Postharvest Biology and Technology 31 (2004) 313317

Research note

Effect of modied atmosphere packaging on chilling-induced peel browning in banana

Thi Bich Thuy Nguyen a , Saichol Ketsa a, , Wouter G. van Doorn b
a

Department of Horticulture, Faculty of Agriculture, Kasetsart University, Chatuchak, Bangkok 10900, Thailand b Agrotechnology and Food Innovations, Wageningen University and Research Centre, P.O. Box 17, 6700 AA Wageningen, The Netherlands Received 17 June 2003; accepted 23 September 2003

Abstract Sucrier bananas (Musa AA Group; cultivar locally known as Kluai Khai) were stored at 10 C, which results in chilling injury (CI). Fruit was held in packages with and without a modied atmosphere (MA). Oxygen levels in the MA packages were about 12% while CO2 concentrations were about 4%. MA packaging resulted in less visible CI (i.e. greyish peel browning). Total free phenolics in the peel of control bananas decreased more rapidly than in fruit held in the MA package. Phenylalanine ammonia lyase (PAL) and polyphenol oxidase (PPO) activities in the peel of control bananas were considerably higher than in MA-packed fruit. Pulp softness, sweetness and avour of MA-packed fruit were better than in control fruit. MA thus reduced CI symptoms. PAL and PPO activities may be causally related to CI-induced peel browning. 2003 Elsevier B.V. All rights reserved.
Keywords: Musa AA Group; Banana; Catechol oxidase; Peel discoloration; Total phenolics; Phenylalanine ammonia lyase; Polyphenol oxidase; Chilling temperature; Modied atmosphere packaging

1. Introduction Banana is extremely susceptible to chilling injury (CI) (Pantastico et al., 1990). Low temperature storage results in pitting and discoloration of the peel and abnormal ripening of the pulp. Browning in bananas, as in other systems, is thought to be due to the enzymatic oxidation of phenolics into quinones, which then polymerise into brown products (Jayaraman et al., 1982).

Corresponding author. Tel.: +66-2-579-0308; fax: +66-2-579-1951. E-mail address: agrsck@ku.ac.th (S. Ketsa).

Modied atmosphere (MA) packaging can reduce CI in banana (Scott and Gandanegara, 1974; Ketsa et al., 2000). This study was carried out to investigate the effect of MA packaging on CI and the related activities of phenylalanine ammonia lyase (PAL) and polyphenol oxidase (PPO). PPO converts free phenolics to quinones and PAL converts phenyalanine to the free phenolic substrates for PPO (Camm and Towers, 1977). We included an ethylene absorbent and a CO2 scrubber in the packages. This resulted in relatively low ethylene and CO2 levels. We previously found that keeping the ethylene concentrations at relatively low levels prevented precocious ripening. We also

0925-5214/$ see front matter 2003 Elsevier B.V. All rights reserved. doi:10.1016/j.postharvbio.2003.09.006

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showed that the CO2 concentrations in MA packages without the scrubber rose to 7%. This resulted in typical CO2 injury, which became visible after storage, when the fruit ripen at room temperature. The peel colour remained faint yellow with some reddish brown spots and the pulp had a hard core and showed inadequate starch conversion. We hypothesised that both PAL and PPO were involved in low-temperature induced peel browning and therefore determined the activities of these enzymes and the concentration of total free phenolics.

2. Materials and methods Sucrier bananas (Musa cavendishii [Musa acuminata] AA Group; locally known as Kluai Khai) were harvested at commercial maturity, in a plantation in the Petchaburi province (Western Thailand). De-handed bananas were placed in corrugated cardboard boxes and transported by refrigerated truck (25 C) to the laboratory within 2 h of harvest. In the laboratory, hands were selected for uniformity of size and colour, i.e. only mature hands were being used. The hands were cleaned in a solution of 0.5% MgSO4 to remove latex from the cut surface, then dipped for 23 min in 500 mg l1 thiabendazole solution to control fruit rots and were allowed to dry at ambient temperatures. Ethylene absorbents were made by soaking pieces of chalk (0.7 cm diameter and 0.5 cm thickness) in saturated potassium permanganate solution and 30 g of the dried material was placed in perforated polyethylene sachets. Carbon dioxide scrubbers were produced by placing 30 g of calcium hydroxide in small paper bags. Six hands of banana were randomly placed in 0.11 mm thick non-perforated polyethylene bags (75 cm wide and 110 cm long), each with three sachets of ethylene absorbent and CO2 scrubber. The bags had no holes and were closed by making a knot (hand twisting). Bags were placed individually in corrugated cardboard boxes (40 cm 48 cm 22 cm). The RH in the bags was higher than 90%. The control fruit were placed in corrugated cardboard boxes without polyethylene bags (and no ethylene absorbent or CO2 scrubber). Boxed bananas were stored at 10 C and 90% RH. The RH in the control boxes was about 90%.

