Physiological and quality responses of Shishe-Kab pomegranates to short-term high CO₂ treatment and modified atmosphere packaging

ABSTRACT

The objective of this study was to monitor quality parameters and to reduce postharvest losses as well as to extend the storage life of pomegranate fruit using pre-storage high CO₂ treatment and active packaging. Short-term high CO₂ treated pomegranate fruits cv. Shishe-kab packed with nano-bags (MAP1 and MAP2) or remained unpacked and stored in cold storage at 5°C for 12 weeks. The results show that the fruit decay, chilling injury, and weight loss values were significantly decreased by short-term high CO₂ treatment in both packed (MAP) and unpacked fruits compared to unpacked control. As a general trend, there were no significant differences in pH, TSS, and TA between treatments at the end of storage period. Slight decreases in aril colour, measured as a* and chroma values were found in packed fruit with MAP2, which was included with the 1-MCP sachet. In conclusion, the short-term high CO₂ shock alone was effective to reduce weight loss, decay, and storage disorders on pomegranate fruit cv. Shishe-kab compared to control, suggesting high CO₂ shock as an alternative treatment to control postharvest decay and also maintaining the postharvest quality of the pomegranate fruits during cold storage.

KEYWORDS:

• Chilling injury
• decay
• MAP
• postharvest
• weight loss

Introduction

Pomegranate (Punica granatum) is one of the oldest known edible fruits originated from central Asia (Pareek et al., 2015). Iran is one of the main producers and also the exporters of some commercial cultivars of this valuable fruit in the world (Tehranifar et al., 2010). However, the global production and consumption of pomegranate have greatly expanded in recent years due to increasing consumer awareness of the potential health benefits of the fruit and consequently more demand for fresh fruit in the market (Aviram et al., 2008). Minimizing postharvest losses of horticultural perishables including tropical and subtropical fruits, which is estimated about 20–50% of the total production in the world, is a very effective way of increasing food availability (Kader, 2004).

In spite of the non-climacteric nature of the fruit, chilling injury, husk scald, water loss, and decay development are the major causes of significant postharvest losses in pomegranate during improper handling conditions (Artés and Tomás-Barberán, 2000; F. Artés, R. Villaescusa, and J.A. Tudela, 2000; F. Artés, R. Villaescusa, and J.A. Tudela, 2000; Kader et al., 1984; Roy and Wasker, 1997). Hence, during the last two decades, research on pomegranate fruit was focused on the application of new postharvest storage technologies included use of fungicides to control decay, and CA (controlled atmosphere) or MA (modified atmosphere) treatments to maintain postharvest quality and to alleviate chilling injury which occur during cold storage of pomegranates (Yahia, 2011). MA and CA storage have been tested with satisfactory results in maintaining pomegranate quality during storage as reported in previous studies (F. Artés, R. Villaescusa, and J.A. Tudela, 2000; Nanda et al., 2001; Pierce and Kader, 2001; Porat et al., 2008). However, the cost of continuously maintaining the low O₂ and/or high CO₂ atmosphere could be prohibitive. The use of short-term controlled atmospheres and using films for modified atmosphere packaging (MAP) could provide a cheaper alternative.

MAP either using plastic bags or shrink film wrapping is beneficial in reducing water loss and quality maintenance of pomegranate fruit, as reviewed by Caleb et al. (2012). Our previous study showed that ‘Shishe-kab’ pomegranate, which is an important commercial Iranian cultivar when treated with hot water, salicylic acid, and calcium chloride and stored at 5°C in low-density polyethylene (LDPE) film of 30 μm thickness had longer storage life because of lower chilling injury symptoms and decay than unpacked fruit (Moradinezhad et al., 2013). Public concern about food safety and environmental issues and the increase of pathogen-resistant populations have enhanced the interest in developing alternative methods to fungicides to control postharvest fruit decay (Nunes, 2012). An alternative is carbon dioxide (CO₂), which is a commonly used food additive, and a compound that is generally regarded as safe. Previous studies have shown that pre-storage short-term high CO₂ treatment effectively maintained the quality of various fruits such as blueberry (Jiang et al., 2011), citrus (Montesinos-Herrero et al., 2012), peach (Bonghi et al., 1999), and persimmon (Besada et al., 2015). Moreover, in a recent study on pomegranate grey mold caused by Botrytis cinerea significantly inhibited by short-term high CO₂ treatment (Palou et al., 2016).

