Effect of Modified Atmosphere Packaging (MAP) on the Stability of Anthocyanins and Degradation of Phenolic Compounds during Postharvest Storage of Pomegranate Fruit

Effects of storage temperature, packaging material, elevated CO 2 and reduced O 2 contents in the package headspace, and their interaction effects on the total content of anthocyanins and degradation of phenolic compounds during long storage of “Malase Torsh Saveh” pomegranate were studied. The study findings showed that only storage temperature and its duration had significant effects on the degradation of phenolic compounds. The content of anthocyanins was affected by the single effect of storage time and its 3-way interaction effect with the storage temperature and the fruit’s surrounding gas composition. Higher contents of both total anthocyanins and browning pigments were recorded at 2˚C storage temperature. The Browning Index (BI) of the pomegranate extracts showed to change quadratically with the variations in the total anthocyanins content but linearly with the variations in the content of browning compounds. Thermal dipping treatment and its interaction with the storage time also had significant effect on the BI values of the extracts obtained from modified atmosphere packaged pomegranates stored at ̊


Introduction
The role of phenolic compounds in food quality, their health-promoting and therapeutic effects, along with their mechanism of actions on human health has How to cite this paper: Lashkari been the topic of numerous researches in recent years. In addition to their health beneficial properties, phenolic compounds are connected with the quality of plant-derived foods such as appearance, color, taste and aroma. Pomegranate fruit (Punica granatum L.) is quite well-known for its nutritious nature and presenting a broad range of health beneficial activities, in particular antioxidative, anti-inflammatory, anti-carcinogenic, antimicrobial, neuro-and cardio-protective properties [1]- [6].
Scientific findings have provided evidence that the type of technologies and treatments being applied during postharvest handling and storage have significant impact on the total contents of phenolic compounds, their biosynthesis, metabolism, biological activities and the involved enzymatic reactions [16] [20] [21] [22] [23]. These effects could be either favorable or unfavorable towards preserving the desired quality of the pomegranate fruit [16]. Therefore, current studies are also focused to find and develop technologies or a combination of them that allow maintaining the original quality of the fresh fruit, i.e., color, flavor, texture and its nutritious compounds including vitamins, sugars, amino acids, antioxidants, and other health-promoting compounds, while avoiding weight loss, fungal decay, and common physiological disorders such as husk scald, pitting, and chilling injury during cold storage.
Various studies have shown that modified atmosphere packaging (MAP) technology by decreasing the oxygen content and/or increasing the carbon dioxide content of the atmosphere surrounding the fresh produce, can delay quality loss, tissue softening, enzymatic activities, and incidence of various physiological disorders and pathogenic developments during long storage of pomegranate fruit [20] [21] [23]- [31]. MAP can also prevent the fruit weight loss and shriveling by creating a higher relative humidity in the surrounding environment of the fruit. Therefore, MAP could be applied or combined with other storage treatments, for instance with various thermal treatments or refrigeration to inhibit potential development of any decay or physiological disorders [16] [32].
Nonetheless, identifying the applicable MAP that obtains the desired outcome in terms of maintaining the quality and nutritional aspects while avoiding decay and unwanted physiological disorders during long storage requires extensive re- shape, appearance, and sound outer skin were sorted for the study the day after.

Chemicals and Other Materials
Unperforated low-density polyethylene (LDPE) and polypropylene (PP) packaging bags were purchased from a local provider. Citric acid, sodium citrate, and sodium hydroxide were procured from Merck KGaA (Darmstadt, Germany).  were checked, and then transferred and stored in the 2˚C and 6˚C cold rooms for the long storage study.

Thermal Dipping Treatment
To investigate the potential effects of thermal dipping treatment on the total anthocyanins and phenolic compound contents of fruits packaged with MA during long storage, 60 pomegranates were selected and divided into two groups. Half of these fruits were dipped in the 46˚C water bath (Tattnauer, NY, USA) for 2 minutes and left to completely air-dry prior to packaging. After they completely dried, they were coded and placed inside the unperforated LDPE bags in pairs (2 pomegranates in each package). The package headspace of both sampling groups was replaced with the gas composition of 5% CO 2 + 5% O 2 (GC1) and got sealed immediately. Both groups were stored at the cold room of 6˚C for a duration of 12 weeks.

