Document Type : Full Research Paper


1 Post-harvest Physiology and Technology, Department of Horticulture, University of Hormozgan, Bandar Abbas, Iran.

2 Department of Horticulture, University of Hormozgan, Bandar Abbas, Iran.

3 Agricultural Engineering Research Department, Isfahan Agricultural and Natural Resources Research and Education Center, AREEO, Isfahan, Iran.

4 Horticulture Crops Research Department, Isfahan Agricultural and Natural Resources Research and Education Center, AREEO, Isfahan, Iran.


Introduction: Pomegranate (Punica granatum L.) is an important horticultural fruit that is generally very well adapted to the Mediterranean climate. Arils are the edible part of this fruit, being rich in anthocyanins and bioactive compounds such as phenolic compounds and flavonoids which act as antioxidants and free radical scavengers. Susceptibility to chilling injury in pomegranate fruits is a major limiting factor in storing fruits at low temperatures. Below 5 °C, pomegranate fruits show symptoms such as surface pitting, browning, discoloration, and decay. The control of temperature is an effective tool for extending the shelf life of fresh horticultural products. Oxidative stress, as caused by an excess of reactive oxygen species (ROS), is usually associated with chilling injury in fruits. Nitric oxide (NO) is an important gas molecule, the involvement of which in many physiological processes can protect plant cells against oxidative stress by reducing the accumulation of ROS. Postharvest studies have shown that the application of NO gas can extend the storage life of a range of horticultural produce by delaying ripening or senescence. Due to the high number of pomegranate cultivars in Iran, limited amounts of information exist on how the qualitative characteristics of arils in the Malas pomegranate can be affected by nitric oxide and different temperatures during storage. The Malas cultivar comprises a large share of pomegranate exports from Iran. In this research, the positive effects of nitric oxide were examined on reducing the chilling injury and maintaining the fruit quality of pomegranate. The application of this treatment at different concentrations and different storage temperatures led to variable effects on the qualitative characteristics of arils in the Malas pomegranate.
Materials and methods: Malas pomegranate fruits were harvested commercially from Isfahan Province and were transferred to the Food Industry Laboratory of Isfahan Natural Resources Research. The fresh arils were separated from fruit tissues and were immersed in solutions of nitric oxide (0, 5 and 10 μM/L) for 5 min. After draining, the arils were placed in packaging films of polyethylene and were immediately stored at 2, 4 and 8 °C for 21 days. Several parameters were measured every 7 days during the storage time. These were the weight loss, total soluble solids, titratable acidity, TSS/TA, acid ascorbic, total phenol, total anthocyanin content, antioxidant activity, MA, ion leakage, POD, PPO activity and sensory evaluation. The current study was carried out as a factorial assay and was based on a completely randomized design with three replications. Data were processed by ANOVA using the SAS software version 9.4. Significant differences were identified using Duncan’s test at 1% probability level.
Results and discussion: Results showed that the total anthocyanin content, total phenol, antioxidant activity, and titratable acidity decreased during storage time. The control group and the treatment with low temperatures significantly reduced the qualitative characteristics of arils during storage. The water content of arils treated with 5 and 10 μM nitric oxide was maintained considerably for 21 days during storage. According to these results, unlike titratable acidity and ascorbic acid which decreased in both treated and untreated fruits, there was an increase in the total soluble solids and POD activity of arils during storage. However, nitric oxide reduced the rate of these changes, whether it be the decrease or increase in the measured parameters. During the experiment, the control samples showed lower values of quality regarding all parameters. The use of nitric oxide in fruits reduced lipid peroxidation and ion leakage significantly, whereas the antioxidant activity increased. The decrease in ion leakage was observed most notably in fruits that were treated with 10 μM nitric oxide. Furthermore, low temperatures managed to disrupt the metabolic balance of reactive oxygen species, leading to the accumulation and destruction of antioxidant enzymes. In the present study, exogenous treatments with nitric oxide at 5 and 10 μM significantly reduced the lipid peroxidation content and electrolyte leakage of arils being stored at cold temperatures, compared to untreated arils. Nitric oxide suppressed the activity of polyphenol oxidase (PPO) and preserved the physical appearance and the internal quality of pomegranate arils. The decrease in phenolic compounds (29.32%) and antioxidant activity (39.91%), besides the increase in lipid peroxidation (38.37%) and ion leakage (36.98%), caused deteriorations in the appearance and organoleptic properties of the control samples. To alleviate these problems, nitric oxide has beneficial effects on maintaining the anthocyanin content of pomegranate arils by partially inhibiting PPO enzyme activity during storage. It prolongs the postharvest life, helps to preserve the quality of pomegranate arils, suppresses the formation of ethylene, reduces the respiratory rate and controls weight loss, in addition to maintaining the firmness of fruits. Delaying the changes in peel color and TSS are also considered as useful effects of nitric oxide on pomegranate arils. Nitric oxide impeded the process of senescence by slowing down PPO-related activities, thereby maintaining the total phenolic content of pomegranate arils.