The active packaging system is more rapid and accurate than traditional MAP. The active system can be an integral part of the package or be a separate component placed inside the package (Yahia, 2009). Different substances that can either absorb or release a specific gas, control the internal atmosphere within the package. It is known that 1-methylcyclopropene (1-MCP), an inhibitor of ethylene action, can also greatly influence the susceptibility of horticultural products to postharvest disorders as reviewed by Watkins (2008) who stated that the effects of 1-MCP on physiological and pathological disorders are beneficial and detrimental depending on the product. In addition, little information about effects of 1-MCP on diseases and disorders resistance of pomegranate is available. Defilippi et al. (2006) found that neither diphenylamine (DPA) nor 1-MCP treatments reduced incidence or severity of scald on ‘Wonderful’ pomegranates.

Although several studies have addressed effectiveness of MAP in maintaining pomegranate fruit quality in different cultivars including Selcuk and Erkan (2016), Nanda et al. (2001), Selcuk and Erkan (2016), Bayram et al. (2009), F. Artés, R. Villaescusa, and J.A. Tudela, (2000), D’Aquino et al. (2010), Moradinezhad et al. (2013), and Ghafir et al. (2009), however, no study has been conducted yet on the effect of short-term CO₂ treatment alone or in combination with active packaging on postharvest quality of Iranian ‘Shishe-kab’ cultivar. Therefore, the aim of this article was to study the effect of pre-storage short-term high CO₂ treatment (12 h) individually or combined with active packaging including 1-MCP sachet on the quality attributes and disorders of pomegranate fruit.

Materials and methods

Fruit preparing, treatments, and packaging procedures

Pomegranate fruits (Punica granatum cv. ‘Shishe-kab’) harvested from similar trees on a commercial orchard in Ferdows, South Khorasan province, Iran during 2015 growing season. Harvesting was carried out manually with maximum care to minimize mechanical damage. Fruits were harvested at full maturity stage, early in November and immediately transported to the Postharvest Laboratory at University of Birjand, Iran. Sunburned, bruised, and injured fruits were discarded. The remaining fruits were prepared for the following treatments in four replications (Table 1). Neither washing nor postharvest chemical treatments were applied to the fruit. A total of 480 uniform fruit (270–320 g) were selected and randomly divided into four lots. Thereafter, each lot was given one of the following four treatments in three replications. 1) control fruits (fruit without any treatment and packaging) were stored in open plastic boxes; 2) short-term high CO₂ fruits were packed in a polyethylene bags (70 μm thickness) and the internal gas in package was removed by a vacuum pump, then added the CO₂ into the package, sealed and kept for 12 h at 18°C; 3) MAP1 fruits treated with CO₂ as high CO₂ treatment and then packed in Nano-bags (40 × 60 cm, Decoo Magic Bag, Decoo Co., Italy); and 4) MAP2 fruits treated with CO₂ as high CO₂ treatment and then packed in Decoo Magic Bag, one 1-MCP sachet (3g/bag, Decoo Co., Italy) was included in the packaged fruit before sealed (Table 1). Each replication comprises two plastic boxes (20 fruit per box), packed in MAP1 and MAP2 or unpacked in control and short-term high CO₂ treatments. The CO₂ level was measured with a gas analyzer (OXYBABY ®6.0, O₂/CO₂, Germany). Fruits were then stored for 12 weeks in cold room at 5 ± 0.5°C at 85 ± 5% RH. Thereafter, fruits were transferred to the lab and were assessed for color, chilling injury (CI), and physicochemical parameters. The rest of fruit from each experimental unit (30 pomegranates) were kept for a further 2 days at 20 ± 2°C to evaluate taste and decay.