Quantification of Total Anthocyanins and Phenolic Compounds' Degradation Products
To measure the total anthocyanin contents and phenolic compound degradation products (or the browning compounds) during storage, first three packages from each sampling group were randomly selected and one pomegranate from each of these packages separated for the study. Also, three pomegranates were randomly selected from each tray of control groups. To obtain the juice, the fruits were half-cut with a clean knife and the juice from the arils was extracted by using a manual stainless-steel pomegranate squeezer. Next the extracted juices were centrifuged (Andreas Hettich centrifuge, Germany) for 20 min at 3000 rpm and the supernatant of the samples were used for the spectrophotometry measurements. For maximum stability of anthocyanins during measurements, the juice supernatants were diluted with citric acid buffer (pH = 3.4) at 1:3 (v/v) ratio. Later, the total anthocyanin contents of the extracts were recorded by measuring the absorbance at 510 nm with the UV/Vis spectrophotometer instrument (Perkin Elmer Lambda EZ 201, MA, USA) and the recorded values were reported as the Absorbance Unit (AU), which is also expressed as the optical density (OD) per unit length (Equation (1)); the cuvette thickness used in this study was 1 cm: where: l = the distance that light travels through the sample (the sample thickness) measured in cm; A λ = absorbance at wavelength λ.
Absorbance at wavelength of 446 nm was recorded by the same spectrophotometer as an indication of polymerization and degradation of phenolic compounds or production of browning compound. Citric acid buffer (pH = 3.4) was used as the reference solution for the absorbance measurements at both 510 and 446 nm wavelengths [20] [21] [25] [26]. All measurements were performed in triplicate.

Browning Index (BI) Calculation
Browning Index of the extracted juice, which reflects the ratio of the total anthocyanin to the development of browning compound or phenolic degradation products was calculated for all sampling groups from Equation (2) [33].

Statistical Analysis
Storage temperature with 2 levels (2˚C and 6˚C), Packaging type with 3 levels (LDPE, PP, and no packaging or None), head space gas composition with 3 levels (air or ambient atmosphere, GC1, & GC2), and storage time with 4 levels (3 weeks intervals) were applied as the experimental design factors. The effect of hot-water dipping pretreatment on the MA packaged pomegranate fruits stored at 6 C was studied on 2 groups for 12 weeks and analyzed separately. All measurements were conducted in 3 replicates and the mean value was reported. JMP Pro software was used to perform the statistical analysis and data visualization. Analysis of variance (ANOVA) at p < 0.05 and Student's t Test (for mean comparison) were applied when appropriate.

Changes in the Total Content of Anthocyanins
Among the multiple factors being studied, only the single effect of storage time was found to have a significant effect on the total content of anthocyanins at p < 0.01 during this study. The results showed a decline from the average absorbance value of 0.917 ± 0.054 (Mean ± SE) at the beginning of the study through the 7th week of the storage followed by a slight increase towards the end of the study (0.49 ± 0.054). The total content of anthocyanins was also affected by the 3-way interaction effects of the storage duration, storage temperature, and the surrounding gas composition. Overall, samples with MA packaging displayed lower values of total anthocyanins around the seventh week of their storage compared to the control samples with no packaging (Figure 2). Among the MA packaged treatments, storage temperature at 2˚C, LDPE packaging, and the initial gas mixture of 5% CO 2 + 5% O 2 (GC1) were associated with higher amounts of total anthocyanin contents by the end of study. As illustrated in Figure 3, the interaction profile also indicates an interaction between the storage temperature and the type of packaging although it was not found to be statistically significant. Besides, higher amounts of anthocyanins were observed on average in the extracts of samples that were stored at 2˚C storage temperature compared to samples that were stored at 6˚C during long storage regardless of their packaging type and their surrounding atmospheric composition.

