Intermittent warming in Tahiti lime treated with an ethylene inhibitor

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1 Postharvest Biology and Technology 29 (2003) 195/203 Intermittent warming in Tahiti lime treated with an ethylene inhibitor Ricardo Alfredo Kluge a,1, *, Maria Luiza L. Jomori a,2, Angelo P. Jacomino b,3, Maria Carolina D. Vitti a,4, Marisa Padula c a Department of Biological Science, Luiz de Queiroz Agricultural College, University of São Paulo, C.P. 9, Piracicaba, SP, Brazil b Department of Crop Production, Luiz de Queiroz Agricultural College, University of São Paulo, C.P. 9, Piracicaba, SP, Brazil c Food Technology Institute, Campinas, SP, Brazil Received 2 July 2002; accepted 20 January 2003 Abstract The storage of Tahiti lime under low temperatures allows the marketing period to be extended, however, the loss of the green skin color and the occurrence of chilling injuries (CI) prevent such improvement to be achieved. The purpose of this work was to verify the efficiency of intermittent warming (IW) associated with the use of 1-methylcyclopropene (1-MCP) during the cold storage of Tahiti lime. Fruit were treated for 12 h with 1-MCP at concentrations of 0 or 1.0 ml l 1 and then were stored at 5 8C. The fruit were submitted to IW (20 8C for 48 h every 7 days, 20 8C for 48 h every 14 days or 38 8C for 24 h every 14 days). Evaluations were carried out after 30, 60 and 90 days of storage (/3 days of simulated marketing at 20 8C). The occurrence of CI in the fruit was not observed after 30 days of storage, however, after 60 days of storage 40/58% of fruit kept continuously at 5 8C and 12.5/20% of fruit intermittent warmed showed CI. All fruit exhibited CI after 90 days. Warming fruit at 38 8C for 24 h every 14 days caused rot development, loss of green skin color, high respiratory rates, as well as accumulation of high levels of ethanol and acetaldehyde in fruit. Application of 1-MCP improved the maintenance of green color of skin. # 2003 Elsevier B.V. All rights reserved. Keywords: Citrus latifolia; Chilling injury; 1-Methylcyclopropene; Ethanol; Acetaldehyde 1. Introduction * Corresponding author. Tel.: / ; fax: / address: rakluge@esalq.usp.br (R.A. Kluge). 1 CNPq fellow. 2 FAPESP fellow. 3 CNPq fellow. 4 CAPES fellow. The maintenance of the green skin color of Tahiti lime fruit (Citrus latifolia, Tanaka) is desirable during storage and marketing. As the green color disappears, the fruit acceptance by the market is gradually reduced. In practice, the Tahiti lime is harvested after reaching full devel /03/$ - see front matter # 2003 Elsevier B.V. All rights reserved. doi: /s (03)00022-x

2 196 R.A. Kluge et al. / Postharvest Biology and Technology 29 (2003) 195/203 opment and marketed while the skin is still green. However, chlorophyll degradation and the carotenoids synthesis progress under room temperature conditions during marketing, causing green color loss (Sinclair, 1984; Baldwin, 1994). This also happens with fruits kept under refrigeration, although the speed of degreening is slower. The best storage conditions for the Tahiti lime are: temperature ranging from 10 to 12 8C and relative humidity from 85 to 95%. Under such conditions, fruit can be stored for 4 /8 weeks. Long periods of temperatures below 8 8C induce chilling injuries (CI) in fruit, characterized by superficial pitting and increase in rot incidence (Hardenburg et al., 1986; Chitarra and Chitarra, 1990; Kader and Arpaia, 1992; Kluge et al., 2001). Treatments in addition to refrigerated storage for fruits and vegetables are being studied with the objective of extending storage and marketing period. Such treatments include intermittent warming (IW), which consists of exposing fruit to one or more periods of high temperature during cold storage. IW increases fruit resistance to low temperatures, enabling fruit to be stored at temperatures below the ones normally recommended (Kluge et al., 2001). In this way, greater benefits are achieved concerning the use of cold temperatures for keeping the product quality during storage. IW has been shown to be effective to reduce CI in several citrus fruits cultivators (Artés, 1995). Cohen et al. (1983) have demonstrated that occurrence of peel pitting in Villa Franca lemons after 4/5 months of storage at 2 8C was 54/59%. Fruit warmed at 13 8C for 7 days after each 21 days period reduced this percentage to 3/4%. This treatment for lemons stored at 2 8C with warming at 13 8C has been used as a commercial method in Israel for storage