Table 1. Treatments tested for quality maintenance and control of storage disorders on ‘Shishe-kab’ pomegranate fruit.

Pre-storage treatments	Packaging	Details
Control	Unpackaged	No treatment, stored in open plastic boxes in air at 5°C
High CO2	Unpackaged	CO2 85% for 12 h at 18°C, then stored in open plastic boxes in air at 5°C
High CO2	MAP1	CO2 85% for 12 h at 18°C, sealed in Decoo Magic Bag, stored in air at 5°C
High CO2	MAP2	CO2 85% for 12 h at 18°C, sealed in Decoo Magic Bag included one 1-MCP sachet, stored in air at 5°C

Physicochemical measurements

Titratable acidity, pH and total soluble solids, TSS/TA ratio

Titratable acidity (TA) was determined by titration of 2 ml of juice with 0.1 M NaOH to an end point of pH 8.2 and results were showed as a percentage of citric acid. The pH was measured at room temperature using a pH meter. Total soluble solids (TSS) was determined with a hand-held refractometer (RF 10, °Brix 0–32, Extech Co., USA) at 25°C, and expressed as °Brix.

Total anthocyanin

Total anthocyanins content (TAC) was determined spectrophotometrically by the pH differential method (Lako et al., 2007). Absorbance was measured at 510 and 700 nm in buffers at pH 1.0 and 4.5 using spectrophotometer (Model Unico 2100, China), and then calculated according to the following Equation (1):

(1)

Results were expressed as mg of cyanidin-3-glucoside per 100 mL of juice, using a molar absorptive coefficient (ε) of 26,900 and a molecular weight of 449.2, and then total anthocyanin content was calculated as follows (2):

(2)

where A = absorbance; MW = molecular weight of cyanidin-3-glucoside; DF = the degree of dilution; = molar absorptive coefficient.

Skin color

Peel color of pomegranate fruits was measured by using colormeter (TES-135A, Taiwan) and recorded as L* (lightness: 0, dark; 100, white), a* (–greenness to +redness) and b* (–blueness to +yellowness). The different color indexes (hue and chroma) were calculated according to the following Equations (3, 4):

(3)

(4)

Weight loss, decay development, and physiological disorders

To determine weight loss, individual fruit were weighed at harvest time (day 0) and after storage period (day 90). The weight loss was calculated as follows (5):

(5)

where W = weight loss (%) of fruit; W1 = initial weight (g) of the fruit at the beginning of storage, and W2 = final weight (g) of the fruit at end of storage period. Weight loss was calculated for each storage temperature on three individual fruits.

The chilling injury (CI) occurrence and its intensity symptoms was recorded visually on a 4-point hedonic scale based on the percentage of husk surface affected by CI symptoms (dehydration, browning, and pitting): 1 = (no CI symptoms), 2 = (1–25% of surface damaged), 3 = (26–50% of surface damaged), 4 = (>51% of the surface damaged). Fruit decay was visually evaluated during the experiment. Any pomegranates with visible mold growth were considered decayed. Fruit decay was expressed as a percentage of fruit showing decay symptoms. The unmarketable fruit was evaluated as a result of overall visual quality based on undesirable fruit after 12 weeks of storage. Any fruits with visible CI and/or decay symptoms were considered unmarketable. The percentage of unmarketable fruit was calculated with the following formula (6):

(6)

where U = unmarketable fruit (%); U1 = number of fruit in each treatment that was undesirable and U2 = number of fruit in each treatment.

Organoleptic evaluation

Sensorial quality for aril taste of the fruit was evaluated by a panel of seven trained subjects after 12 weeks of storage. The evaluation was scored on a scale of 1–5, where a score of 5 indicated the fruit was very good (evident harvest freshness, bright pink juicy arils, and absence of off-flavor), and a score of 1 was considered a very bad degree (complete dislike, desiccated fruits with tough brown peel, brown colour arils with low juiciness and becoming dry). A score of 3 (like moderate with retention of freshness, colour, and juiciness of arils) and above was considered acceptable for marketing.

