Evaluation of modified atmosphere packaging system developed through breathable technology to extend postharvest life of fresh muscadine berries

Abstract Muscadine grapes (Vitis rotundifolia Michx.) are delicate in nature with short shelf life. Postharvest technologies like modified atmosphere packaging (MAP) with reduced oxygen (O2) and elevated carbon dioxide (CO2) could increase the postharvest storage life with better quality. In the current experiment, physical and biochemical quality attributes of black and bronze cultivars of muscadine grapes ('Supreme' and 'Granny Val', respectively) were evaluated in active MAP. Fruit were packed in plastic trays, sealed with impermeable film, and CO2 was introduced into the package. The MAP was created by a rigid microperforated plastic patch coated with a proprietary semipermeable resin, which was applied over a hole in the tray; packages with the same size hole without a patch were the control. Fruit were stored at 4°C for 42 days (6 weeks). MAP resulted in significantly lower decay incidence and better retention of fruit firmness for up to 28 days of storage in both cultivars as well as reducing color changes in 'Supreme' fruit. Although MAP did not affect the biochemical quality of muscadine grapes, total antioxidants increased initially and then decreased during storage, irrespective of packaging treatments. A significant linear increase in total phenolic content was also found during storage, regardless of treatments applied. Overall, the results of the current study demonstrate that MAP can be an affective technology to increase storage duration of muscadines with better retention of physical quality, without affecting the biochemical attributes.

Muscadines are native to the southeastern USA and are successfully grown there because of the plant's hardy nature and resistance to the endemic pests and diseases of the region.Muscadine plants have natural resistance against Pierce's disease, which is a major limiting factor for growing common grapes (Vitis vinifera L.) in Florida (Andersen et al., 2020).Unlike common grapes that are harvested in bunches, muscadines do not form bunches and thus the ripe berries are individually harvested (Khalil, Rajwana, Razzaq, Brecht, et al., 2023).
Muscadine grapes have a short shelf life and about 50% of the berries can suffer from decay within 7-10 days at ambient temperature (Walker et al., 2001).Notable reduction in decay incidence of muscadines was achieved when the berries were stored at 0 or 4.5°C in comparison to 20°C (Takeda et al., 1983).At refrigerated or lowtemperature storage, muscadines can be stored up to 2-3 weeks (Perkins-Veazie et al., 2012), but they are susceptible to postharvest softening, shriveling, weight loss, and decay when stored longer (Walker et al., 2001).
Being nonclimacteric, muscadines must be allowed to ripen completely before harvest and they have a very low respiration rate compared to climacteric fruit.Although the quality of the fruit deteriorates during storage due to physiological breakdown, the decay caused by microorganisms is the major cause of fruit losses during storage.Common grapes can be stored for about 6 months in cold storage along with fumigation with sulfur dioxide (SO 2 ) or by using SO 2 pads in packaging to reduce decay (Thompson, 2015).However, SO 2 fumigation is not recommended for muscadine because of its bleaching effect on the berries and off-flavor development (Smit et al., 1971;Takeda et al., 1983).Moreover, usage of SO 2 is restricted in many countries because of health concerns, especially for people allergic to sulfites.Therefore, alternative methods of decay control need to be explored to extend muscadine grape's storage life.
Controlled atmosphere (CA) storage involves altering gas concentrations, especially reducing the oxygen (O 2 ) and elevating the carbon dioxide (CO 2 ), in addition to low temperature, which can increase the storage duration of perishable fruit and vegetables (Thompson, 2018).
Metabolic activity of fruit is reduced under CA storage conditions that increase shelf life.We previously reported that muscadines were quite tolerant of high CO 2 concentrations (up to 30%) during postharvest storage and that CA storage maintained better fruit quality and reduced decay incidence compared to air storage for up to 42 days at 4°C (Shahkoomahally et al., 2021).Smittle (1990) reported that muscadines can be stored for 6 weeks in a CA of 5% O 2 plus 15% CO 2 at 1.1-2.2°C.
The use of modified atmosphere packaging (MAP) for extending storage life of fruit and vegetables is a well-established technology.
Recent studies have shown promising effects of MAP on the storage life and quality of various perishable fruits and vegetables like raspberry, mulberry, figs, persimmon, banana, bell pepper, and jujube (Huynh et al., 2023;Islam et al., 2022;Kızıldeniz et al., 2023;Li et al., 2023;Liamnimitr et al., 2018;Lwin et al., 2022;Wang et al., 2023).Using MAP involves designing a package to control gas exchange with the outside environment so that the product respiration and the package permeability interact to achieve a beneficial steady-state atmosphere within the package that, like CA, consists of reduced O 2 and elevated CO 2 .The reduced O 2 can result in attenuated product respiration, which slows metabolism by restricting the supply of adenosine triphosphate (ATP), as well as potentially reducing biosynthesis of ethylene, which requires O 2 ; elevated CO 2 primarily benefits produce storage by reduction decay due to its fungistatic effect (Brecht et al., 2020).There are two types of MAP systems, active and passive (Zagory & Kader, 1988).In active MAP, the desired atmosphere is quickly established by flushing the package with a gas mixture during sealing, while in passive MAP the steady-state atmosphere is slowly established by the interaction between product respiration and package permeability; the same interaction maintains the atmosphere previously established in active MAP (Lange, 2000).
MAP has been reported to retain better quality during storage of many common grape cultivars (Artés-Hernández et al., 2003, 2004;Martínez-Romero et al., 2003;Yamashita et al., 2000).For muscadine, there was remarkably less decay when the berries were held in vented plastic clamshell packages that were wrapped in polyethylene bags, although the resulting modified atmosphere (MA) was not reported (Walker et al., 2001).A problem with MAP is that the response of product respiration to changes in temperature is much greater than the response of the plastic film permeability to the same changes (Beaudry et al., 1992).The BreatheWay® MAP system (Curation Foods, Inc., Santa Maria, CA, USA) addresses this issue by coating a rigid microperforated plastic patch with a semipermeable resin, the permeability of which dramatically differs above and below a designed phase transition temperature (Clarke, 2011).
We recently reported on the use of BreatheWay® MAP to improve the shelf life of pink tomatoes (Batziakas et al., 2022).
Therefore, the current study was conducted to investigate the impact of an active MAP on muscadine quality during storage including the biochemical composition of the fruit.In the current experiment, we used BreatheWay® MAP technology to achieve the desired MA, and its impact on physicochemical quality of muscadine grapes was evaluated.The novelty of this study is that the MAP was maintained through a self-adjusting breathable technology, and its impact was evaluated on both the physical and biochemical quality of muscadine grapes, which are entirely different from common vinifera grapes.

