK and C3 combinations were observed to have a respiration rate peak at the 24th day. Certainly, the subsequent decrease in respiration rate indicated that the mangoes began to enter the senescence stage [22]. However, C5 and C7 combinations still increased slowly during the subsequent storage period, indicating that C5 and C7 combinations could reduce the peak respiration rate of mangoes and delay senescence [24]. Phakdee et al. showed that MAP treatment could decrease the respiration rate and ethylene production [25]. The microenvironment of high carbon dioxide and low oxygen slowed down the physiological metabolism rate of mangoes and maintained the antioxidant capacity [26]. Ethylene is a natural plant hormone, which plays an important role in regulating the whole process from seed germination to senescence. The 1-aminocyclocarboxylic acid-1-carboxylic acid synthase (ACC synthase, ACS) is a key restriction enzyme in the process of methionine synthesis of ethylene, which can regulate the production of ethylene in fruits [27]. Zaharah et al. found that the trend of ACS content in mangoes was similar to the ethylene production [28]. For the five experimental combinations, the ACS content showed a trend of fluctuation downward and then a gradual increase ( Figure 1B). The C5 combination reached the lowest value on the 6th day, then continued to rise and was significantly higher than the other treatment combinations in the final storage period. On the 18th day, the ACS content of the CK and C3 combination reached the lowest value, and then increased rapidly. The ACS content of the C7 combination was always lower than the initial value of 738.73 ng L −1 FW, and the content was lowest from the 24th to 30th day. These results suggested that MAP treatment reduced the ACS content with varying degrees during half of the storage period. This may be due to the controlled atmosphere treatment and low-temperature treatment, which affected the activity and synthesis of enzymes [29]. Khan reported that Japanese plums had similar results after MAP treatment, which reduced the activity of ACS and inhibited the production of ethylene [30]. During the subsequent storage process, only the C7 combination maintained a low level of ACS content and reduced the metabolic activity of the mangoes.

MRI, FMC, and ML
Low-field nuclear magnetic resonance (LF-NMR) uses the characteristics of hydrogen atoms in a magnetic field to qualitatively or quantitatively determine the moisture migration or pore structure. The LF-NMR is an effective method to evaluate the freshness of fruits and vegetables, and MRI can be used to explore the moisture distribution and migration [31]. The moisture distribution of mango pulp of different carbon dioxide-MAP combinations during storage at 13 • C is shown in Figure 2A. The red, yellow, and green in the figure represent the high, medium, and low moisture content, respectively. As the storage time increased, the red area of the MRI image of mango pulp gradually decreased with less brightness and more greenness. During the storage period from the 6th to 18th day, the high-FMC area of mango pulp was significantly reduced and moved to the mango peel. Only a small amount of high-FMC area (red area) appeared on the mango peel at the end of storage, indicating that mangoes lost a great amount of free moisture during storage [17]. The results shown in Figure 2B are consistent with the trend of 2A, and the FMC of mangoes are continuously decreasing. Compared with the CK combination, the FMC dropped by 7.29% during the entire storage period. Although the values of FMC in the C5 and C7 combinations were not the highest, the content of the two combinations decreased less than the other treatment combinations. The results showed that MAP treatment could reduce the FMC loss of mangoes effectively, which is probably due to the reduction of mangoes transpiration by MAP [32], and the effect of controlled atmosphere treatment on mango's physiological metabolism also helped to maintain moisture [33].
The mass loss of mangoes increased with storage time is shown in Figure 2C. The increase in respiration rate between 18 to 24 days accelerated the ML during this period. Zerbini et al. also reported that higher respiration rate can lead to the consumption of stored substances in tissues, resulting in mass loss [27]. In addition, the natural evaporation of mangoes also decreased the mass loss [8]. From the perspective of different treatment combinations, the results obtained were not significantly different, except that the ML in the C7 combination was significantly lower than other treatments on the 12th day. However, the mass loss of the C7 combination was the least during the whole storage process, indicating that this treatment could effectively reduce the material consumption of mangoes in the physiological metabolic process and moisture loss [34]. + 79% N 2 ; C3: 3% CO 2 + 7% O 2 + 90% N 2 ; C5: 5% CO 2 + 5% O 2 + 90% N 2 ; C7: 7% CO 2 + 3% O 2 + 90% N 2 . The "d" in Figure 2A represents the number of days). Different superscript letters (a-c) indicate a significant difference between treatment and control at p ≤ 0.05.