of fruit for a period longer than 6 months (Cohen, 1988). Schirra and Cohen (1999) have verified in Olinda orange that IW during storage, with cycles of 3 weeks at 3 8C by 2 weeks at 15 8C delayed the onset of CI by approximately 10 weeks with respect to storage continually at 3 8C and greatly enhanced resistance to CI development. Investigations with Fortune mandarins showed that IW at 10 8C for 3 days every 4 days at 2 8C retarded CI development if compared with storage constantly at 2 8C. Favorable effects of IW have also been seen in Valência and Clementine mandarins warmed at 20 8C during 6 h every week (Martinez-Javega and Cuquerella, 1984). Beneficial effects of IW were observed in other commodities, such as tomatoes (Artés and Escriche, 1994; Kluge et al., 1998a,b), cucumbers and zucchinis (Wang, 1993), peaches (Ben-Arie et al., 1970; Kluge et al., 1996), plums (Kluge et al., 1997), eggplants, okras and sweet peppers (Kluge et al., 1998a,b). The compound 1-methylcyclopropene (1-MCP) binds to the ethylene receptor and this acts as an antagonist to ethylene. With the receptors inactivated, the tissue no longer responds to ethylene even if it is present. It has great potential for commercial use by controlling the ripening process in fruits and vegetables, as well as senescence in flowers (Sisler and Serek, 1997). According to McMurchie et al. (1972) fruit showed two system of ethylene production (system 1 and system 2). System 1 would be responsible for the low rate of ethylene production present in preclimacteric fruit. Furthermore, most if not all ethylene production by nonclimacteric fruit can be classified as system 1. System 2 is responsible for the high rate of ethylene production observed during the climacterium. Its main characteristic is that this ethylene production is autocatalytic. Although nonclimacteric fruits, such as citrus, have only system 1 for ethylene production, this does not mean that there is no ethylene influence on fruit ripening (Vendrell and Palomer, 1997). Goldschmidt (1997) states that ethylene, even at low concentrations in nonclimacteric fruits, is involved in ripening-related events, such as the chlorophyll degradation in the skin. It has already been shown that 1-MCP increases the storage of nonclimacteric commodities, such as strawberries (Ku et al., 1999) and broccoli (Ku and Wills, 1999). Research with Shamouti orange showed that 1-MCP effectively inhibited the ethylene, as indicated by blocking of the degreening process (Porat et al., 1999). 1-MCP have demonstrated to delay ripening in climacteric fruits, such as plums (Abdi et al., 1998), bananas (Jiang et al., 1999), apples (Fan et al., 1999), avocados (Feng et al., 2000) and peaches (Kluge and Jacomino, 2002).

3 R.A. Kluge et al. / Postharvest Biology and Technology 29 (2003) 195/ The purpose of the present study was to investigate the effect of IW on storability of Tahiti lime treated with 1-MCP. 2. Material and methods 2.1. Plant material The Tahiti acid limes used in this work came from the production region of Mogi-Guaçú, SP, Brazil. The fruit were selected according to size (50 mm diameter 9/3 mm) and color (intense green) MCP application and storage Fruit were treated with 1-MCP at concentrations of 0 or 1.0 ml l 1 (0 and 1.0 ppm, respectively) inside hermetic boxes, with 186-l capacity. The commercial product SmartFresh TM was used to produce the concentrations above. When SmartFresh TM is dissolved in water it releases 1-MCP so that 1.6 mg of commercial product generates a 1-MCP concentration on the order of 1 ml l 1. In this way, predetermined quantities of SmartFresh TM were weighed and placed into sealed flasks. Twenty milliliter of distilled water at 50 8C were added to the flasks, which were stirred until the compound completely dissolved. Then, the flasks were opened in the chambers, which were immediately sealed to avoid gas loss. Fruit were exposed to this treatment for a 12 h period at 20 8C. After 12 h, the chambers were opened and fruit were transferred to a refrigerated chamber at 5 8C. The fruits were divided into four lots, the first of which was stored uninterrupted at 5 8C; the second one intermittently warmed at 20 8C for 48 h every 7 days; the third lot was submitted to warming at 20 8C for 48 h every 14 days and the fourth lot was submitted to warming at 38 8C for 24 h every 14 days. These warming procedures consisted in transferring the fruit to chambers set to the prescribed temperatures Assessments Fruit were evaluated after 30, 60 