Changes in the Degradation of Phenolic Compounds
The results of this study showed that the single effects of storage time and temperature have had significant effects on the degradation of phenolic compounds and formation of browning pigments at p < 0.05. On average higher amount of phenolic compounds degradation products was observed by the end of study (0.332 ± 0.024) compared to the beginning of the study (0.278 ± 0.024) throughout the 10-week storage time. Likewise, degradation of phenolic compounds was enhanced at 2˚C storage temperature with the average absorbance value of 0.323 ± 0.017 at 446 nm compared to the corresponding values of 0.264 ± 0.017 when samples were stored at 6˚C temperature ( Figure 4). None of the 2 & 3-way interaction effects was found to be significant. Yet, the interaction profile ( Figure 5) displayed an interaction between the storage temperature and the type of packaging that may not be statistically significant. As shown in Figure 5, storage temperature of 2˚C, UPP packaging, and the initial gas mixture of 5% CO 2 + 5% O 2 (GC1) induced higher amounts of degradation of phenolic compounds and subsequent formation of browning pigments among pomegranate fruits with MA packaging. Extracts of pomegranates that were stored with no packaging and surrounded by the air or ambient atmosphere of the cold room  Each error bar is constructed using 1 standard error from the mean. Packaging type: PP, polypropylene; LDPE, low-density polyethylene; NONE, no packaging is used. Gas composition: NRM, air or ambient atmosphere; GC1, initial MAP with 5% CO2 and 5% O2; GC2, initial MAP with 10% CO2 and 5% O2.
showed the least amounts of browning pigments on average at the end of study (Figure 4, bottom).

Changes in the Browning Index (BI)
The results of the data analysis indicate that the Browning Index was affected by the single effect of the storage time, its 2-way interaction effects with the type of packaging, and its 3-way interaction effects with the storage temperature and the surrounding atmospheric composition at p < 0.01. The average BI value was increased from 0.31 ± 0.02 at the beginning of the study to 0.69 ± 0.04 by the end  of study. The increase occurred during the first 4 weeks of the storage and remained around the same value during the rest of the 10-week study. Extracts of pomegranates with no packaging that were surrounded by the ambient atmosphere during storage displayed the lowest values of BI (0.53 ± 0.06) at the end of the storage compared to the treatments with MA packaging. Additionally, the extracts of pomegranates with PP packaging showed the highest BI value (0.63 ± 0.06) at the end of study (Figure 6, top). Overall higher BI values were associated with storage temperature at 2˚C. Control samples with no packaging that were surrounded by the ambient atmosphere showed higher BI values when stored at 6˚C. In contrast to air-stored samples, the extracts of MA packaged pomegranates displayed higher BI values when stored at 2˚C cold room ( Figure  6, bottom).

