Improvement Shelf-Life Extension of Apple by Prestorage Thermal Treatment, CaCl2 and Gamma Irradiation

Improvement Shelf-Life Extension of Apple by Prestorage Thermal Treatment, CaCl2 and Gamma Irradiation Salem, E.A and Zeiad Moussa Food Irradiation Department, National Center for Radiation Research and Technology Atomic Energy Authority, Cairo, Egypt Plant Pathology Research Institute Agricultural Research Center, Giza, Egypt Received: 16/6/2013 Accepted: 30/6/2013 ABSTRACT This study was conducted to evaluate the efficiency of physical and chemical methods to extend the shelf life of apple fruits by control the blue mold disease causing by Penicillium expansum. Apple fruits are subjected to different temperatures between 38, and 50 C for 24 hr. and stored at 0 C for 4 months. Increasing in temperature caused decreasing in firmness and blue mold incidence percentage (%) caused by P. expansum. At 50 C the treated apple fruits gave sharp softness and inhibition of blue mold incidence (%) caused by P. expansum exposing for 4 days and cold storage at 0 C for 4 months and 5 days at 20 C. Dipping apple fruits in CaCl 2 at 2% and 4% decreased blue mold incidence (%) caused by P. expansum and increased apple fruits firmness at 2 and 4 months storage periods. Also, CaCl 2 treatments gave insignificant change in total soluble solid (TSS%) and in titratable acidity (TA%) of apple fruits. Gamma irradiation doses above 1 kgy significantly decreased firmness of apple fruits with the decrement being higher at higher doses. Key word: Apple fruits, heat treatment, CaCl 2 dipping, gamma irradiation, blue mold INTRODUCTION Apples are a major worldwide fruits. Worldwide production of apples was about 75.5 million Metric Tons FAO (1). Postharvest pathogens cause major losses in apple production. More than 90 fungal species have been described as causative agents of apple decay during storage Pianzzola et al., (2). Blue mold caused by Penicillium expansum is the most important postharvest disease of apple Romano et al., (3) and Gholamnejad et al., (4). This disease causes shortage of shelf-life and economic loss of apple. The cool storage is not enough to reduce the yield loss. Therefore, there is a need to treatment other than fungicides to reduce the loss of yield before cool storage Mostafavi et al., (5). Physical and chemical pre-storage treatments are safe treatments to manage this disease. Heat treatments cause change in fruit ripening, such as inhibition of ethylene synthesis and action of cell wall degrading enzymes, due to changes in gene expression and protein synthesis Paull and Chen, (6). Hot water treatments are also used as alternative method to control postharvest diseases. Ripe fruits are highly susceptible to disease infection. Some means of controlling it are necessary Margonsan et al., (7). Maxin et al. (8) suggested that possible applications for hot-water dipping of apples at harvest, after short-term cold storage or after the opening of controlled-atmosphere storage rooms in order to improve fruit quality during succeeding storage periods. Dipping of apple fruits in CaCl 2 indirectly reduced Penicillium expansum activity due to ameliorating physiological disorders Conway et al., (9). CaCl2 solution at 1000 ug of Ca 2+ /ml inhibited germ tube growth by 41% relative to control. Also, calcium salt reduced Colletotrichum acutatum dry weight in liquid culture media and treated apple fruits exhibited 30% smallest lesions than control

Biggs, (10). The application of Cacl 2 (4%) leads to retention of fruit color, texture and reduced fig fruit molds Irfan et al., (11). Gamma radiation usually used as additional treatment to fruit cold storage to reduce weight loss, delay ripening and in shelf-life extension. Kovacs et al., (12) reported that low dose (1 kgy) gamma radiation on apples and pears induced softening in the fruit through dissolution of middle lamella, wrinkling of cell membranes. On the other hand, higher doses resulted in a fruit tissue injury Shi et al., (13). The possible double advantage of low irradiation doses (0.3 and 0.6 KGy) combined with cold storage which significantly retained phenol contents, antioxidant activity, firmness, weight loss and total soluble solids. Gamma rays is a suitable method to reduce apple quality losses and for better postharvest preservation Mostafavi et al., (5). This study investigated physical and chemical treatments to control blue mold disease of apple fruits and extend their shelf-life. MATERIALS AND METHODS Anna Apple fruits (Mallus domestica, Borkh) collected from local markets in Sharkia Governorate. Fungal species: Penicillium expansum was