Gas concentrations were determined with a gas chromatograph equipped with a ame ionisation detector (Shimadzu GC-14) for ethylene and a thermal conductivity detector (Shimadzu GC-RIA) for O2 and CO2 . The syringe was punched through the plastic lm of the bags. The hole in the plastic was sealed with tape and the same hole was used at later sampling dates. After every 6 days of storage, three control boxes and three boxes with MAP bags were taken out of storage for determination of visible CI symptoms, total free phenolics and activities of PPO and PAL. The six hands in each control box and each bag were taken out, three hands were immediately used for CI assessment and biochemical analysis, three other hands were left at room temperature (about 28 C) for 4 days, after which CI symptoms were determined again. The colour of Sucrier bananas, upon chilling, turned to greyish brown, different from the dark browning observed during ripening at non-chilling temperatures. Surface browning was rated on a scale of 15 (Nguyen et al., 2003): 1, indicates no chilling injury, 2, mild injury, 3, moderate injury, 4, severe injury and 5, very severe injury. For measurement of total free phenolics and the activities of PAL and PPO, we took peel from the middle part of 10 ngers from one hand. The peel was taken from the whole circumference of the fruit and was 4 cm long. This was replicated with two other hands. The peel of the 10 ngers from each hand was mixed. Free phenolics were determined in fresh tissue, in the three biological replications and absorbance was determined three times from each extract. For PAL and PPO analysis the acetone powder of peel from each of the three hands was extracted. Absorbance was measured 45 times. Total free phenolic content was estimated colorimetrically (Singleton and Rossi, 1965). Frozen tissue was homogenised in ethanol, ltered and centrifuged and phenolics were measured with the Folin Ciocalteu reagent. PAL was extracted and assayed by the method of Zucker (1968). Frozen tissue was homogenised in acetone, ltered, extracted again in cold ethanol and ltered once more. Acetone powder was dried in a desiccator and added to cold 0.2 M sodium borate buffer at pH 8.8. The beaker was shaken for 30 min at 4 C and the suspension was ltered and centrifuged.

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During the preparation of the enzyme extract, the temperature was kept at 4 C. The assay medium contained 3 ml of enzyme extract and 2 ml of phenylalanine. The mixture was incubated at 37 C for 1 h and the reaction was stopped by adding 0.5 ml of 5 N HCl. PAL activity was determined by measuring absorbance at 290 nm. One unit of PAL activity was dened as the change in absorbance per millilitre of enzyme extract. PPO was extracted and assayed using the method of Luh and Phithakpol (1972). The extraction method was the same as that of PAL, except that 0.1 M citrate buffer at pH 6.2 was used. The assay medium contained 10 ml of enzyme extract and 5 ml of catechol. PPO activity was determined by measuring absorbance at 420 nm. One unit of PPO activity was dened as the change in absorbance after 1 min of measurement, per millilitre enzyme extract. Protein content was estimated using the Bradford (1976) method. Specic activity of the enzyme was expressed as units per milligram of protein. Eating quality was tested in fruit taken out of storage after 12 and 18 days, then left for 4 days at room temperature (28 C). A panel of six experts used a scale of 1 to 5, where 1 indicates extremely low quality and 5, extremely good quality. Where possible, means were compared using Duncans new multiple range test (DMRT) and Wilcoxon rank-sum test. Correlations were calculated by using Pearsons correlation coefcients.

