Maturation Physiology under Modified Atmosphere of Prata Banana Treated Postharvest with 1-Methylcyclopropene

Maturation Physiology under Modified Atmosphere of Prata Banana Treated Postharvest with 1-Methylcyclopropene S. de Melo Silva 1, a, O. do C. de Oliveira Neto 2, R.E. Alves 3 and E. de Oliveira Silva 3 1 DCFS/CCA/UFPB, C.P 0, CEP 58.397-000 Areia-PB, Brazil 2 CFT/UFPB, 58.2-000, Bananeiras-PB, Brazil 3 Pesq. Dr. Embrapa Agroindústria Tropical, CP. 3711- CEP 511-110 Fortaleza-CE, Brazil Keywords: color development, respiratory profile, 1-MCP Abstract The aim of this research was to evaluate the influence of 1- methylcyclopropene (1-MCP) and modified atmosphere packaging on the maturation of banana cultivar Prata harvested in the maturation stage 1. Banana hands were treated at room temperature with 0 and 1-MCP in sealed plastic chambers of 0.19 m 3, during 2 hours. Following the exposition to 1-MCP, a set of three fruits were packed in polystyrene trays and packed using Xtend TM film (Stepac, L. A., Israel) for modified atmosphere generation and kept under ambient (AA) and modified atmospheres (MA) at 15 C and 90±2% of Relative Humidity and room temperature (23±2 C and 85±2% RH). At each evaluation period, three replications (1 set of three fruit/rep) from each treatment were used. 1-MCP application delayed the onset of respiratory peak, also maintained fruit gloss and retarded the increase in the a* and b* values, which is a result of the also delayed transition from the green to the yellow skin color. However, the development of the skin yellow color was very irregular at 15 C storage. On the other hand, 1-MCP treatment resulted in a more intense skin yellow color development for fruits kept at room temperature. INTRODUCTION Ripening of climacteric fruits, such as banana, is initiated either by natural evolution of endogenous ethylene or by commercial exogenous ethylene ripening procedures (Golding et al., 1998). In contrast, increased storage life is closely related to maintenance of low ethylene levels (Sisler and Serek, 1997). The gaseous compound, 1- methylcyclopropene (1-MCP), has been reported to inhibit ethylene action, and therefore, repining. 1-MCP brings about its action by competitive inhibition of ethylene receptors. Thus, 1-MCP has potential for the commercial control of ripening and senescence of harvested bananas. Modified atmosphere by flexible films is a versatile technology that is applied to a wide range of fruits and vegetables (Kays, 1997; Beaudry, 0). Fruit quality and shelf life are related to the rate of respiration of the fresh produce. In turn, the rate of respiration is a result of the genetic nature of the produce, its maturity stage, temperature and the influence of oxygen uptake, and ethylene and carbon dioxide production (Kader, 1986). A storage life similar to that of fruit kept under refrigeration can be achieved at ambient temperature by packing fruits using modified atmosphere (MA), mainly when combined with ethylene absorbents such as potassium permanganate (Scott et al., 1970). However, the general benefits of using MA associated to ethylene scrubbers is limited because since temperature increasing can adversely influence fruit quality and shelf life as CO 2 levels became too high and/or O 2 levels became too low, leading to anaerobic respiration (Kays, 1997). The ethylene catalyzed rise in respiration that may increase this problem should be inhibited by 1-MCP. The objective of this work was to evaluate the influence of the treatment with 1- a silvasil@cca.ufpb.br Proc. III rd IS on Trop. and Subtrop. Fruits Eds.: M. Souza and R. Drew Acta Hort. 86, ISHS 10 371