isolated from apple fruits showing blue mold, these fruits kept in clean and sterile plastic bags at room temperature for isolation. Samples sterilized with 1% sodium hypochlorite for 2 min then washed using sterilized distilled water for several times and dried using filter paper. Fungal isolation was carried out from the inner tissues neighbouring the infected ones. Segments were separately transferred to Petri plates contain potato dextrose agar (PDA) media and incubated for 7 days at 27 C Waller, (14). The fungus was identified according to Raper and Thom (15) at Mycological Lab. 2 (ML2), Faculty of Science, Zagazig University, Egypt. Effect of heat treatment on flesh firmness of apple fruits Undamaged apple fruits were subjected to the desired temperatures 38, 40, 42, 44, 46, 48 and 50 C for 24 hrs by thermostatically controlled walk in chambers equipped with air circulation. All boxes were then removed from heating rooms and immediately stored at 0 C for 4 months. The firmness was measured by a penetrometer (McCormic fruit firmness pressure tests FTO-11#CO-111 Ibs) in N. Three replicate of 5 apples were used for each tested temperature degree. Effect of heat treatment on blue mold of apples during cold storage: Apple fruits were wounded on two opposite sides to a depth of 2 mm and subsequently were inoculated by immersion for 15 seconds in a conidial suspension (1x10 5 spores/ml) of a virulent isolate of Pencillium expansum isolated from decayed apples and identified as previously mentioned. One hour after the inoculation, seven of the eight groups of inoculated apples (48 apples/group) were placed in three tray packed boxes with perforated polyethylene bags as liners before being heated. Relative humidity was modified inside the boxes by placing wet tissues in tray cavities over polyethylene liners and fruits were subjected to the desired temperature 38, 40, 42, 44, 46, 48 and 50 C for 1, 2, 3 and 4 days at heating rooms. The eight group control was inoculated and placed without heat treatment. All boxes were then removed from heating rooms and immediately stored at 0 C for 4 months. The results were calculated according to the method described by Morcos (16) as follows: The external rotted area Severity of infection % = X 100 Total area of fruit No of rotted fruits Decay % = X 100 Total fruits

Effect of dipping apple fruits in calcium chloride on blue mold incidence, firmness, total soluble solids (T.S.S) and on titratable acidity (T.A%): As previously mentioned in heat treatment after inoculation of apple fruits seven of the eight groups of inoculated apples (48 apples/ group) were immersed in 2% and 4% calcium chloride concentrations for 5 min. and stored for 2 and 4 months in cold storage. The eight group control immersed in water and stored for 2 and 4 months in cold storage. After storage periods blue mold incidence and fruits firmness determined as previously mentioned. Total soluble solids (T.S.S%): Determined using hand refractometer, according to A.O.A.C. (17). Titratable acidity (TA%): Titratable acidity was expressed as percentage of citric acid (g citric acid/100 ml juice), according to A.O.A.C. (17). Irradiation treatment: Irradiation treatment was done by subjecting apple fruits to gamma irradiation from Co 60 source at the National Center for Radiation Research and Technology (NCRRT), Nasr City, Cairo, Egypt. The dose rate was 0.9 kgy/hr at the time of experiment. Experimental design and statistical analysis: All treatments in this study were arranged in complete randomized design. The obtained data were subjected to analysis of variance using the general linear module procedure of SAS (18). Appropriate treatment means were separated using Duncan's multiple range test Duncan, (19). RESULTS Effect of heat treatment on flesh firmness of apple fruits: Data in Table (1) show that as temperature degree increase apple fruit firmness decrease. Sharp decrease in firmness obtained at 48 C and 50 C where the most soft fruits were those held at 50 C for 24 hr. (20N). The firmness of fruits flesh held at 38 C was 36 N as relatively equal to that of control treatment 38 N. Table (1): Effect of heat treatment on apple fruits firmness at 38, 40, 42, 44, 46, 48 and 50 C for 24 hr. Heat degree ( C) Firmness (N) 38 36 a 40 35 b 42 34 c 44 34 c 46 31 d 48 28 e 50 20 f Unheated control 38 a Effect of heat treatment on blue mold incidence (%) on apples during cold storage: Data in Table (2) show blue mold incidence(%) caused by Penicillium expansum after prestorage heating for 1, 2, 3 and 4 days at 38, 40, 42, 44, 46, 48 and 50 C and storage at 0 C for 4 months followed by 5 days at 20 C.