Statistical analysis

The obtained data were statistically analyzed as a completely randomized design experiment with four replications. Data were analyzed using GenStat program (Version 12.1, VSN, International, Ltd., UK, 2009). The difference between mean values of parameters was investigated by using LSD’s test to examine if differences were significant at p ≤ 0.05.

Results and discussion

Fruit weight loss

The results showed that the highest amount of weight loss (7.2%) was found in unpackaged control (Table 2). No significant effects were found between other treatments. Interestingly, high CO₂ treated fruit that was kept unpacked in the air had similar weight loss to high CO₂ + MAP1/MAP2 packed fruits. This shows that high CO₂ shock alone significantly reduced respiration rate of Shishe-kab pomegranates in unpacked fruit. Generally, the weight loss of the fruits progressively is increased over the storage time. The weight loss in pomegranates is mainly caused by water transpiration and CO₂ loss during respiration. It has been reported that low O₂ and/or high CO₂ reduced respiration and ethylene production rates. So that the reducing weight loss by CO₂ treatment could be because of lower respiration which leads to lower water transpiration. The modification of gas inside the package reduced the respiration rate of the fruit so that the water loss decreased. Moreover, the high relative humidity inside the bag is an important factor in suppressing the water loss from fruit; the modified gas condition might be a secondary effect cause of reducing the transpiration (Srilaong et al., 2002). MAP may also prevent weight loss and fruit shriveling by creating a higher relative humidity in the surrounding environment of the products (Zagory et al., 1989). 1-MCP did not affect fruit weight loss.

Table 2. Effect of pre-storage short-term high CO₂ treatment and active packaging on weight loss (WL), decay, chilling injury index (CI), unmarketable, and organoleptic assessment of pomegranate fruit cv. ‘Shishe-kab’ after 12 weeks of storage at 5°C.

Pre-storage treatments	Packaging	WL (%)	Decay (%)	CI (%)	Unmarketable fruit (%)	Organoleptic* (Taste)
Control	Unpackaged	7.20a	13.20a	1.95a	26.00a	3.50a
High CO2	Unpackaged	1.40b	1.60b	1.02b	14.20b	3.80a
High CO2	MAP1	0.80b	0.70b	1.22b	12.90b	3.60a
High CO2	MAP2	0.50b	1.00b	1.40b	15.00b	3.30a

Mean values in each column followed by the same letter are not significantly different by the LSD (p ≤ 0.05).

* A score of 3 and above was considered acceptable for marketing.

Decay development

As expected, the highest percentage of decay (13.2%) was found in unpacked control fruit (Table 2). No significant differences were found between other treatments. Control unpacked pomegranate were subjected to microbial decay, while high CO₂ shock inhibited the growth of decay organisms on both unpacked and MAP fruits. Decay was successfully controlled by CO₂ treatment, as previously shown (Pierce and Kader, 2003). In addition, in ‘Mollar de Elche’ sweet pomegranates stored at 2 or 5°C for 12 weeks in unperforated polypropylene film of 25 μm thickness had less incidence of decay mainly due to Penicillium spp (F. Artés, R. Villaescusa, and J.A. Tudela, 2000). Microbial growth is retarded at high concentration of carbon dioxide in various products, due to an increased lag phase and generation time during the logarithmic phase of microbial growth (Farber et al., 2003). Inhibition of organisms by CO₂ may result from dissolved CO₂ in the aqueous phase of food products, which causes a decrease in the intracellular pH, inhibits enzymatically catalyzed reactions and enzyme synthesis, and interacts with the cell membrane (Molin, 2000). These results were according to Eksteen and Truter (1985) on avocado who stated that controlled atmosphere improves the quality of avocado.

In contrary to findings of Guo et al. (2012) who stated 1-MCP treatment decreased the decay rate of pomegranate fruit, in our study using 1-MCP sachet did not prevent fruit decay rate further. This inconsistency is likely due to the different method of 1-MCP application as Guo et al. (2012) were gassed fruits with 1-MCP before storage whereas we used 1-MCP sachet inside package during the storage period.