Changes in pH, TA, & Total Soluble Solids (TSS)
Storage time showed to have significant effects (at p < 0.01) on the pH values of  the treatments over the course of study. The pH values displayed an increase from 3.45 ± 0.05 at the harvest to 3.67 ± 0.17 during the first month of storage that remained around the pH value of 3.64 ± 0.19 till the end of 10 weeks of storage. pH was not influenced significantly by the storage temperature; however, it was affected by the 2-& 3-way interaction effects of storage time with packaging type & the surrounding gas composition (p < 0.01). Overall, the control samples that were surrounded by the ambient atmosphere and had no packaging showed pH values closer to the average pH value at the harvest (pH = 3.53 ± 0.03).
Whereas extracts from pomegranates that had unperforated PP packaging and Figure 5. Interaction profile of temperature, packaging type, gas composition effects on the development of phenolic compounds degradation products of MA packaged pomegranates. Packaging type: PP, polypropylene; LDPE, low-density polyethylene. Gas composition: GC1, initial MAP with 5% CO2 and 5% O2; GC2, initial MAP with 10% CO2 and 5% O2.
packaged with the initial gas mixture of 5% CO 2 + 5% O 2 (GC1) were associated with higher values of pH at the end of 10 weeks of storage with the average pH values of 3.66 ± 0.03 and 3.63 ± 0.02 respectively (Figure 7, top). TA values were significantly affected by the single effect of gas composition and its interaction with the storage duration (p < 0.01). The average TA values displayed slight decrease from the average TA value at the harvest (1.02% ± 0.032%) until the 7th week of storage (0.918% ± 0. 032%) and then started to slowly increase toward the end of the study (0.974% ± 0.032%). As shown in Figure 7 (bottom), the control samples that were surrounded by the ambient atmosphere and had no packaging showed higher amounts of TA, while the samples with PP packaging and the initial gas mixture of 5% CO 2 + 5% O 2 (GC1) showed the lowest values for TA by the end of study. These results are in agreement with the study findings on pH changes throughout the storage. Also, pomegranates that were packaged with the initial gas mixture of 10% CO 2 + 5% O 2 (GC2) exhibited the lowest pH values (pH = 3.5) and the highest TA (TA =   respiratory activities of pomegranate fruits during the long storage.

Effects of Thermal Dipping (TD) Treatment
The results show that the single effect of thermal dipping (TD) pretreatment was not significant on the total content of anthocyanins, degradation of phenolic compounds, and Browning Index during the long storage. However, as shown in

Discussion
The findings of this study showed an overall decrease in the total contents of anthocyanins during 10 weeks of storage, in which multiple factors might have been involved. Some of the factors that affect anthocyanins stability or their susceptibility to degradation are temperature, increased sugar, pH, acidity level, metal chelation, and copigmentation with other flavonols and tannins [19] [34].
Likewise, regulation and activity of phenylalanine ammonia-lyase (PAL), which is the key enzyme in biosynthesis of flavonoids can be another influential factor [16]. Therefore, the overall decline in the contents of anthocyanins during the Nonetheless, a different pattern in the contents of anthocyanins during the cold storage of MA packaged "Hicaznar" and "Hicrannar" cultivars was reported. Selcuk & Erkan (2014) reported an increase in the total anthocyanin contents of "Hicrannar" pomegranates during the first 100 days of storage at 6˚C that followed by a decrease over the last 20 days of the study in both MAP and control treatments through 120 days of storage [28]. Total anthocyanin contents in "Assaria" pomegranates that were subjected to several storage treatments also showed an increase during the first month of storage with continued decrease towards the end of 4 months of cold storage at 5˚C [22]. "Ruby" pomegranates  (2018) observed a delayed anthocyanins synthesis and lower anthocyanin concentrations in MA packaged "Hicaznar" pomegranates [30]. The difference in the observed anthocyanins contents of these cultivars during long storage can be attributed to the inherent differences among various cultivars given that the same trend has been recorded for both control and MA packaged pomegranates during long storage that indicates the potential continued postharvest metabolic activities including biosynthesis of anthocyanins in the fruit despite of being refrigerated and/or subjected to the MA packaging.
The current study results also showed higher contents of browning pigments due to the degradation of phenolic compounds when pomegranates stored at 2˚C compared to 6˚C storage temperature. These findings can be attributed to the enzymatic activities of polyphenol oxidases (PPO) and peroxidases (POD) that catalyzed enzymatic browning in fruit tissues as the results of environmental stresses such as low storage temperatures and mechanical injuries [16]. Likewise, elevated CO 2 contents in the surrounding atmosphere of MA packaged pomegranates can be another contributing factor that enhanced polymerization of phenolic compounds in the tissues and arils of MA packages pomegranates compared to the control (air-stored) samples. These observations are consistent with the findings reported for "Mollar de Elche" pomegranates when packaged with CO 2 -enriched MA and stored at 5˚C for 12 weeks [25]. The results of this study also recorded higher amounts of browning pigments over the cold storage that can be an indication of PPO and POD enzymatic activities as the results of minor incident of chilling injury in the tissues that often led to development of browning pigments in pomegranates when stored at low temperatures for a long time.
As shown in Figure 9, the variations in the browning index (BI) values during long storage is more influenced by the variation in the total contents of anthocyanins rather than degradation of other phenolic compounds, which is an interesting finding and implies that anthocyanins are the main phenolic compounds in the arils. Likewise, these results indicate minor development of browning pigments as the result of degradation of phenolic compounds other than anthocyanins during 10 weeks of storage that is even more noticeable at 2˚C compared to 6˚C storage temperature. Storage temperature of 2˚C is associated with higher contents of both total anthocyanins and development of browning pigments that imply the potential effect of low temperatures on the gene regulations, expression and physiological activity of the enzymes that are involved in the biosynthesis of phenolic compound such as anthocyanins and those that are participating in the enzymatic browning as the results of the incidence of physiological disorders (e.g. chilling injury) due to environmental stresses such as long storage at low temperatures or elevated CO 2 contents in the surrounding atmosphere of the fruit.
As presented in Figure 10, the values of the BI of the extracts change quadratically with the variations in the contents of total anthocyanins but linearly   with the variations in the contents of the browning compounds. Consequently, the prediction profiles can be used to determine the optimum anthocyanin contents and storage conditions based on the desired BI value for the anticipated applications such as fresh consumption, processed food products for instance juices, jams, jellies, or as a value-added ingredient e.g., natural colorant or a bioactive compound.
The study results also revealed that thermal dipping (TD) treatment can affect the BI value of the pomegranate extracts by enhancing degradation of anthocyanins, their polymerization, and subsequent formation of browning compounds throughout the storage. The increase in the BI value of the samples that received TD treatment prior their packaging is more visible since the ninth week of the study. Similarly, the significant 2-way interaction effect of TD with the storage time on the pH, TA, and TSS changes during long storage of MA packaged pomegranates indicates the higher metabolisms of soluble solids, mainly sugars (such as glucose, fructose, arabinose) and organic acids (e.g., citric acid) by the pomegranates that have been subjected to the thermal treatment compared to the control group. Nonetheless, the TSS/TA ratio of pomegranates was not significantly affected (data not shown here) by TD treatment after 12-weeks of storage at 6˚C.