There are a clear reduction in blue mold incidence (%) with increasing in heating temperature degree. As heating temperature increased the blue mold incidence (%) decreased. The level of blue mold incidence (%) was decreased as the heating storage period was prolonged. The lowest level of mold incidence (%) was achieved at 50 C heating degree in particular after 4 days at the same degree where the blue mold incidence (%) eliminated. Table (2): Blue mold incidence (%) caused by Penicillium expansum on apples upon prestorage heating treatment at 38, 40, 42, 44, 46, 48 and 50 C for 1, 2, 3 and 4 days cold storage at 0 C for 4 months and 5 day at 20 C. Heat treatment C Blue mold incidence (%) / heating storage periods (day) 1 2 3 4 Control 100 a 100 a 100 a 100 a 38 74.50 b 28.91 b 21.83 b 14.78 b 40 71.81 b 26.45 c 17.57 c 10.69 c 42 60.34 c 21.23 d 13.08 d 7.29 d 44 55.01 d 18.76 e 7.99 e 5.50 e 46 53.69 d 16.46 e 5.31 e 2.90 e 48 46.14 e 13.41 e 2.11 f 0.0 50 42.66 f 10.03 f 1.36 f 0.0 Effect of dipping apples fruits in CaCl 2 on blue mold incidence after storage: Data in Table (3) proved that apple fruits were dipped in calcium chloride at 0, 2% and 4% concentrations for 5 min. and storage for 2 and 4 months at cold storage. After 2 months storage period, dipping apple fruits in calcium chloride caused reduction in blue mold incidence (%) caused by Penicillium expansum from 100 to 55.95 and 54.30 at 2% and 4% calcium chloride concentrations, respectively. Also, after 4 months storage periods dipping apple fruits in CaCl 2 reduced blue mold incidence from 100 to 62.30 and 60.17 at 2% and 4% CaCl 2 concentrations, respectively. The level of blue mold incidence after 2 months lower than which of after 4 months storage. Table (3): Effect of dipping apples fruits in CaCl 2 on blue mold incidence (%) caused by Penicillium expansum at different storage periods. CaCl 2 concentration Blue mold incidence (%) 2 months cold storage period 4 months cold storage period Control (water) 100 a 100 a 2% CaCl 2 55.95 b 62.30 b 4% CaCl 2 54.30 c 60.17 c. Effect of dipping CaCl 2 and storage periods on firmness of Apple fruits: Data in Table (4) showed that apple fruits were dipped in CaCl 2 at 0, 2% and 4% concentrations for 5 min. and storage for 2 and 4 months at cold storage. After 2 months storage period, dipping apple fruits in CaCl 2 cause increasing in fruit firmness to 40 and 41 at 2% and 4% CaCl 2 concentration, respectively. Also, after 4 months storage periods fruit firmness gave 39 at 2% and 4% CaCl 2 concentrations. The value of apple fruits firmness increased by dipping in CaCl 2 and these value decreased by increasing storage periods.