MCP in combination with modified atmosphere by X-tend film on the physiology and extension of the postharvest life of Prata banana stored at room temperature and 15 C. MATERIAL AND METHODS Bananas (Musa ssp.) cultivar Prata were harvested during the commercial harvesting season from an orchard in Bananeiras, Paraíba State, Brazil, in the maturation stages 1 (physiologically ripen fruit, but with totally green skin). Fruits were harvested, no later than 8, and Banana hands were treated in sealed plastic chambers of 0.19 m 3 (90 x cm), with and 10. L -1 1-MCP during 2 h, at room temperature (2±1 C). 1-MCP was applied as a gas, according to Fan et al. (1999) procedures. Bananas from the treatment control (without 1-MCP) were also sealed in chambers under similar conditions. Upon removal from the chambers, fruits from each treatment were stored at room temperature (2±1 C) and 80% RH during 16 days. Rate of carbon dioxide production for each treatment was measured hourly in a flow-through system by ventilating approximately 1000 g of fruit enclosed in a 5-L glass jar with CO 2 -free air. Each hour the system was closed and the CO 2 produced was collected in 0.1 N KOH solutions, which was afterwards tritrated with 0.1 N HCl solution. At each evaluation period, four replications (1 set of three fruit/rep) from each treatment were evaluated. For color changes evaluation, following the exposition to 1-MCP, a set of three fruits were packed in polystyrene trays and packed using Xtend TM film (Stepac, L.A., Israel) for modified atmosphere generation and kept under ambient (AA) and modified atmospheres () at 15 C and 90±2% of Relative Humidity (RH) and room temperature (23±2 C and 85±2% RH). Objective skin color was measured with a portable Minolta Chromameter CR-, according to CIELAB system, which express color in three parameters: L*, that corresponds to lightness; a*, that defines the transition from green (-a*) to red (+a*); and b*, that represents the transition from blue (-b*) to yellow (+b*). The more distant from the center (=0), the more saturated is the color (Calvo, 1989). Ripening of fruit was also visually assessed by subjective skin color evaluation, which was measured by 10 judges, using a 1 to 7 scale, with 1 = totally green, 2 = breaker; 3 = <25% color change; = 25 to % color change; 5 = >% but <100% color change; 6 = 100% color definition; and 7 = 100% color definition with light brown spots (over ripen). A complete randomized design, with four replications (three fruits/rep), was used, and arranged in a factorial scheme (2 x 6), with atmospheres evaluated, (AA, ), 6 evaluation periods. MCP doses (0 and ) and temperatures were evaluated separated. Data were expressed as the average of four replications/treatment. Statistical analyses, regressions and/or LSD, were performed using SAS procedures (SAS Inst., Inc., 1988). Tukey s multiple range test (P 0.05) was used to discern between treatment classifications when F values were significant for main effects. Unless stated otherwise, only results significant at P 0.05 are discussed. RESULTS AND DISCUSSION Control Prata banana (without 1-MCP) ventilated with air began to ripen on day 8 and showed characteristic respiration climacteric pattern and normal yellow color development. The maximum respiratory rate (climacteric peak), reached on day 11, was 98.7 mg CO 2.kg -1.h -1. 1-MCP application delayed the onset and reduced the intensity of the respiratory peak (Fig. 1). The initial rate of CO 2 production of Prata banana treated with 1-MCP was lower than the control. These results clearly show the effect of 1-MCP as an efficient inhibitor of ethylene action and suppressor of respiration, as also shown by Golding et al. (1998) in bananas subgroup Cavendish, cultivar Williams. The rate of CO 2 production for fruits treated with 10 was lower than those treated with 1-MCP. It is generally accepted that endogenous ethylene production has a continuing role in integrating the many biochemical changes associated with ripening (Seymour, 1993). The results herein demonstrate that 1-MCP suppressed the rate of carbon dioxide production and retarded the onset of the respiratory peak (Fig. 1). Therefore, it can be that 1-MCP blocked the normal adjustment of response of ethylene production in Prata 372