and 90 days of storage (/3 days of simulated marketing period at 20 8C). CI was determined by the percentage of affected fruit and CI severity was evaluated. Fruit were classified into five categories, according to the surface area affected, as follows: 0/0, 1/1/ 5, 2/5/25, 3/25/50 and 4//50%. The skin color was evaluated by Minolta Chroma Meter CR-300, which provided measurements of the hue angle (h8) and chroma (C*). To measure respiratory rate ten fruit from each treatment were placed into 5-l hermetic containers, where they remained for 3 h. A rubber septum was placed on each pot lid, through which a gas sample was taken. A sample from each pot was collected with the aid of a 1-ml syringe and then was analyzed with a CO 2 and O 2 analyzer. Results were expressed in mg CO 2 kg 1 h 1. The soluble solids concentration (SSC) was determined as percentage, titratable acidity (TA) as citric percentage, and ascorbic acid content as mg 100 g 1. The technological index (TI) was determined through the equation: TI / (SSC /% juice)/100ti is an important variable to the citrus industry. Higher values of TI mean better quality for juice manufacture (Chitarra and Chitarra, 1990). Ethanol and acetaldehyde were determined by juice head-space analysis by gas chromatography as previously described by Cohen et al. (1990) Data analysis Analysis of variance was performed using SAN- EST software (Zonta and Machado, 1984) according to a completely randomized experimental design. Mean comparisons were performed by Tukey test at P 5/0.05). 3. Results No visible symptoms of CI were observed in fruit of all treatments after 30 days of storage. CI in fruit was observed only after 60 days of storage and symptoms were visible only after the chilled fruit have been removed from cold storage to

4 198 R.A. Kluge et al. / Postharvest Biology and Technology 29 (2003) 195/203 Table 1 Percentage of CI in Tahiti lime after 60 days of storage at 5 8C (/3 days at 20 8C) Storage temperature 1 1-MCP (ml l 1 ) Fruits affected (%) 2 Category 0 (0%) 1 (1/5%) 2 (5/25%) 3 (25 /50%) 4 (/50%) Percentage of fruits 2 Constant a 40.0 c 45.5 a a 60.0 b 40.0 a IW b 87.5 a 12.5 b b 82.5 a 17.5 b IW b 90.0 a 10.0 b b 80.0 a 20.0 b F-test ** * ** / / / **, * F-test significant at P 5/0.01 and P 5/0.05, respectively. 1 Constant, storage continuously at 5 8C; IW-1, IW at 20 8C for 48 h every 7 days; IW-2, IW at 20 8C for 48 h every 14 days. 20 8C (Table 1). In this period, CI was characterized by small black superficial pits on the fruit skin. The incidence of CI varied from 40 to 58% in fruit kept continually at 5 8C while fruits intermittently warmed showed 12.5 /20% of CI, with no effect of 1-MCP being observed on such disorders. After 90 days of storage, all fruit from all treatments were affected by CI with several black superficial pits in more than 50% of the skin. Fruit warmed for 24 h at 38 8C every 14 days did not exhibit CI but nearly 40% of fruit developed rot caused by Penicillium digitatum. There was a change in color from harvest to after 30 days of cold storage, considering that initial values of h8 and chroma were and 32.04, respectively. Also, there were no significant differences between treatments in relation to color of fruit after 30 days of storage, except that fruit submitted to IW for 24 h at 38 8C every 14 days that exhibited the greater losses (Table 2). Such results were expected, considering that the speed of metabolic reactions is directly related to the temperature in which it occurs. Thus, it was observed that fruit warmed at the higher temperature showed more yellowing than non-warmed fruit. Fruit intermittently warmed at 38 8C showed hue angle (h8) values between 116 and 1188 after 30 days of storage and between 108 and 1098 after 60 days. The remaining treatments showed h8 varying from 120 to 121 and 112 and 1188 after 30 and 60 days, respectively. Hue angle values greater than 908 represent greener fruits, whereas the nearer values were to 908, the more yellow the fruits. Chroma defines the color intensity, with values around zero for neutral colors (grey) and values around 60 for bright colors (McGuire, 1992). In this way, as for the present work, higher chroma values mean higher yellow color intensity (Table 2). After 60 days of storage, fruit treated with 1- MCP showed higher h8 value if compared with non-treated fruit, except that fruit intermittently warmed at 38 8C which showed the lowest h8 values (Table 2). This behavior was similar to the chroma values. After 90 days, all