Table (4): Effect of dipping apples fruits in CaCl 2 and storage periods on firmness of fruits. CaCl 2 concentration Fruits firmness (N) 2 months storage period 4 months storage period Control (water) 39 a 38 a 2% CaCl 2 40 b 39 b 4% CaCl 2 41 c 39 b Effect of dipping in CaCl 2 and storage periods on total soluble solids (T.S.S%) and on titratable acidity (T.A%) of apple fruits: Data in Table (5) illustrated that dipping apple fruits in CaCl 2 at 2% and 4% give insignificant changes in total soluble solids of apple after 2 months and 4 months storage periods, since total soluble solids (T.S.S%) changed from 13.50 in control to 13.16 and 13.75 at 2% and 4% CaCl 2 concentrations, respectively in 2 months storage period. Also after 4 months the value changes from 13.23 in control to 12.83 and 13.26 in 2% and 4% CaCl 2 concentrations, respectively. The same results obtained on titratable acidity (T.A%) of apple fruits after dipping in CaCl 2, insignificant result obtained after 2 months control 1.17 give 1.17 and 1.04 at 2% and 4% CaCl 2 conc., respectively and after 4 months control 0.99 give 1.03 and 1.10 at 2% and 4% CaCl 2 concentrations, respectively. Table (5): Effect of dipping on CaCl 2 and storage periods on total soluble solids (T.S.S%) and on titratable acidity (T.A%) of apple fruits. CaCl 2 concentration Total soluble solids at different storage periods Titratable acidity at different storage periods 2 months 4 months 2 months 4 months Control (water) 13.50 a 13.23 a 1.17 a 0.99 a 2% CaCl 2 13.16 a 12.83 a 1.17 a 1.03 a 4% CaCl 2 13.75 a 13.26 a 1.04 a 1.10 a Effect of gamma radiation on apple fruits firmness: Data in Table (6) mentioned that effect of different gamma radiation doses 0.5, 1, 1.5 and 2 kgy on apple fruits firmness, since as gamma radiation dose increase apple fruits firmness decreased. The firmness of apple fruits at 0.5 kgy relatively equal to that of control but softness of apple obtained at higher gamma radiation doses. Table (6): Effect of different gamma radiation doses on apple fruits firmness (N). Gamma radiation doses Firmness (N) Control 38 a 0.5 kgy 36 a 1 kgy 34 b 1.5 kgy 31 c 2 kgy 28 d

DISCUSSION Penicillium expansum is an important postharvest pathogen that not only causes decay on apple and pear fruit but also produces the carcinogenic mycotoxin patulin in spoiled fruit and processed fruit. Although, synthetic fungicides are effective to protect against fruit decay. Their potential effects on human health and on the environment Hiromi et al., (20). The results of the present study indicated that as heat treatment of apple fruits increase firmness of fruits decrease and sharp firmness decrease obtained at 48 C and 50 C also the firmness of fruits flesh held at 38 C as relatively equal to control. Also, the obtained result showed that as heating temperature increase blue mold incidence (%) decreased and the lowest blue mold incidence (%) obtained at 48 C and 50 C. The same results confirmed by Wang et al., (21) who found that heat treatment had effects on storage life and quality and fruit physiology of apple. They found that at 38 C is the most appropriate for reduction of mold incidence of apple. The main effect of heat treatment is proved to be due to reduction of ethylene release. Liping et al., (22) explained the mode of heat treatment action to peach fruits at 35, 38 and 43 C for 2 days and stored in 0 C for 4 weeks followed by 3 days shelf life at 27 C they found that heat treatment stimulate fruit metabolism during first week of cold storage. Respiration rates, ethylene production and peroxidase and polyphenol oxidase activities were initially higher in treated fruits than in control but reverse effects was obtained during subsequent cold storage and shelf life. El-Deep (23) confirmed that the same effect of heat on firmness of apple and blue mold incidence caused by Penicillium expansum. In addition, Maxin et al. (8) revealed that blue mold was significantly reduced by dipping apples in hot water around 50 C. Temperatures above 52 C caused serious heat scald on the fruit surface and gave rise to increasing levels of fruit rot caused by P. expansum. Regarding effect of dipping apple fruits in CaCl 2 on blue mold incidence the obtained results showed that calcium chloride in 2% reduced blue mold incidence from, 100 to 55.95 and 62.30 after 2 months and 4 months storage periods, respectively. Also CaCl 2 in 4% reduced blue mold incidence from 100 to 54.30 and 60.17 after 2 months and 4 months, respectively. The same results obtained by Chardonnet et al. (24) indicate that Ca reduced fungal growth, inhibiting pectolytic enzymes and decrease plant tissues susceptibility to decay. Also, Youness, (25) found that different calcium salts slightly restricted growth of pathogenic fungi attacking fruits. Johnson and Dover (26) indicated that CaCl 2 treatments increased the firmness of apple fruits at harvest and also in fruit stored in controlled atmosphere for 4 or 6 months. El-Deep (23) confirmed that CaCl 2 treatments by dipping increased the firmness of apple fruits and reduced rate of softening after storage. Raese and Drake (27) cleared that, calcium chloride treatment increased Ca concentration in fruit cortex and peel of Anjou pear (Pyrus communis). Mayr and Schroder (28) demonstrated that CaCl 2 sprays increased Ca content in Boskoop and apples compared to the unsprayed ones. Regarding effect of CaCl 2 on total soluble solids (T.S.S) and on titratable acidity (T.A) Our result illustrated that dipping apple fruits in 2% and 4% CaCl 2 give insignificant changes in total soluble solids and on titratable acidity (T.A) of apple. The same result confirmed by Moon et al. (29) who mentioned that, no differences in soluble solids content were observed among foliar spray and postharvest dipping with liquid Ca fertilizer on pear fruits. Also, El-Deep (23) cleared that dipping apple fruits in CaCl 2 solutions give insignificant changes in total soluble solids and on titratable activity (TA).