| Fruit supply
Individual berries of two muscadine grape cultivars, bronzecolored 'Granny Val' and black 'Supreme', were harvested, based on their color development, from a commercial muscadine vineyard in Wray, Georgia, USA.The berries were transported in insulated coolers to the postharvest laboratory at the Horticultural Sciences Department, University of Florida in Gainesville, FL (approx.250 km or 3 h), where they were held overnight at 4°C.The following morning, damaged or diseased berries were removed, and only healthy and uniform berries were selected for further use.

| Fruit MAP
Muscadine berries were packed in 650-ml gas-impermeable polypropylene plastic trays (Model 1/8 Gastronorm H45, Orved S.p.A., Venice, Italy) with dimensions of 14.5 × 9.5 × 4.3 cm (L × W × D) that were heat-sealed over the tray opening with a gas-impermeable 85μm thick laminated polypropylene film (ORVED OPA PP) using an ORVED VGP tray sealing machine.Berries were placed in a single layer inside the package and the fruit weight inside each package ranged from 181 to 227 g.For the active MAP treatment, a CO 2 gas supply was connected to the sealing machine to flush during tray sealing and establish a targeted 6% CO 2 inside the packages.We had previously determined that 6% CO 2 is sufficient to maximize decay control in muscadines, while low O 2 is relatively ineffective (Shahkoomahally et al., 2021).The CO 2 flushing procedure ensured immediate establishment of the desired gas composition inside the package so that the fruit quality would not be affected by delay in developing the MAP due to the low respiration rate of muscadines (i.e., this was an active MAP system).On one side of the plastic tray, a hole of 4 mm diameter was made and based on preliminary tests, approximately half of the area of the hole was covered with plastic tape.For the MAP, a microporous patch with polymer coating (BreatheWay®, Curation Foods) was then pasted over the hole (Figure S1) to maintain the desired modified atmosphere (MA) inside the packages, which can adapt to temperature as well as respiration changes within the package.This technology helps in maintaining the desired gas mixture inside the package.Identical packages with the same hole size without a microporous patch were used as the control.This procedure was employed based on preliminary tests to restrict air movement in the packages to limit water loss without modifying the package atmosphere with regard to the respiratory gases.