fruit were completely yellow and unsuitable for marketing (data not shown). Fruit submitted to IW for 24 h at 38 8Cevery 14 days exhibited greater respiratory rates than other treatments after 30 and 60 days of storage (Table 3). The reduction of respiratory rate from 30 days to 60 days was expected, taking into consideration that lime is a nonclimacteric fruit, with respiration gradually decreasing after harvest. In relation to ethanol and acetaldehyde contents, it was observed that fruit submitted to IW for 24 h at 38 8C every 14 days showed alterations in the respiratory metabolism, with higher levels of products from fermentation being observed when compared with fruit from the remaining treat-

5 R.A. Kluge et al. / Postharvest Biology and Technology 29 (2003) 195/ Table 2 Effect of treatments on hue angle (h8) and chroma of Tahiti lime after 30 and 60 days of storage at 5 8C (/3 days at 20 8C) Storage temperature 1 1-MCP (ml l 1 ) Skin color 2 h 8 Chroma 30 days 60 days 30 days 60 days Constant a b ab a a c IW a b a a a b IW a b ab a a b IW b c a b c a F-test ** ** NS * NS, *, **, F-test non significant or significant at P 5/0.05 and 0.01, respectively. 1 Constant, storage continuously at 5 8C; IW-1, IW at 20 8C for 48 h every 7 days; IW-2, IW at 20 8C for 48 h every 14 days, AI-3, IW at 38 8C for 24 h every 14 days. Table 3 Effect of treatments on respiratory rate of Tahiti lime after 30 and 60 days of storage at 5 8C (/3 days at 20 8C) Storage temperature 1 1-MCP (ml l 1 ) Respiratory rate (mg CO 2 kg 1 h 1 ) 2 30 days 60 days Constant ab 6.33 b b 6.97 b IW b 5.40 b b 5.01 b IW b 5.72 b b 5.63 b IW a a a a F-test ** ** **, F -test significant at P 5/ Constant, storage continuously at 5 8C; IW-1, IW at 20 8C for 48 h every 7 days; IW-2, IW at 20 8C for 48 h every 14 days, AI-3, IW at 38 8C for 24 h every 14 days. In general, no differences were observed in the physicochemical characteristics among treatments in which fruit were submitted to IW at 20 8C for 48 h every 14 or 7 days, as well as those stored at 5 8C continuously (Tables 5 and 6). However, fruit submitted to IW for 24 h at 38 8C every 14 days had a reduction in the TA during storage and the lowest juice percentage after 60 days of storage (Table 5). The lower juice percentage caused reduction on technological index in these fruit. This represents a disadvantage in relation to quality of fruit to industry. These fruit also had a loss of ascorbic acid content, having declined approximately 30% from harvest values during 30 days of storage and more than 50% after 60 days (Table 6), considering that concentration of ascorbic acid at beginning of experiment was mg kg 1. This represents a drastic reduction on nutritional quality. ments (Table 4). An informal sensory evaluation confirmed the presence of odd smells in fruit, produced by the fermentative process. Therefore, this IW regime was too drastic, affects the fruit quality. The ethanol and acetaldehyde amounts were similar among the other treatments. 4. Discussion The present study showed that IW at 20 8C for 48 h every 7 or 14 days reduced CI after 60 days of storage. No injuries due to chilling were observed after 30 days of refrigerated storage in any of the treatments used. The occurrence of chilling symp-

6 200 R.A. Kluge et al. / Postharvest Biology and Technology 29 (2003) 195/203 Table 4 Effect of treatments on ethanol and acetaldehyde amounts of Tahiti lime after 30 and 60 days of storage (/3 days at 20 8C) Storage temperature 1 1-MCP (ml l 1 ) Ethanol (ml l 1 ) 2 Acetaldehyde (ml l 1 ) 2 30 days 60 days 30 days 60 days Constant b b 8.67 b 9.00 b b b 9.67 b 8.67 b IW b b 6.67 b b b b 7.99 b 9.33 b IW b b 7.00 b ab b b 8.54 b b IW a a a a a a a a F-test ** ** ** ** **, F -test significant at P 5/ Constant, storage continuously at 5 8C; IW-1, IW at 20 8C for 48 h every 7 days; IW-2, IW at 20 8C for 48 h every 14 days, AI-3, IW at 38 8C for 24 h every 14 days. toms depends on both, the temperature and the exposure time to this temperature. In the present work, the 30 days exposure period at 5 8C was not enough to induce CI in fruit and, thus, the temperature of 5 8C can be used for Tahiti lime during 30 days of cold storage, without risk of CI. Fruit submitted to IW at 38 8C for 24 h every 14 days showed undesirable alterations in some physicochemical characteristics, such as reduction in ascorbic acid (vitamin C), acidity, juice percentage and technological index. A higher respiratory rate, as well as higher ethanol and acetaldehyde