Postharvest Biology and Technology 77 (2013) 1927

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Postharvest Biology and Technology

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Residual effects of low oxygen storage of mature green fruit on ripening processes and ester biosynthesis during ripening in bananas
Yoshihiro Imahori a, , Kohei Yamamoto a , Hiroshi Tanaka a , Jinhe Bai b
a b

Laboratory of Postharvest Physiology, Graduate School of Life and Environmental Sciences, Osaka Prefecture University, 1-1 Gakuen-cho, Nakaku, Sakai, Osaka 599-8531, Japan USDA-ARS Horticultural Research Laboratory, 2001 South Rock Road, Ft. Pierce, FL 34945, USA

a r t i c l e

i n f o

a b s t r a c t
Mature green banana (Musa sapientum L. cv. Cavendish) fruit were stored in 0.5%, 2%, or 21% O2 for 7 days at 20 C before ripening was initiated by ethylene. Residual effects of low O2 storage in mature green fruit on ripening and ester biosynthesis in fruit were investigated during ripening for up to 6 d at 20 C. Concentrations of ethanol in mature green fruit did not change during storage in both 21% and 2% O2 atmospheres, but increased in fruit stored in 0.5% O2 . The activities of alcohol dehydrogenase (ADH) in 2% and 21% O2 atmospheres remained very low throughout the storage period, but signicantly increased with 0.5% O2 . After transferring fruit to regular air and trigging ripening with ethylene, yellowing of peel, fruit softening and hydrolysis of starch in fruit stored in low O2 atmospheres were slower than in the control. Fruit stored in low O2 also showed a delayed onset of the climacteric peak. The activities of ADH were lower in the low O2 stored fruit than in the control fruit. Productions of ethyl acetate, isoamyl acetate, and isobutyl acetate were remarkably suppressed by low O2 storage. Alcohol acetyltransferase activity increased gradually with storage time in all treatments, being signicantly lower in fruit with low O2 pretreatments. The results indicate that low O2 plus room temperature storage can extend storage life of bananas with the sacrice of a low production of ester volatiles. 2012 Elsevier B.V. All rights reserved.

Article history: Received 4 August 2012 Accepted 12 November 2012 Keywords: Banana Low-oxygen atmosphere Alcohol acetyltransferase Alcohol dehydrogenase Ester Volatile

1. Introduction Bananas are among the most important fruit in world trade. As a climacteric fruit, bananas are commercially picked from the tree at the mature green stage, and then shipped to distant markets, usually by air or sea. Ripening treatment by ethylene gassing is usually applied by distributors or retailers directly before retail display. To protect mature green fruit from ripening during storage and transportation, a common practice is to pre-cool fruit to 13 C and maintain the low temperature during the entire storage period. However, refrigeration is costly, and an alternative low-cost method that delays ripening would be useful, particularly for small operators and in developing countries (Wills et al., 1990). Application of controlled atmospheres (CA) and modied atmospheres (MA) can extend the storage life of mature green bananas (Yahia, 1998). The benecial effects of CA storage include extending shelf-life and maintaining quality by decreasing metabolism and suppressing postharvest decay (Imahori et al., 2004). Palomer et al. (2005) reported that CA storage slows respiration, peel de-greening and changes in sugars, and minimizes the susceptibility of bananas to crown rot. In a low oxygen pretreatment, the exposure of mature

Corresponding author. Tel.: +81 72 254 9418; fax: +81 72 254 9418. E-mail address: imahori@plant.osakafu-u.ac.jp (Y. Imahori). 0925-5214/$ see front matter 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.postharvbio.2012.11.004

green banana fruit to low O2 for 2 days was effective in delaying ripening during storage and after ethylene treatment (Wills et al., 1982, 1990; Pesis et al., 2001). Most commodities require a minimum of 1% O2 in CA or MA storage to avoid anaerobic metabolism. At O2 -stress atmospheres, acetaldehyde which is produced through pyruvate decarboxylation by pyruvate decarboxylase (PDC) is converted to ethanol by alcohol dehydrogenase (ADH) using NADH, hence, ethanol is usually the major end-product (Imahori et al., 2004). Accumulated ethanol in fruit drives the biosynthesis of esters to ethyl esters, thus decreases production of typical fruit aromas, such as butyl acetate, isobutyl acetate and isoamyl acetate, and causes off-avors (Bai et al., 1990). Aroma, besides other properties such as texture and appearance, plays an important role in the quality assessment of fruit. This parameter inuences consumer acceptability of food (Jayanty et al., 2002). Although more than 250 volatile components have been identied in banana (Jayanty et al., 2002), the banana fruity top notes are from volatile esters, such as isoamyl acetate and isobutyl acetate (Macku and Jennings, 1987; Bai et al., 1990; Wendakoon et al., 2006). The volatiles are formed by esterication of alcohols and carboxyl groups catalyzed by alcohol acetyltransferase (AAT), using alcohol and acetyl CoA as substrates (Lara et al., 2003). It has been reported that activity of AAT increases with advanced maturity of fruit but can be inhibited by low O2 concentrations during storage of fruit, such as CA storage, decreasing