| Fruit storage
After packing, the berries were stored in a cold storage room at 4 ± 0.1°C and 95% relative humidity (RH) to represent the typical temperature of many commercial storage facilities and of household refrigerators of consumers.Trays were kept on wire shelf units with a plastic curtain covering the top and sides of each shelf unit to avoid direct contact with air from the room fans.The experiment was laid out in such a way that each of the three replicates per treatment from both cultivars was evaluated after every 2 weeks in storage for 6 weeks.

| Concentrations of carbon dioxide and oxygen
Concentrations of CO 2 and O 2 inside three packages per treatment and cultivar were recorded twice per week during storage using a PBI Dansensor CheckMate 9000 Gas Analyzer (PBI, Ringsted, Denmark).A needle was inserted through the tray wall to the inside of the package to take gas readings and after taking each reading, the hole in the packaging was sealed with plastic tape.

| Evaluation of physical parameters
Weight loss on a fresh weight basis was evaluated by recording the weight of trays of berries at the beginning of the experiment and at the time of final evaluation.Decay incidence was calculated from the decayed berries out of the total number of berries in each tray and expressed as a percentage.
To record color and firmness, 10 berries from each of three replicates at each evaluation time were selected randomly.Color was recorded from two opposite sides of each berry using a chromameter (Konica-Minolta, CR-400, Osaka, Japan) and expressed as L*, a*, and b* values, with the a* and b* values being converted to hue angle and chroma.Firmness was recorded using a FirmTech 2 (Bioworks Inc., Wamego, KS, USA) instrument and 10 berries were subjected to the compression test to calculate the firmness from each replicate, which is expressed by the instrument as the resistance per unit distance of compression (g per mm −1 ).

| Evaluation of biochemical attributes
For evaluation of various biochemical attributes, 10 berries from each of three replicates at each evaluation time were blended after removal of seeds (leaving the edible portion of peel and flesh) and centrifuged at 19,319 × g n for 20 min at 4°C.The resulting supernatant was used to determine total soluble solids (TSS) and titratable acidity (TA).The TSS was recorded using an automatic temperaturecompensated refractometer (Reichert, Inc., Reichert r2i300, NY, USA).To record TA, 3.0 mL of fruit juice was titrated against 0.1 N NaOH to an endpoint of pH 8.2 using an automatic titrator (Metrohm 814 USB Sample Processor, Herisau, Switzerland).The methods for TSS and TA are as described by Shahkoomahally et al. (2021).The TA was expressed as the percentage of acid in the fruit juice based on the molecular weight of tartaric acid, the predominant organic acid in muscadine.

| Total antioxidants, total phenolic contents, and anthocyanins
Total antioxidants from the juice were determined using the ferric reduction antioxidant power (FRAP) assay method described by Benzie and Strain (1996).Trolox (6-hydroxy-2,5,7,8-tetramethylchro man-2-carboxylic acid) was used as the standard solution and the absorbance from each plate having standards and sample was recorded at 595 nm using a microplate reader (Epoch 2; BioTek Instruments, Inc., Winooski, VT, USA).Total antioxidants were expressed as mg kg −1 on a fresh weight basis for Trolox equivalents.
Total phenolic content (TPC) was determined using the Folin-Ciocalteu reagent method and the absorbance was recorded at 765 nm, using the method described by Singleton (1966) with some modifications.Gallic acid (GA) was used to make a standard curve and TPC was expressed as GA equivalents on a fresh weight basis and expressed as mg kg −1 .
Anthocyanins were determined using the pH differential method (Giusti & Wrolstad, 2001).Sodium acetate (0.40 M, pH 4.5) and potassium chloride (0.025 M, pH 1.0) buffers were used to determine anthocyanins.The absorbance of samples was recorded at 510 and 700 nm using a BioTek microplate reader and anthocyanins were calculated using the formula described by Shahkoomahally et al. (2021).