In conclusion, the application of nitric oxide was observed to reduce ion leakage and PPO activity in pomegranate arils, while also maintaining the quality of arils. Ultimately, the use of 10 µM nitric oxide at 8 °C can be suggested as the most optimum treatment herein.


Abdollahi, R., Asghari M., Esmaiili M. and Abdollahi, A., 2013, Postharvest nitric oxide treatment effectively reduced decays of selva strawberry fruit, International Journal of Agriculture and Crop Sciences, 6(6): 353-355. (in persian with english abstract)
Abe, K. and Ogata, K., 1978, Chilling injury in eggplant fruits. V. Changes of K ion leakage and contents of phospholipids during storage and effects of phenolic compounds on K ion leakage, phospholipids content and ultrastructural changes of eggplant fruit sections, Journal of the Japanese Society for Horticultural Science, 47:111-120.
Amal, s.h., Atress, El-Mogy., Aboul-Anean, M.M., and Alsanius, B.W., 2010, Improving Strawberry Fruit Storability by Edible Coating as a Carrier of Tymol or Calcium Chloride, Journal of Horticultural Science & Ornamental Plant, 2(3): 88-97.
Antunes, M.C. and Sfakiotakis, E. M., 2008, Changes in fatty acid composition and electrolyte leakage of 'Hayward' kiwifruit during storage at different temperatures, Food chemistry, 10:891-896.
AOAC, 2008, Official methods of analysis of the association of official analytical chemists, Vol. II. Arlington, VA: Association of Official Analytical Chemists.
Ayala-Zavala, J.F., Wang, S.Y., Wang, C.Y. and Gonzalez-Aguilar, G.A., 2007, High oxygen treatment increases antioxidant capacity and postharvest life of strawberry fruit, Food Technology and Biotechnology, 45:166-173.
Barman, K., Siddiqui, M., Patel, V. and Prasad, M., 2014, Nitric oxide Reduces Pericarp Browning and Preserves Bioactive Antioxidants in Litchi, Sci. Hortic, 171: 71-77.
Bogdan, C., 2001, Nitric oxide and the regulation of gene expression, Trends in Cell Biology, 112: 66–75.
Chance, B. and Maehly, A.C., 1955, Assay of catalases and peroxidases, Methods Enzymol, 11:764-755.
Chaturvedula, V., Sai, P. and Indra, P., 2011, Bioactive Chemical Constituents from Pomegranate (Punica granatum) Juice, Seed and Peel-A Review, International Journal of Research in Chemistr and Environment, 1:1-18.
Duan, X., Su, X., You, Y., Qu, H., Li, Y. and Jiang, Y., 2007, Effect of nitric oxide on pericarp browning of harvested longan fruit in relation to phenolic metabolism, Food Chem, 104: 571–576
Dushni, M. and Zakaye, M.R., 2008, Physiology and post-harvest technology, First edition. Hamadan University Press, Hamedan, 658 pages.