Chilling injury (CI) index

As the results showed, there were no significant differences between high CO₂ treated fruits in both unpackaged or packaged (MAP1 and MAP2) treatments, while unpacked control significantly had the higher amount of chilling injury (Table 2). However, high CO₂ treatment prevents chilling injury after prolonged cold storage in unpackaged pomegranates compared to the unpackaged control. The CI index in control (1.95) was higher than high CO₂ treated unpackaged pomegranates (1.02). This was in contrary to findings of Faubion et al. (1992) who stated that carbon dioxide shock was not effective in delaying chilling injury in the ‘Hass’ avocado. It has also been reported that MAP using unperforated polypropylene films of 25 μm thickness at 5°C for 12 weeks was very effective in controlling chilling injury in ‘Mollar de Elche’ pomegranates (F. Artés, R. Villaescusa, and J.A. Tudela, 2000). Therefore, it seems that Decoo Magic Bags used in our experiment were not suitable for controlling chilling injury in ‘Shishe-kab’ pomegranates and/or this cultivar is susceptible to chilling injury during prolonged cold storage. In our previous report regarding Shishe-kab pomegranate (Moradinezhad et al., 2013), which is an Iranian commercial cultivar, we noted that Shishe-kab like some other cultivars are susceptible to chilling injury when they stored in cold storage at 5° for long term, hence, we finish the storage time at week 12 to prevent from further CI occurrence. Fruits that were treated with high CO₂ and packed with 1-MCP sachet inside the package (MAP2) had similar chilling injury values to other treatments. The results were in agreement with the findings of Defilippi et al. (2006) who stated that 1-methylcyclopropene (1-MCP) treatment did not reduce the severity of scald on ‘Wonderful’ pomegranates. Although 1-MCP reduced chilling injury in avocado as reported by Hershkovitz et al. (2005).

Unmarketable fruit

Unpacked control showed the highest percentage of unmarketable fruits (26%), while high CO₂ unpacked and MAP1 or MAP2 treatments showed the lowest percentage of unmarketable fruits (Table 2). It has been reported that high CO₂ leads to reduced enzymatic browning in lettuce tissue by inhibition of phenolic production and polyphenol oxidase activity (Siriphanich and Kader, 1985). Eksteen and Truter (1985) also reported that controlled atmosphere storage improves the shelf life of avocados.

The potential for flesh browning of fruits is primarily a function of the browning enzyme polyphenol oxidase (PPO) (Kahn, 1975). Bower et al. (1989) studied the effect of CA, CO₂ shock and regular atmosphere (RA) storage in PPO activity in Fuerte avocados. They found that both CA and CO₂ shock treated fruit showed a lower incidence of physiological disorders than the control fruit. The results of our experiment showed that using Nano-bags and/or 1-MCP sachets in combination with high CO₂ shock did not further impact on reducing the amount of damaged unmarketable fruits. In other words, high CO₂ shock treatment alone was effective in controlling decay of pomegranates.

Organoleptic evaluation

No significant differences were found between the treatments from organoleptic aspects of pomegranate fruits (Table 2). Smith (1992) also recorded that there was no indication that the CO₂ caused any undesirable changes in quality of the fruit and it’s organoleptic. 1-MCP sachet did not affect fruit taste. Overall, no off-flavor or bad smell were observed even after 12 weeks of cold storage as judged by panelists. However, we did not measure any alcoholic taste.

Skin colour

The results showed that there were no significant differences in the b* value of pomegranate arils among different treatments (Table 3). This was in accordance to Tavasoli Talarposhti et al. (2016) results who stated that there were no differences in the b* value of aril color between different modified atmospheres. Also, there were no significant differences in the L* and hue values of pomegranate arils among different treatments (Table 3). However, the aril color of high CO₂ treated pomegranates in both unpackaged (30.5) and MAP1 (24.5) treatments became more intensely red (chroma increased) and was higher than control (21.5) and MAP2 (17.2) fruits. This was in agreement with the findings of Nunes et al. (1995) in strawberry and Tavasoli Talarposhti et al. (2016) in pomegranates who stated that fruit stored in the high CO₂ atmosphere were redder (higher chroma) than fruit stored in air. 1-MCP affected a* and chroma of arils. The a* (16.6) and chroma (17.2) values of aril significantly decreased in 1-MCP treated fruits. The results in this work are in accordance with the results of Hershkovitz et al. (2005) who observed a significant decrease in chroma value of avocado when used 1-MCP+MAP at low storage temperature.