| Statistical analysis
Statistical analysis was performed as analysis of variance with two factors (MAP treatments × storage days) using MINITAB® 16 statistical software (Minitab Inc., PA, USA).The data were treated for multiple comparisons with least significant differences test (Fisher's LSD) at p ≤ .05level of significance.

| Concentrations of carbon dioxide and oxygen
Muscadines were packed in sealed trays with BreathWay® patches to develop MAP.In MAP, the CO 2 concentration decreased from 6.29% initially to a minimum of 4.01%, averaged over the two cultivars, then started increasing gradually, whereas the control trays did not show any significant change in CO 2 during the entire storage duration (Figure 1).For 'Granny Val', a 1.76-fold decrease in CO 2 concentration was recorded after 14 days of storage.Later, CO 2 started slowly increasing from 14 to 42 days of storage, reaching 4.75%.Like 'Granny Val', 'Supreme' also followed a similar decreasing trend of CO 2 concentration in the beginning, but the minimum CO 2 concentration for 'Supreme' (4.37%) was recorded after 7 days of storage in MAP.This minimum CO 2 concentration was 1.44-fold less as compared to Day 1 and started increasing afterward; on the last day of storage, it was 1.44-fold higher than Week 1 reaching 6.28% of CO 2 .Regardless of F I G U R E 1 Effect of MAP on the package headspace CO 2 levels for (a) 'Granny Val' and (b) 'Supreme' muscadine grape berries over 42-day storage at 4°C.Data presented here are the mean values of three packages and different letters indicate significant differences between treatments and sampling times (p ≤ .05).

| Physical parameters
Berry weight loss was not affected significantly in response to MAP (Figure 3a) but reduction in decay incidence was highly significant (Figure 3b).The firmness of the berries of both grape cultivars declined significantly during storage -by approximately 14.6 and 15.1% for 'Granny Val' and 'Supreme', respectively (Figure 4).Berry firmness of 'Supreme' was not affected significantly by MAP, whereas MAP resulted in significantly higher firmness retention for 'Granny Val' during the early part of the storage duration (Figure 4).

| Biochemical attributes
There were no significant differences among treatments and storage days for the TSS of both muscadine grape cultivars (Figure 5),  whereas TA increased during storage in both cultivars (Figure 6).
Regardless of treatment, the TA values for 'Granny Val' and 'Supreme' were 1.14-and 1.24-fold higher, respectively, at 42 days than on Day 0. The MAP did not affect the TA of 'Supreme' while in 'Granny Val', the TA of the MAP berries remained lower (i.e., closer to the initial value) than the control.

| Total antioxidants, total phenolic contents, and anthocyanins
Total antioxidants in both cultivars were not affected significantly by the packaging treatments, but there were highly significant changes during storage (Figure 7).Both cultivars showed an increasing trend in total antioxidants initially, which decreased later during storage.
In 'Granny Val', total antioxidants increased up to 14 days and afterward decreased linearly until the last day of storage.In 'Supreme', a linear increase in total antioxidants was recorded up to 28 days that decreased later until 42 days.Averaged over the treatments, the maximum antioxidant values for 'Granny Val' and 'Supreme' were 1.25-fold higher on Day 14 and 1.34-fold higher on Day 28, respectively, than on Day 0. Like antioxidants, TPC was unaffected by MAP; however, there were significant linear increases in TPC during storage for both grape cultivars (Figure 8).Regardless of MAP treatment, the maximum TPC values were found on the last day of storage when they were 2.55-fold and 1.55-fold higher than on Day 0 for 'Granny Val' and 'Supreme', respectively.There were no significant differences in the anthocyanin contents of 'Granny Val' or 'Supreme' grapes in response to MAP treatments; however, the amounts of anthocyanins differed hugely between the two cultivars (Figure 9).For bronze 'Granny Val' berries, the anthocyanin content during storage varied between about 1.2 and 0.4 mg kg −1 while for black 'Supreme', the anthocyanin content increased significantly from about 500 mg kg −1 on Day 0 to about 800-900 mg kg −1 on Day 28, with no further change occurring afterward.