Banana. It may be that, since 1-MCP was applied to fruit at the maturity stage 1, it occupied the ethylene receptors irreversibly and blocked their normal responses, mainly when maintained at 15 C. Molecular studies suggest that in tomato the number of binding sites may increase during normal ripening, and the ethylene perception may be upregulated in response to ethylene production, in a similar way that ethylene biosynthesis is subject to positive feedback regulation (Klee and Tieman, 1997). It is largely accepted that 1-MCP suppresses ethylene perception by receptors, inhibiting ripening (Sisler and Serek, 1997). This suggestion is supported in here, since timing of onset and rate carbon dioxide production at climacteric peak was affected by 1-MCP treatment. In tomatoes, 1- MCP prevents the accumulation of a number of mrnas coding for the expression of ACC synthase, ACC oxidase and the ethylene receptors involved in positive feedback regulation of autocatalytic (System 2) ethylene production (Nakatsuma et al., 1998). Golding et al. (1998) have suggested, for banana Williams treated with 0 nl.l -1 1- MCP, that when new ethylene receptors are synthesized or are capable of ethylene reception after 1-MCP treatment, a continuous presence of propylene activates the ethylene receptors. In this study, on the other hand, for Prata banana treated with nl.l -1 1-MCP, ripening was unevenly resumed eventually; however, the reversibility of the effect of 1-MCP by following ethylene treatment was not tested. The b* values increased after 6 days storage, for treatment control (Fig. 2), independent on temperature. Fruits maintained under modified atmosphere tended to present constant b* values during storage, as a result of the maintenance of green color. The a* values for fruits of treatment control changed from negative (-a*) to positive (+a*) during storage (Fig. 3), characterized by green color disappearance and synthesis of other pigments. Fruits treated with 1-MCP, on the other hand, tended to present negative values until the end of storage period, indicating that the pathways for chlorophyll degradation and carotenoids synthesis were affected by 1-MCP treatment, and therefore, at least indirectly, in Prata banana they are influenced by ethylene. For, 1- MCP treated fruits, the a* value started to be positive following 1 days storage at room temperature. Fruits kept at 15 C, a* value was positive (changed from green to yellow) following 2 days storage. Based on L* values (Fig. ), banana non-treated with 1-MCP (control) lost fruit gloss during storage, independent on temperature or atmosphere utilized. Fruits exposed to 1-MCP maintained constant L* values during storage. These results suggest that that 1- MCP was effective in keeping fruit gloss. Golding et al. (1998) reported that in Cavendish bananas, the role of ethylene is of a catalyze, accelerating and coordinating the processes related to pigment synthesis and chlorophyll degradation. In Prata banana, the skin color evolution, for treatment control (without 1-MCP), progressively increased from green (Grade 1) to yellow with light brown spots (Grade 7), during storage at 15 and 23 C, independent on atmosphere. On the other hand, 1-MCP significantly delayed skin color evolution of Prata banana during storage. Similar results were also reported for Cavendish Banana, treated with 1-MCP (Golding et al., 1998; Jiang et al., 1999; Harris et al., 00). This effect was much more evident when 1-MCP treatment was associated with modified atmosphere packaging (Fig. 5). However, the development of the skin yellow color was very irregular, persisting the green coloration, mainly in the extremities of 1-MCP treated fruit. This irregular skin color development can occur as a function of the positional difference between the rates of novo-synthesis of ethylene receptors present in the skin and in the pulp of banana, which differ with respect to ethylene evolution and ethylene response (Oetaker and Yang, 1995). 1-MCP applied in association with modified atmosphere (MA) packaging was the most efficient condition in delaying skin color evolution in Prata banana (P>0.01). The positive effect of MCP MA in delaying yellow color development was much more evident when fruits were maintained at 15 C. Fruits exposed to 1-MCP presented stronger yellow pigmentation, as compared to the control. This may be due to the synthesis of other pigments or due to a reduction of the rate of degradation/oxidation of the already synthesized pigments (Gooding and Goad, 373