El-Dien Salama, M., Ayaad, H.M., Aboul-Anean, H.E. and Fahmy, H.M., 2012, Effect of edible coating as a carrier of essential oils and ultraviolet light (UV-C) on improving quality of minimally processed Manfalouty pomegranate, Research Journal of Agriculture and Biological Sciences, 8(2): 315-324.
Emamifar, A., 2014, Assess the impact of oral aloe vera gel as a coating on microbial characteristics, physicochemical and sensory strawberries fresh during storage, Journal of Food Science and New Technologies, 2(6): 15-29.
Ghasemnezhad, M., Sherafati, M. and Payvast, A., 2011,Variation in phenolic compounds, ascorbic acid and antioxidant activity of five coloured bell pepper (Capsicum annum) fruits at two different harvest times, Journal Funct Foods, 3:44–49.
Hakim, K.A, Sarkar, M.A.R., Rahman, S.M., Ibrahim, M. and Ibrahim, M.K., 2013, Effect of post-harvest treatments on physiochemical characters during storage of two banana (Musa spp. L.) cv. Sabri and Amritasagar, International Journal of Biosciences, 3:168-179.
He, J.Y., Jiang, L.H, Wang, Y.M., joyce, D.C., Ji, Z.L. and Lu, W.J., 2008, Role of phenylalanine ammonia-lyase in heat pretreatment-induced chilling tolerance in banana fruit, Physiologia Plantarum, 132:318–328.
Hodges, D.M., DeLong, J.M., Forney, C.F. and Prange, R.K., 1999, Improving the thiobarbituric acid-reactive-substances assay for estimating lipid peroxidation in plant tissues containing anthocyanin and other interfering compounds, Planta, 207, 04-611.
Holcroft, D. M. and Kader, A. A., 1999, Carbon dioxide-induced changes in colour and anthocyanin synthesis of stored strawberry fruit. HortScience, 34, 1244-1248.
Hong, K., Gong, D., Xu, H., Wang, S., JiaZ., Chen, J. and Zhang, L., 2014, Effects of salicylic acid and nitric oxide pretreatment on the expression of genes involved in the ethylene signalling pathway and the quality of postharvest mango fruit, New Zealand Journal of Crop and Horticultural Science, 42:205-216.
Lai, T., Li, B., Qin, G. and Tian, S., 2011, Oxidative damage involves in the inhibitory effect of nitric oxide on spore germination of Penicillium expansum, Current Microbiology, 62:229–234.
Li, X.P., Wu, B., Guo, Q., Wang, J.D., Zhang, P., and Chen, W.X., 2014, Effects of nitric oxide on postharvest quality and soluble sugar content in papaya fruit during ripening, Journal of Food Processing and Preservation, 38:591-599.
Lopez-Rubira, V., Conesa, A., Allende, A. and Artes A, 2005, Shelf life and overall quality of minimally processed pomegranate arils modified atmosphere packaged and treated with UV-C, Postharvest biology and Technology. 37: 174–185.
Mirdehghan, S. H. and Rahemi, M., 2002, Reduction of chilling injury in the pomegranate (Punica granatum) fruit by intermittent warming, Iranian Journal of Agricultural Science, 33(1), 75-80. (In Persian with English abstract)
Mirdehghan, S. H., Rahemi, M., Serrano, M., Guillen, F., Martinez-Romero, D. and Valero, D., 2007., The application of polyamines by pressure or immersion as a tool to maintain functional properties in stored pomegranate arils,, Journal of Agriculture Food Chemistry, 55, 755–760.
Muanda, F.N., Soulimani R., Diop, B. and Dicko, A., 2011, Study on chemical composition and biological activities of essential oil and extracts from Stev reia baudiana Bertoni leaves, LWT - Food Science and Technology, 44: 1865-72.
Pizzocaro F., Torreggiani D., and Gilardi G. 1993. Inhibition of apple polyphenol oxidase (PPO) by ascorbic acid, citric acid and sodium chloride, Journal of Food Processing and Preservation, 17, 21–30.
Rahemi, M ., 2006, Post-harvest physiology (An introduction to the physiology and handling of fruit and vegetables), Shiraz University Press.(Iran)
Saadatian, M., Ahmadiyan, S., Akbari, M. and Balouchi., Z., 2012, Effects of Pretreatment with Nitric oxide on Kiwifruit Storage at Low Temperature, Adv. Environ. Biol, 1902-1909.