Fruit Firmness and Pectin Content Analysis
Mango fruits' firmness and pectin content are related to fruit quality and denote important parameters of the characteristics of mango products. Figure 3A illustrated that the protopectin content of mangoes gradually decreased during the storage at 13 • C for 30 days. However, not all experimental combinations performed better than the CK combination. The degradation rates of the C3 and C5 combinations' protopectin were faster, and the content was significantly lower than CK combination during storage period. The C7 combination maintained the highest content from the 0th to 24th day and was significantly higher than the other treatment combinations, with the best performance. The demethylation of pectinmethylesterase (PME) and the hydrolysis of polygalacturonase (PG) made the protopectin substances continue to be degraded and increased the content of soluble pectin [35]. Wang et al. proposed that the solubility of pectin during storage also led to an increase in soluble pectin content [36]. The soluble pectin content of all groups was significantly lower than the CK combination, but the difference between treatment combinations was not significant, in most cases. Under the conditions of the C7 combination, the soluble pectin content of mangoes rose the slowest. This indicated that the pectin that constitutes the cell structure was degraded by enzymes due to the after-ripening effect, and the mangoes continued to soften and gradually mature [37]. In this study, MAP treatment significantly delayed the increase in soluble pectin and the decrease in protopectin content ( Figure 3A,B), which may have contributed to increasing mango firmness ( Figure 3C) and keeping commercial value. C3: 3% CO 2 + 7% O 2 + 90% N 2 ; C5: 5% CO 2 + 5% O 2 + 90% N 2 ; C7: 7% CO 2 + 3% O 2 + 90% N 2 ). Different superscript letters (a-d) indicate a significant difference between treatment and control at p ≤ 0.05.
As the storage time increases, the percentage of mango firmness loss continued to increase ( Figure 3C). The firmness of the CK, C3, C5, and C7 combinations decreased to 68.87%, 83.92%, 83.39%, and 79.63% of the initial value at the end of storage at 13 • C, respectively. During the 0th to 6th day and after 18 days, the fruit firmness of the CK combination decreased rapidly and was significantly faster than the other experimental combinations. This indicated that the MAP treatments could retain higher mango firmness during storage, which was mainly due to the greater retention of the protopectin material and the moisture content, as with the above results. The C5 combination had the best effect on maintaining the firmness of mango, but it was not significantly better than the C3 and C7 combinations.

Color Characteristics
The values of L*, a*, b*, and ∆E of fresh mango pulp were 63.94 ± 0.56, −8.50 ± 0.06, 17.73 ± 0.25, and 42.46 ± 0.34, respectively. The color of mango pulp stored at 13 • C for 30 days changed from green to light yellow gradually, which can be quantified by the increase in b* value. This was consistent with the results of Ebrahimi et al. [1]. The C7 combination maintained the best quality, and the b* value was the lowest among all treatment combinations after 30 days of storage ( Table 1). The results showed that high carbon dioxide and low oxygen conditions could more effectively inhibit the yellowing of mango pulp during postharvest storage. This effect could be explained by the ability of carbon dioxide to inhibit chlorophyll degradation, as reported for dragon fruit [22] and blue honeysuckle fruits [38]. Besides, MAP treatment inhibited the physiological metabolism of mangoes and delayed the ripening process, which was also one of the important reasons.

VC and MDA Content
Vitamin C is an important indicator to measure the quality of fruits, and it is also a non-enzymatic antioxidant substance that scavenges free radicals in fruits [39]. As shown in Figure 4A, the VC content of the mangoes' quality index gradually decreased during the entire storage time at 13 • C for all treatments. During the storage period of 0-6 days, the VC content of the C7 combination mangoes was significantly the highest (p < 0.05). The effects of different carbon dioxide-MAP treatments on the VC content during low-temperature storage were different. The higher the carbon dioxide content in the initial gas component, the earlier the inhibitory effect of VC loss. Overall, compared with CK combination, MAP treatment could maintain the VC content of mangoes. Each experimental combination had different advantages in different stages of storage, which indicated that free radicals generated by physiological metabolism were eliminated by VC at different times [13]. The reason for this phenomenon may be due to the initial gas composition that led mangoes to undergo different physiological processes [32].  The MDA content is considered to be one of the main lipid peroxidation products, which reflects the actual degree of membrane-lipid-peroxidation induced by reactive oxygen species (ROS) [40]. The MDA content of mango pulp tissues gradually increased with the progress in the storage period at 13 • C ( Figure 4B). However, the MDA content of controlled atmosphere groups was lower than the control after 18 days of storage. As far as different carbon dioxide-MAP combinations were concerned, fruits stored under C7 combination exhibited significantly (p < 0.05) lower MDA content than the other combinations at 30 days of storage ( Figure 4B). The increase in ROS content of mangoes after harvest usually led to enhanced lipid peroxidation, which destroyed the membrane structure. The destruction of the cell membrane structure of mangoes made the tissue soft, and their storage potential, quality, and marketability gradually declined [41]. The lower MDA production in MAP storage fruits may be due to the controlled atmosphere maintained the normal cell activities of mangoes, reduced the production of ROS, and decreased the degree of peroxidation of membrane lipids by ROS [11]. Finally, combined with all the observations in this study, the C7 combination had the greatest impact on the metabolism and quality of mangoes. In addition, VC could eliminate ROS or other oxidizing substances and reduced the peroxidation of mango pulp. Overall, combined with all the observations in this study, the C7 (7% CO 2 + 3% O 2 + 90% N 2 ) treatment had the greatest impact on mangoes metabolism and quality.

Conclusions
The difference in carbon dioxide in the controlled atmosphere had a significant impact on the physical, chemical, and physiological properties of green mango. The respiration rate of mangoes showed a typical trend of climacteric fruits, with respiratory peaks appearing in the late storage period. MAP treatment and low-temperature had an effect on the ACS content, which made a significant difference between treatment groups. The change in firmness was mainly due to respiration, enzymatic hydrolysis of pectin, and free moisture loss. As storage time increased, the degree of lipid peroxidation in the pulp gradually deepened, the MDA content continued to increase, and VC content gradually decreased. Compared with the CK combination, MAP combinations retained firmness, free moisture, and nutrient compounds at 13 • C during 30 days storage. In short, the C7 (7% CO 2 + 3% O 2 + 90% N 2 ) combination should be the first choice for the preservation of mangoes to maintain the quality and reduce nutrient loss. In addition, it provided guidance for industrial production and could improve the potential market for the consumption of high-quality fruits.

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