| DISCUSS ION
Muscadine grapes, being a delicate fruit, cannot be stored for more shelf life of numerous perishable commodities.Moreover, higher CO 2 and lower O 2 have been reported to increase the storage life of muscadine, primarily due to the CO 2 benefits (Shahkoomahally et al., 2021).In the current study, a MAP that was developed through a patented membrane technology (BreatheWay®, Curation Foods, USA) can adapt to temperature fluctuations and respiration changes.
MAP was tested in cold storage to determine the impact on shelf life, quality, and composition of two muscadine grape cultivars.In our study, the CO 2 concentration in MAP was increased from almost 0% to about 4%-6% and the O 2 was decreased from 20.8% to about 14%-15%.These gas conditions proved to be beneficial environment for muscadine grape storage, particularly in reducing fungal decay.
It was found that MAP can reduce decay incidence significantly without having any impact on berry weight loss.The elevated concentration of CO 2 maintained by MAP in the current work was previously shown to help in reducing disease incidence of muscadine during CA storage (Shahkoomahally et al., 2021).Exposure to elevated CO 2 has also been reported to reduce decay incidence of common table grapes in some other studies (Candir et al., 2012;Romero et al., 2019).
Weight loss was not significantly different in the control and MAP treatments as berries in both treatments were stored in sealed trays under cold storage.Similar results of insignificant weight loss have also been reported in some other studies of packaged table grapes (Martínez-Romero et al., 2003;Valverde et al., 2005).
Among various color parameters, L* and Hue are important to describe changes in postharvest fruit quality (Valverde et al., 2005).In the current experiment, L* and Hue decreased linearly during storage for 'Granny Val' as the skin darkened slightly (Figures S2 and   S3), but MAP resulted in better retention of L* during storage, which agrees with the results of Shahkoomahally et al. (2021).Changing of the color from bright to dull could be due to changes in the TPC (Himelrick, 2003), which increased during storage.Berry firmness of 'Granny Val' was better maintained in MAP for 14 days at 4°C, but was otherwise not affected significantly by MAP in either cultivar as the firmness declined over the entirety of storage, which could be related to the decrease in total pectin during storage of muscadines as previously reported (Himelrick, 2003).
The TSS and TA are important parameters to determine the organoleptic quality of dessert fruit like muscadine.In the current study, TSS was not affected by MAP treatment and did not change significantly during storage, but TA was maintained slightly lower in MAP as it increased during storage.No differences in TSS during storage have also been reported in different studies on both table grapes and muscadine (Barchenger et al., 2015;Romero et al., 2019).Marsh et al. (2004) reported an increase in malate content during storage of kiwifruit at 4°C as compared to 0°C.Thus, storage temperature can affect the ratio of the major acids in fruit.This could be a possible reason for the increase in acidity of muscadine grape berries stored at 4°C in the current study.
Fruit suffer from stress during storage as nutrient and water supply from the parent plant is discontinued, which results in activating defense mechanisms.Different enzymatic and nonenzymatic antioxidants are produced under such postharvest storage stress (Xu et al., 2009).
Phenylalanine ammonia-lyase (PAL) is among the enzymes whose activity can increase in response to stress, resulting in increasing TPC (Nia et al., 2021;Shah et al., 2022).In the current study, an increase in TPC and antioxidants during storage could be due to the defense mechanism of the fruit against postharvest storage stress.Moreover, TPC during storage could also increase due to enhanced supply of carbon skeleton and degradation of organic acids (Maurer et al., 2017).After a certain period of storage, the decrease in antioxidants observed here might be due to fruit aging and senescence, which had previously been observed in grapes (Khalil, Rajwana, Razzaq, Farooq, et al., 2023;Nia et al., 2021).
Anthocyanins are pigments and their virtual absence in 'Granny Val' is not surprising being a bronze cultivar.'Supreme', a black muscadine cultivar, contained higher anthocyanin content than 'Granny Val', which also increased during storage.A similar increase in some cultivars of muscadine was also reported by Marshall-Shaw et al. (2014) who, like in the present study, also found a positive correlation between TPC and anthocyanins.