Shi, H.T., Li, R.J., Cai, W., Liu, W., Fu, Z.W., Lu, Y.T., 2012, In vivo role of nitric oxide in plant response to abiotic and biotic stress, Plant Signaling and Behavior, 7: 437–439.
Shuhua, Z., Lina, S., Mengchen, L. and Jie, Z., 2008, Effect of nitric oxide on reactive oxygen species and antioxidant enzymes in kiwifruit during storage, Journal of the Science of Food and Agriculture, 13:2324-2331.
Shui, G. and Leong, LP. 2002, Separation and determination of organic acids and phenolic compounds in fruit juices and drinks by high-performance liquid chromatography, Journal of Chromatography A, 977(1): 89-96.
Singh, D. and Singh, R.K, 2004, Processed products of pomegranate, Nat Prod Radiance, 3: 66–68.
Singh, Z., Khan, A.S., Zhu, S. and Payne, A.D., 2013, Nitric oxide in the regulation of fruit ripening: challenges and thrusts. Stewart Postharvest Review, 9: 1–11.
Varasteh, F., Arzani, K., Barzegar, M. & Zamani, Z., 2012, Changes in anthocyanins in arils of chitosan-coated pomegranate (Punica granatum L. cv. Rabbab-e-Neyriz) fruit during cold storage. Food Chemistry, 130: 267-272.
Wang, Y., Luo, Z., Du, R., Liu, Y. and Mao, L., 2013, Effect of nitric oxide on antioxidative response and proline metabolism in banana during cold storage, Journal of Agriculture Food, 61: 37.8880-8887.
Yang, A.P., Cao, S.F., Yang, Z.F., Cai, Z.T. and Zheng, Z.H., 2011, γ-aminobutyric acid treatment reduces chilling injury and activates the defense response of peach fruit, Food Chemistry, 129:1619-1622.
Zaharah, S.S. and Singh, Z., 2011, Postharvest nitric oxide fumigation alleviates chilling injury delays fruit ripening and maintains quality in cold-stored ‘Kensington Pride’ mango, Postharvest Biol Technol, 60:202–210
Zanardo, D.I.L., Zanardo, F.M.L., Ferrarese, M.D.L.L., Magalhaes, J.R. and Filho, O.F., 2005.Nitric oxide affecting seed germination and peroxidase activity in canola (Brassica napus L.), Physiol. Mol. Biol. Plants, 11: 81-86.
Zhang, M., Tao, Q., Huan, Y.J., Wang, H.O. and Li C.L, 2002, Effect of temperature control and humidity on the preservation of Jufeng grapes, International Agrophysics, 16: 277-280.
Zheng, X., Tian, S., Gidley, M.J., Yue, H. and Li, B., 2006, Effects of oxalic acid on control of postharvest browning of litchi fruit, Food Chemistry, 96:519-523.
Zhou, Y., Li, S. and Zeng, K., 2016, Exogenous nitric oxide‐induced postharvest disease resistance in citrus fruit to Colletotrichum gloeosporioides, Journal of the science of food and agriculture, 96:505-512.
Zhu, L.Q., Zhou, J., Zhu, S.H. and Guo, L.H., 2009, Inhibition of browning on the surface of peach slices by short-term exposure to nitric oxide and ascorbic acid, Food Chemistry, 114:174-179.
Zhu, S., Sun, L. and Zhou, J., 2009. Effects of nitric oxide fumigation on phenolic metabolism of postharvest Chinese winter jujube (Zizyphus jujuba Mill. cv. Dongzao) in relation to fruit quality, LWT - Food Science and Technology, 42:1009-1014 114:174-179.
Zhu, S., Sun, L., Liu, M. and Zhou, J., 2008, Effect of nitric oxide on reactive oxygen species and antioxidant enzymes in kiwifruit during storage, Journal of the Sci of Food and Agriculture, 88:2324–2331.