1970; Gross et al., 1976). It seems that although fruit treated with 1-MCP eventually resumed color development, it was irregular. According to Golding et al. (1999) color evolution may occur dependent or independent from ethylene action. The data herein, however, indicate that, at least indirectly, ethylene is related to color development in Prata. Among other changes, ripening of banana is accompanied by a change in peel color from green to yellow. It is generally accepted that continued production and action of ethylene are required for integration of these biochemical events. Peel degreening is influenced by ethylene produced by the pulp (Dominguez and Vendrell, 1993) and is thought to be mediated by a multienzyme system in which chlorophyllase unmasks stable carotenoids present in mature peel (Gross et al., 1976). In this work, 1-MCP delayed color evolution (Fig. 5) and disrupted degreening. Peel color for the control treatment progressively evoluted from green (grade 1) to yellow with small brown spots (grade 7) for fruit kept at 15 and 2 C, independent on atmosphere of storage. Incomplete and uneven yellowing of the peel was a feature of 1-MCP treated fruit. Although yellowing is initiated by ethylene, completion of the process involves enzymes whose biosynthesis may be irreversibly disrupted by 1-MCP. Sisler and Serek (1997) related that 1-MCP binds irreversibly to ethylene receptors and suggests that plants eventually overcome inhibition by making new receptors. When new ethylene receptors are synthesized or are capable of ethylene reception after 1-MCP treatment, probable color evolution will occur. Collectively, based on color changes, treatment with 1-MCP kept constant the l* values, maintaining fruit gloss, and retarded the increase in the a* and b* values, which is a result of the also delayed transition from the green to the yellow skin color. However, the development of the skin yellow color was very irregular, mainly at 15 C storage. On the other hand, 1-MCP treatment resulted in a more intense skin yellow color development for fruits kept at room temperature. In conclusion, this research has confirmed that respiratory profile and color evolution changes associated with ripening of Prata banana may be dependent on functioning ethylene receptors. Although banana treated with 1-MCP eventually resumed ripening, there was some disturbance to the biochemical changes associated with normal ripening. These changes include uneven peel degreening, and reduction of respiration rate, probably resulting from a disruption of the normal regulation of ethylene production. ACKNOWLEDGEMENTS Financial supports from the Conselho Nacional de Pesquisa (CNPq) from Brazil and from Rohm Haas Company are greatly appreciated. Literature Cited Dominguez, M. and Vendrel, M. 1993. Ethylene biosynthesis in banana fruit: evolution of EFE activity and ACC levels in peel and pulp during ripening. J. Hort. Sci. Ashford. 68: 70. Golding, J.B., Shearer, D., McGlasson, W.B. and Wyllie, S.G. 1999. Relationships between respiration, ethylene, and aroma production in ripening banana. J. Agri. Food Chem. Washington 7:166 1651. Golding, J.B., Shearer, D., Wylle, S.G. and McGlasson, W.B. 1998. Application of 1- MCP and propylene to identify ethylene-dependent ripening processes in mature banana fruit. Postharvest Biol. Tech. Brugges. 1:87 98. Goodwin, T.W. and Goad, L.J. 1970. Carotenoids e Triterpenoids. p.5 358. In: A.C. Hulme (ed.), The biochemistry of fruits and their products. London: Academic Press. vol.1. Gross, J., Cannon, M., Lifshitz, A. and Costas, C. 1976. Carotenoids of banana pulp, peel and leaves. Food Science Technology 9:211 21. Harris, D.R., Seberry, J.A,, Wills, R.B.H. and Spohr, L.J. 00. Effect of fruit maturity on efficiency of 1-methylcyclopropene delay the ripening of bananas. Postharvest Biol. Tech. Brugges. :3 8. Jiang, Y., Joyce, D.C. and Macnish, A.J. 1999. Extension of the shelf life of banana fruit 37