| CON CLUS ION
The current study provides evidence of improved quality retention of muscadine grapes during storage by using MAP.Fruit stored in MAP had lower decay with better berry firmness and color retention, especially up to 28 days of storage at 4°C.Even after 42 days of storage, the decay incidence was significantly lower in MAP, whereas the biochemical attributes were not affected.Further studies should be carried out with MAP and muscadine using even higher CO 2 concentrations than the current experiment as muscadine grapes are highly tolerant of elevated CO 2 levels.
', a linear increase in values of L*, b*, chroma, and hue was recorded up to 28 days, which decreased slightly afterward, irrespective of treatment.The L* remained lower in MAP as compared with the control and, on the last day of storage, the difference was 4.0 units.The a* value decreased linearly during the storage irrespective of the applied treatments, but MAP fruit retained 37.6% higher a* value until the last day of storage.The values of b*, chroma, and hue were retained at lower values in MAP with a difference of 3.4-, 1.4-, and 1.5-fold, respectively, at the end of storage.

F I G U R E 3
Effect of MAP on the weight loss (a) and decay incidence (b) for 'Granny Val' and 'Supreme' muscadine grape berries after 42 days of storage at 4°C.Data presented here are the mean values and different letters within column clusters indicate significant differences between treatments (p ≤ .05).Effect of MAP on color parameters of 'Granny Val' and 'Supreme' muscadine grape berries over 42-day storage at 4°C.
than about 2 weeks without postharvest interventions.Refrigerated storage in MAP has been proven effective in maintaining better F I G U R E 4 Effect of MAP on berry firmness for (a) 'Granny Val' and (b) 'Supreme' muscadine grape berries over 42-day storage at 4°C.Data presented here are the mean values and different letters indicate significant differences between treatments and sampling times (p ≤ .05).Effect of MAP on total soluble solids for (a) 'Granny Val' and (b) 'Supreme' muscadine grape berries over 42-day storage at 4°C.Data presented here are the mean values and different letters indicate significant differences between treatments and sampling times (p ≤ .05).

F I G U R E 6
Effect of MAP on titratable acidity for (a) 'Granny Val' and (b) 'Supreme' muscadine grape berries over 42-day storage at 4°C.Data presented here are the mean values and different letters indicate significant differences between treatments and sampling times (p ≤ .05).Effect of MAP on total antioxidants for (a) 'Granny Val' and (b) 'Supreme' muscadine grape berries over 42-day storage at 4°C.Data presented here are the mean values and different letters indicate significant differences between treatments and sampling times (p ≤ .05).

F I G U R E 8
Effect of MAP on total phenolic content (TPC) for (a) 'Granny Val' and (b) 'Supreme' muscadine grape berries over 42-day storage at 4°C.Data presented here are the mean values and different letters indicate significant differences between treatments and sampling times (p ≤ .05).Effect of MAP on total anthocyanins for (a) 'Granny Val' and (b) 'Supreme' muscadine grape berries over a 42-day storage at 4°C.Data presented here are the mean values and different letters indicate significant differences between treatments and sampling times (p ≤ .05).
storage days, MAP resulted in 6.49-and 7.48-fold higher CO 2 than the control for 'Granny Val' and 'Supreme', respectively.The concentrations of O 2 for both cultivars did not change significantly during the entire storage duration in the control trays without MAP (Figure2).Overall, the O 2 level decreased during storage in MAP for both cultivars.For 'Granny Val', the O 2 level de- Data presented here are the mean values and different letters within column indicate significant differences between treatments (p ≤0.05).