Trentham (30) illustrated that mature apples fruits immersed for 2 min in (0, 2%, 4% or 6%) aqueous solutions (w/v) CaCl 2 were stored at 0 C. Histological samples showed that cuticles of apples fruits were also affected at higher CaCl 2-treatment becoming more condensed and uniform. All tissues, including cuticle, were stained magenta red, indicating a possible chemical alteration of cuticle and underlying tissues by Ca. Also, Irfan et al. (11) cleared that CaCl 2 (4%) reduced the molds of fig fruits and delayed ripening of figs and was useful in prolonging the postharvest shelf-life. Our results indicate that gamma irradiation doses, 1, 1.5 and 2 kgy decreased firmness of apples fruits. These results are in agreement with Al-Bachir. (31) who reported that gamma irradiation immediately increased the softening of apple fruits due to that gamma irradiation had an effect on decreasing protopectin and total pectin or to a change of in soluble pectic materials to soluble forms or may be due to degradation of cellulose and pectin and increasing the activity of pectin methyl esterase enzyme. Furthermore, Mostafavi et al. (5) illustrated that low irradiation doses (0.3 and 0.6 KGy) combined with cold storage is a way to minimize apple quality losses during nine month storage period. Lesion diameter of blue mold of non-irradiated apple was significantly increased with increasing storage period. As discussed above, increasing temperatures decreased firmness and blue mold incidence. Dipping apple fruits in CaCl 2 at 2% and 4% decrease blue mold incidence and increase firmness. Gamma rays above 1 KGy significantly decrease firmness of apple. REFERENCES (1) FAO,( 2011): Food and Agriculture organization of United Nations, FAOSTAT, Food and Agricultural commodities production. http://faostat.fao.org/site/339/default.aspx (2) Pianzzola, M. J. M. Moscatelli, and S. Vero (2004): Characterization of Penicillium Isolates associated with blue mold on apple in uruguay. Plant Disease, Vol. 88 (No. 1), 23 28. (3) Romano M.L., Gullino M.L., Garibaldi A. (1983): Evaluation of the sensitivity to several fungicides of post-harvest pathogens in North-western Italy. Meded. Fac. Landbouw. Univ. Gent. 48: 591 602. (4) Gholamnejad, J.; Etebarian, H.R.; Roustaee, A. and Navaz Sahebani, N. (2009): Biological control of apples blue mold by isolates of Saccharomyces cerevisiae. J. Plant Protection Research, Vol. 49 (No. 3), 270 275. (5) Mostafavi, H.A.; Mirmajlessi, S.M.;, Seyed, M.M.; Fathollahi, H. and, Askari, H. (2012): Gamma radiation effects on physico-chemical parameters of apple fruit during commercial post-harvest preservation, Radiation Physics and Chemistry, 81, 666 671. (6) Paull, R. and Chen, N. (2000): Heat treatments and fruits ripening postharvest. Biol. Technol., 21: 21-37. (7) Margonsan, D.A., Smilanick, J.L., Simmons, G.F. and Henson, D.J. (1997): Combination of hot water and ethanol to control postharvest decay of peaches and nectarines. Plant Dis., 81: 1405-1409. (8) Maxin, P.; Weber, R.W.S.; Lindhard Pedersen, H. and Williams, M. (2012): Hot-water dipping of apples to control Penicillium expansum, Neonectria galligena and Botrytis cinerea: effects of temperature on spore germination and fruit rots. Europ.J.Hort.Sci., 77 (1). S. 1 9, (9) Conway, W.S., Sams, C.E., McGuire, R.G. and Kelman, A. (1992): Calcium treatment of apples and potatoes to reduce postharvest decay. Plant. Dis., 76: 329-334. (10) Biggs, A.R. (1999): Effect of calcium salts on apples bitter rot caused by two Colletotrichum spp. Plant Dis., 83 (11): 1001-1005.

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