Titratable acidity, pH, total soluble solids, TSS/TA

There were no significant differences among treatments in total soluble solids and titratable acidity (Table 4). This may be the result of the high buffering capacity of pomegranate juice. Juice pH also did not change significantly among treatments (Table 4). This was in accordance to Holcroft et al. (1998) findings who stated that carbon dioxide had no effect on pH, TSS, and TA of pomegranate juice.

Table 3. Effect of pre-storage short-term high CO₂ treatment and active packaging on aril colour attributes of pomegranate fruit cv. ‘Shishe-kab’ after 12 weeks of storage at 5°C.

Pre-storage treatments	Packaging			Aril
		L*	a*	b*	h°	C*
Control	Unpackaged	17.7a	21.3a	2.88a	8.9a	21.5b
High CO2	Unpackaged	21.9a	29.6a	7.37a	14.2a	30.5a
High CO2	MAP1	16.5a	23.3a	4.74a	10.8a	24.5a
High CO2	MAP2	17.4a	16.6b	3.29a	12.1a	17.2c

Mean values in each column followed by the same letter are not significantly different by the LSD (p ≤ 0.05).

Table 4. Effect of pre-storage short-term high CO₂ treatment and active packaging on chemical attributes and anthocyanin of pomegranate fruit cv. ‘Shishe-kab’ after 12 weeks of storage at 5°C.

Pre-storage treatments	Packaging	pH	TSS (%)	TA (%)	TSS/TA	Anthocyanin (mg^−l)
Control	Unpackaged	5.91a	16.67a	0.56a	29.76a	15.60a
High CO2	Unpackaged	5.97a	16.52a	0.50a	33.04a	16.20a
High CO2	MAP1	5.71a	17.33a	0.69a	26.25a	16.35a
High CO2	MAP2	5.90a	17.17a	0.62a	27.69a	15.42a

Mean values in each column followed by the same letter are not significantly different by the LSD (p ≤ 0.05).

Total anthocyanin

Fruit anthocyanin was not affected by treatments (Table 4). Our results agree with those reported by Gil et al. (1996), who found no significant change in total anthocyanin content in arils from ‘Mollar de Elche’ pomegranates harvested in early October during MAP storage at 1°C up to 7 days. However, F. Artés, R. Villaescusa, and J.A. Tudela (2000) reported that packed pomegranate fruits ‘Mollar de Elche’ cultivar in polypropylene perforated (PPP) and unpackaged control at 5°C showed an increase in total anthocyanin content after 12 weeks of cold storage period.

Conclusions

The lowest physiological disorders were obtained in MAP and high CO₂ treatments after prolonged cold storage. Our results also confirm the review of Watkins (2008) who stated that the effects of 1-MCP on physiological and pathological disorders are beneficial and detrimental depending on the product. In summary, the pre-storage short-term high CO₂ shock was an effective treatment to reduce weight loss, decay, chilling injury, and the total damaged unmarketable pomegranate fruit cv. Shishe-kab and can be used as an alternative to fungicide and chemical treatments. In addition, it is a cheaper and quick method than MAP. Pomegranate fruits also are susceptible to dipping as it develops the pathogens activities like grey mould rot caused by Botrytis cinerea that usually starts from the calyx and also on the affected region under a moist condition during storage. Therefore, the short-term high CO₂ gassing can be used as a useful and low-risk method than dipping in pomegranates. However, further study is needed to assess the effect of short-term high CO₂ treatment in different concentration/duration on Shishe-kab pomegranate and also for a longer storage period than 12 weeks.