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production of acetate esters (Fellman et al., 1993; Chervin et al., 2000). The negative impacts of CA storage, ultra-low-oxygen storage in particular, on volatile emission and biosynthesis is well documented, especially for apples and pears (Lara et al., 2003). The increased ethanol levels and/or ADH activity in fruit stored under a hypoxic atmosphere may be retained during subsequent ripening in air, and in some cases may lead to off-avors (Lara et al., 2003). Thompson (1998) reported that exposure of bananas to less than 1% O2 for an unspecied period causes low O2 injury in the form of a dull yellow or brown peel discoloration, failure to ripen and off aromas. Differences in aroma compounds were found between airstored and nitrogen-stored post-ripening bananas (Klieber et al., 2002). The objective of our research was to determine the tolerance of mature green bananas to low oxygen concentrations for short periods at 20 C, and to examine the effects of low oxygen pretreatments on the biosynthesis of ester in banana fruit during subsequent ripening at 20 C, with the general purpose of assessing the inuence of such storage conditions on fruit quality during commercial shelf life of the fruit.

(ADH) was measured throughout storage and post-storage ripening. Every treatment at each sampling day included three replicates and two fruit per replicate. For non-destructive measurements of attributes, surface color, respiration rate, rate of ethylene production, two fruit per replicate labeled for the last sampling day (post-storage day 7) were used on all experimental days. For destructive measurements, two fruit per replicate at each sampling day were peeled, sliced to 0.5 mm thick discs, quartered, and then mixed. Measurements were taken from the fresh tissue or snap frozen in liquid nitrogen and storage at 80 C for up to 30 days. 2.2. Measurement of ethanol content Ethanol levels were determined according to the method of Sugiura and Tomana (1983). Frozen tissue (5 g) was homogenized with a chilled pestle and mortar in 10 mL cold acetone. The homogenate was quickly transferred to a cold screw-capped glass bottle and stored at 20 C. The homogenate was ltered through Advantec No. 2 lter paper. One microliter of the ltrate was injected into GC (Hitachi model 163, Hitachi) equipped with FID and a stainless column (3 mm 1.0 m) containing 50/80 mesh Porapak Q. The ow rate of the nitrogen carrier gas was 40 mL min1 . The oven temperature was 140 C. The ethanol concentrations in the sample were calculated from the comparison of peak areas with those of standards. Amounts of ethanol were expressed as g per 100 gram fresh weight of esh. 2.3. Volatile compounds analysis Frozen pulp tissue (5 g) was homogenized in a chilled mortar and pestle with 20 mL of 50 mM potassium phosphate buffer (pH 7.0). The homogenate was transferred into a conical ask and closed with a silicon cap. After incubating the sample at 30 C for 60 min, 1 mL of headspace gas was taken with a glass syringe and injected into a GC (GC-8A, Shimadzu) equipped with FID and polyethylene glycol 9000 column (3 mm 1.0 m). The ow rate of the nitrogen carrier gas was 27 mL min1 . The oven temperature was 90 C. The volatile compounds of banana were qualied and quantied by comparing the retention time and peak size (area) of the authentic standards. 2.4. Extraction and assay of enzymes Extraction and assay of ADH were conducted by following the method of Ke et al. (1994) with some modications. Briey, frozen pulp tissue (5 g) was homogenized in a chilled mortar and pestle with 20 mL of 100 mM MES buffer (pH 6.5) containing 2 mM dithiothreitol and 1% (w/v) polyvinylpyrolidone (PVP). The homogenate was ltered through two layers of Miracloth (Calbiochem) and the ltrate was centrifuged at 13,000 g for 20 min at 4 C. Quantication of enzyme activity was performed by monitoring the oxidation of NADH at 340 nm at 30 C using a spectrophotometer (Model V530, Jasco). Enzyme activities were expressed as moles of substrate used per minute per gram of protein. AAT was extracted by following Wendakoon et al. (2006) with some modications. Briey, frozen pulp tissue (5 g) was homogenized in a chilled mortar and pestle with 20 mL of 100 mM potassium phosphate buffer (pH 8.0) containing 5 mM dithiothreitol and 1% (w/v) PVP. The homogenate was ltered through two layers of Miracloth (Calbiochem) and the ltrate was centrifuged at 13,000 g for 20 min at 4 C. AAT activity was assayed according to the method described by Lara et al. (2003) with slight modications. The reaction mixture contained 0.1 M phosphate (pH 8.0), 5 mM MgCl2 , 0.25 mM acetyl-CoA, 5 mM isobutyl alcohol, 10 mM