Conclusion
In summary, the total contents of anthocyanins in "Malase Torsh Saveh" pomegranates declined through 10 weeks of storage in all treatments. Among samples with modified atmosphere (MA) packaging, pomegranates with LDPE packaging and the initial gas composition of 5% CO 2 + 5% O 2 in their package headspace maintained higher contents of total anthocyanins when stored at 2˚C. Nonetheless, higher incidence of browning pigments was also recorded at 2˚C, possibly as the results of occurrence of minor chilling injury throughout the cold storage. Lesser browning pigments were developed among samples with MA packaging through the first seven weeks of storage, however, their contents started to increase towards the end of study. The browning index (BI) values of the extracts showed an overall increase throughout the storage and they were more influenced by the variations in the content of total anthocyanins in the extracts rather than the content of browning pigments, which indicates minor development of browning pigments as the result of phenolic compounds degradation during long storage. Pre-storage thermal dipping (TD) as a complementary treatment along with MAP showed no significant effects on the total content of anthocyanins, degradation of phenolic compounds, or development of browning compounds during the cold storage. However, BI values of MA packaged pomegranates that had received TD prior to the storage started to significantly increase after 2 months of storage at 6˚C. and it was funded by University of Tehran, College of Agriculture and Natural Resources, Karaj, Tehran. The author also thanks Dr. Banafshe Lashkari, PhD, from University of Waterloo for her assistance in article retrieval.

Ethics Statement
The author declares that this research (a) is the author's own original work, (b) has not been published previously, and (c) is not under consideration for publication elsewhere.

Conflicts of Interest
The author declares no known conflicts of interest regarding publication of this paper.