CI symptom (scores)

1 0 6 12 18
Time (d)

24

30

Fig. 1. Development of chilling injury of banana peel without ( ) and with MA packaging ( ) stored at 10 C. Data are mean of the scores of three hands per MAP package or control box. The data of three MAP packages/controls were then averaged. Chilling injury was determined directly after storage, prior to shelf life. Vertical bars indicate S.D. When no bar is shown S.D. was zero.

3. Results and discussion Ethylene concentration in the polyethylene bags containing bananas slightly increased during the rst
Table 1 Eating quality determined using a scale of 15 for each parameter Parameters Day 12 Control Softness Sweetness Sourness Flavour 2.43 3.21 4.43 3.21 MA 4.00 4.14 4.21 4.21

week and then remained steady at about 0.4 l l1 . The oxygen concentration in the bags sharply decreased to 12% during the rst week and slowly increased to 13% thereafter, whereas the CO2 concentration rapidly increased to about 4% and stayed at that level. As the control boxes had rather large holes and the cool room was ventilated, the concentrations of ethylene, CO2 and O2 were close to ambient (results not shown). The peel of control bananas showed greyish brown discoloration after 6 days of storage. This CI

Day 18 Difference a a ns a Control 2.60 3.20 3.40 2.80 MA 3.80 3.80 4.20 3.80 Difference a a a a

A higher score indicates better quality. Bananas were stored at 10 C for 12 or 18 days and then ripened at room temperature (28 C) for four days. Scores were compared by the Wilcoxon rank-sum test. a Indicates signicant difference (P < 0.01) whereas ns means not signicant.

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symptom was initially mild, but steadily increased until 30 days of storage. When the fruit were transferred to ambient temperature for ripening, CI symptoms became rapidly worse as the peel became dark yellow, whereas non-chilled controls stayed bright yellow (data not shown). CI symptoms in bananas held in MA packages were not observed prior to 18 days. The discoloration was mild at this time-period and slowly increased thereafter. The Wilcoxon rank-sum test showed a highly signicant difference (P < 0.05) in visible peel CI between bananas with and without MA packaging (Fig. 1). When taken out of storage the peel did not show rapid darkening. After 12 and 18 days of storage, scores for softness, sweetness and avour were higher in MA-packed fruit (Table 1). The results thus suggest that the MA packaging alleviated low temperature-induced peel browning. This corroborated ndings of Scott and Gandanegara (1974) and Ketsa et al. (2000). It is not clear, at present, to what extent the various factors that were different in the package atmosphere, compared with the control, contributed to this effect. During storage, total free phenolics content in the peel of control bananas was considerably lower than that of bananas with MA packaging. The total free phenolic level in the peel of control bananas also decreased more rapidly than that in bananas with MA packaging (Fig. 2A). The decrease in the total phenolic compounds of control bananas correlated with the degree of CI. PPO activity in the peel of control bananas stored at 10 C increased at the beginning of storage. It increased later in the peel of bananas with MA packaging (Fig. 2B). During the rst two weeks of storage, PAL activity in the peel of bananas with and without MA packaging slowly increased (Fig. 2C). Subsequently, PAL activity rapidly increased in the peel of control bananas whereas it slowly increased in the peel of bananas with MA packaging. The strong inverse relationship between PAL activity and total phenolics indicates that the turn-over of free phenolics was more rapid free phenolics its synthesis. It is concluded that MA packaging with an ethylene absorbent and CO2 scrubber alleviated CI in bananas stored at 10 C. MA packaging resulted in lower PPO and PAL activities in the peel, which may partially

1.8
Total phenolics (mg/g FW)
(A)

1.5 1.2 0.9 0.6

(B)

PPO activity (units/mg protein) PAL activity (units/mg protein)

1.0

0.8

0.6

0.4
(C)

0.8 0.6 0.4 0.2 0 6 12 18 Time (d) 24 30

Fig. 2. Total phenolics (A) and the activity of PPO (B) and PAL (C) in the peel of bananas without () and with MA packaging () stored at 10 C. Data are the means of three biological replications. The parameters were measured directly after storage, prior to shelf life. Vertical bars indicate S.D.

explain the alleviating effect of MA on peel browning during chilling conditions. Acknowledgements The authors are thankful for the nancial support from the Ford Foundation and Thailand Research Fund (TRF).

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