by 1-methylcyclopropene in combination with polyethylene bags. Postharvest Biol. Tech. Brugges. 16:187 193. Kays, S.J. 1997. Postharvest physiology of perishable plant products. Athens, Avi. p.532. Klee, H.J. and Tieman, D. 1997. Potential application of controlling ethylene synthesis and perception in transgenic plants. p.289 287. In: A.K. Kanellis, C. Chang, H. Kende, D. Grierson (eds.), Biology and Biotechnology of the Plant Hormone Ethylene. Kluwer Academic, Dordrecht. Nakatsuka, A., Murachi, S., Okunishi. H., Shiomi, S., Nakano, R., Kubo, Y. and Inaba, A. 1998. Differential expression and internal feedback regulation of 1- aminocyclopropene-1-carboxylate synthase, 1-aminocyclopropene-1-carboxylate oxidase, and ethylene receptor genes in tomato fruit during development and ripening. Plant Physiol. 118:1295 15. Oetaker, J.H. and Yang, S.F. 1995. The role of ethylene in fruit ripening. Acta Hort. 398:167 178. Sisler, E.C. and Serek, M. 1997. Inhibitors of ethylene responses in plants at the receptor level: recent developments. Physiology Plantarum 100:577 582. Seymour, G.B. 1993. Banana. p.83 106. In: G.B. Seymour, J. Taylor and G. Ticker (eds.), Biochemistry of Fruit Ripening. Chapman and Hall, London. Figures CO 2 Production (mg.kg -1.h -1 ) 100 80 Prata Banana Without 1-MCP 1-MCP 10 1-MCP 0 0 5 10 15 25 35 Postharvest Period (Days) Fig. 1. Production of CO 2, at room temperature, of banana cultivar Prata, harvested in the maturity stage 1 (physiologically developed fruit, but with skin totally green) and treated with 1-MCP (0,, and 10) during 2 hours, at room temperature (23±1ºC). 375

AA Prata b* Index 10 y 1AA = 35,069-0,0787x + 0,1123x² R 2 = 0,90 y2 = 37,512-1,282x + 0,96x² R 2 = 0,82 10 Fig. 2. Changes in b* index of banana fruit, cultivar Prata, harvested in the maturity stage 1 (physiologically developed fruit, but with skin totally green) and treated with 1-MCP (0 and ) during 2 h, kept at 15 C and 23 C, under ambient (AA) and modified () atmospheres. AA a* Index 16 12 8 0 - -8-12 -16 16 12 8 0 - -8-12 -16 y 1AA = -21,797 + 6,159x - 0,27x² R 2 = 0,85 y 2 = -19,762 + 3,9369x - 0,0962x² R 2 = 0,87 y 1AA = -21,89 + 8,3339x - 0,517x² R 2 = 0,88 y 2 = -19,51 + 6,1007x - 0,32x² R 2 = 0,92 y1aa = -10, - 1,6x + 0,387x² R 2 = 0,9 Prata y 1AA = -6,8292-2,6771x + 0,5258x² R 2 = 0,7 Fig. 3. Changes in a* index of banana fruit, cultivar Prata, harvested in the maturity stage 1 (physiologically developed fruit, but with skin totally green) and treated with 1-MCP (0 and ) during 2 hours, kept at 15 C and 23 C, under ambient (AA) and modified () atmospheres. 376

70 AA l* Index 70 y 1AA = 8,18 + 5,65x - 0,6382x² R 2 = 0,81 Fig.. Changes in L* index of banana fruit, cultivar Prata, harvested in the maturity stage 1 (physiologically developed fruit, but with skin totally green) and treated with 1-MCP (0 and ) during 2 h, kept at 15 C and 23 C, under ambient (AA) and modified () atmospheres. Color Evolution Grade (1-7) 7 6 5 3 2 1 7 6 5 3 2 1 y 1AA = -0,8782 + 1,527x - 0,0717x² R 2 = 0,93 y 2 = -0,3679 + 1,152x -0,01x² R 2 = 0,95 AA y 1AA = -1,0833 + 2,1979x - 0,155x² R 2 = 0,95 y 2 = -0,6333 + 1,8105x -0,1129x² R 2 = 0,96 y 1AA = 2,0885-0,3596x + 0,077x² R 2 = 0,93 y 2 = 1,19 + 0,1896x - 0,0062x² R 2 = 0,91 Prata y 1AA = 1,6833-0,0652x + 0,0515x² R 2 = 0,9 y 2 = 1,681 + 0,061x + 0,017x² R 2 = 0,92 Fig. 5. Subjective color evolution grade (1 to 7) of banana fruit, cultivar Prata, harvested in the maturity stage 1 (physiologically developed fruit, but with skin totally green) and treated with 1-MCP (0 and ) during 2 hours, kept at 15 C and 23 C, under ambient (AA) and modified () atmospheres. 377