amounts, were also verified. These alterations resulted in unmarketable of fruit after 30 days of storage. On the other hand, fruit submitted to IW at 20 8C every 7 or 14 days showed virtually no significant differences in the physicochemical analyses performed. Although IW has been shown to be effective to reduce CI in several commodities, the greatest difficulty in creating optimum conditions to application of this technology lies in the need to operate with temperatures, duration and IW frequency that may change from cultivator to cultivator, fruit maturity stage and growing conditions (Artés, 1995). The 1-MCP had been showed to be effective to retard ripening of several climacteric fruit due to be an efficient inhibitor of the ethylene action (Serek et al., 1995). Nonclimacteric fruits also have ripening events related to ethylene action. Manning (1994) has reported that changes in gene expression are clearly associated with the ripening phase of strawberry, including up regulation of anthocyanin biosynthesis genes. Similarly, several ripening associated genes have been described in citrus (Alonso and Grannel, 1995; Alonso et al., 1995). Changes in gene expression have also been verified in the rin tomato mutants, which in many respects, resembles nonclimacteric fruits (Picton et al., 1993). Porat et al. (1999) have demonstrated that 1- MCP increase CI and cause accumulation of the volatile acetaldehyde, ethyl acetate and ethanol in Shamouti orange. In present study, 1-MCP treatment did not increased CI symptoms and acetaldehyde and ethanol amounts. Thus, the effect of 1-MCP appears to be related to citrus species. This effect may be beneficial in some specific citrus species such limes and pomelos, which are preferably marketed green. On the other hand, this effect may be detrimental such in Shamouti orange (Porat et al., 1999). In this experiment the delay of fruit degreening caused by 1-MCP in Tahiti lime have confirmed that this treatment can be effective to storage of this cultivator considering that fruit treated were more greener that fruit not treated after 60 days of

7 R.A. Kluge et al. / Postharvest Biology and Technology 29 (2003) 195/ Table 5 Effect of treatments on SSC, TA, juice percentage and technological index (TI) of Tahiti lime after 30 and 60 days of storage at 5 8C (/3 days at 20 8C) Storage temperature 1 1-MCP (ml l 1 ) SSC (%) 2 60 days 30 days Constant IW IW IW F-test NS NS TA (% citric acid) 2 Constant a 5.25 a a 5.50 a IW a 5.76 a a 6.17 a IW a 5.62 a a 5.57 a IW b 3.99 b b 3.83 b F-test * ** Juice percentage 2 Constant a ab IW a a IW a a IW b b F-test NS * TI [(TSS/% juice )/100] 2 Constant a 3.21 a a 3.03 a IW a 3.62 a a 3.85 a IW a 3.09 a a 3.80 a IW b 2.05 b b 1.56 b F-test * ** NS, *, **, F-test non significant or significant at P 5/0.05 and 0.01, respectively. 1 Constant, storage continuously at 5 8C; IW-1, IW at 20 8C for 48 h every 7 days; IW-2, IW at 20 8C for 48 h every 14 days, AI-3, IW at 38 8C for 24 h every 14 days. 2 Mean separation in column by Tukey test at P 5/0.05 for each variable. Table 6 Effect of treatments on ascorbic acid content (mg kg 1 )of Tahiti lime after 30 and 60 days of storage at 5 8C(/3 days at 20 8C) Storage temperature 1 storage. Goldschmidt (1997) stated that the chlorophyll degradation is an event that can be regulated by ethylene. Consequently, the loss of green skin color of Tahiti lime could be blocked by inhibitors of ethylene action. Others inhibitors of ethylene action, besides 1-MCP, such silver and norbornadiene (NDB), have inhibited the loss of chlorophyll from green harvested Shamouti orange. The loss of chlorophyll from green harvested Shamouti, as compared with Valência orange is correlated with several fold higher levels of ethylene evolutions (Goldschmidt et al., 1993). Acknowledgements 1-MCP (ml l 1 ) Ascorbic acid (mg kg 1 ) 2 30 days 60 days Constant a a a a IW a a a a IW a a a a IW b b b b F-test * ** *, **, F-test significant at P 5/0.05 and 0.01, respectively. 1 Constant, storage continuously at 5 8C; IW-1, IW at 20 8C for 48 h every 7 days; IW-2, IW at 20 8C for 48 h every 14 days, AI-3, IW at 38 8C for 24 h every 14 days. The authors thank Dr Walter Pereira, Rohm and Haas, for providing the SmartFresh TM and Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), for providing the fellowship to main author and Fundaçâo de Amparo á Pesquisa do Estado de Sâo Paulo- FAPESP, for providing the fellowship to second author.

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