2. Materials and methods 2.1. Plant materials and treatments Hands of mature green banana (Musa sapientum L. cv. Cavendish) fruit were obtained from a local wholesale market in Sakai, Japan. They were harvested at a commercial plantation in the Philippines and shipped at 13 C following commercial routes to the laboratory within 5 days of harvest. The fruit were sorted for absence of visual defects and uniformity of weight and size. Fruit (ngers) separated from the banana hands were submerged into 20 C water for 5 min, and then dried by paper towel. Six out of 114 fruit were randomly taken for day 0 sample analysis (two fruit per replicate three replicates), and the rest were divided into three groups for three gas treatments, 0.5, 2, and 21% O2 . For each treatment, 36 fruit were evenly divided and placed in three 7-L glass jars to represent three replicates. Fruit in the jars were stored in the dark at 20 C and exposed to a given gas combination with a ow-through system at a ow rate of 250 mL min1 . The gas mixtures with different O2 levels were obtained by mixing humidied pure O2 and pure N2 . After 7 days, all gas sources for the owthrough system were replaced by 10 L L1 ethylene in air for 24 h to trigger ripening. Finally, fruit were ripened in ethylene-free air for an additional 7 days at 20 C. A subsample (two out of twelve fruit per replicate) was taken at days 1, 4 and 7 during storage, and days 1, 4 and 7 during post-storage ripening. Oxygen concentration in the ow-through system was monitored using a gas chromatograph (GC) (Yanaco model G80, Yanaco), equipped with a thermal conductivity detector (TCD) and a stainless steel column (3 mm 1.0 m) containing molecular sieve 5A. The ow rate of the carrier helium gas was 40 mL min1 . The oven was set at 60 C and the injector was kept at 125 C. Ethylene concentration was checked using a GC (GC-8A, Shimadzu) equipped with a ame ionization detector (FID) and a glass column (3 mm 0.5 m) containing 80/100 mesh activated alumina. The ow rate of the carrier N2 gas was 40 mL min1 . The oven was set at 40 C and the injector was kept at 140 C. Ethanol content was measured during storage of mature green fruit at day 0, 1, 4 and 7, and surface color, respiration rate, rate of ethylene production, rmness, eating quality, content of ethanol, isoamyl acetate, isobutyl acetate and ethyl acetate, and alcohol acetyltransferase (AAT) activity were measured during post-storage ripening at days 1, 4 and 7. Alcohol dehydrogenase

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Ethanol (g100g-1FW)

5,5 -dithiobis (nitrobenzoic acid) (DTNB) and an appropriate amount of the enzyme extract. Quantication of enzyme activity was performed by monitoring the increase in absorbance at 412 nm at 30 C, using a spectrophotometer (Model V-530, Jasco). One activity unit (U) was dened as the increase of absorbance per minute, and the results were expressed as U per gram of protein. Protein in the extracts was determined by the method of Bradford (1976) with bovine serum albumin as the standard.

20

15

10 21%O2 2%O 2 0.5%O 2 0 0 25 1 2 3 4 5 6 Days in storage at 20C 7 8

2.5. Measurement of soluble solids content (SSC) The pulp tissue (5 g) was homogenized in a mortar and pestle and uid was taken after ltering through two layers of Miracloth (Calbiochem) and the SSC reading was taken with a digital refractometer (PR-101, Atago, Tokyo, Japan) calibrated against water.

B
21%O2 2%O 2 0.5%O 2

2.6. Measurement of surface color, rmness and eating quality Fruit color was determined visually and by using a color difference meter (ND-1001 DP, Nippon Denshoku, Tokyo, Japan), that expresses the color as Hunter L*, a*, b* values. Hunter L*, a*, b* values were taken on six different surface portions of each fruit and the average from two fruit per replicate were used for calculations. L*b*/a*, a yellowing index was calculated to present de-greening or yellowing of fruit surface, the higher index value, the more developed yellow color (Kanellis et al., 1989). The visual appearance was assessed as described by Pesis et al. (2005). Briey, the peel color was expressed as an index on an eight-grade scale: 1 = green; 3 = breaker; 5 = yellow, green tip; 7 = yellow, ecked with brown; 8 = over-ripe. Firmness evaluation was carried out using a manual fruit rmness tester (FT 327, EFFEGI) tted with an 8 mm diameter tip. The measurement was taken from opposite sides in middle of intact fruit, complete with peel. Eating quality was tested in fruit after 6 days at 20 C after treatment. A taste panel of ve experts used a scale of 15, where 1 indicates extremely low quality and 5, extremely good quality.

ADH(molNADHmin-1mg-1protein)

20

15

10

3 4 5 6 Days in storage at 20C

Fig. 1. Changes in ethanol content (A) and alcohol dehydrogenase activity (B) in mature green banana fruits stored in 21% O2 ( ), 2% O2 ( ) and 0.5% O2 ( ) at 20 C. Data represent means S.E. from three replicate samples.

3. Results 3.1. Effect of low oxygen treatments on ethanol contents and ADH activity of mature green banana fruit The concentrations of ethanol in mature green fruit did not change during storage in 2% or 21% O2 at 20 C. In contrast, the concentration in the 0.5% O2 treatment at 20 C increased 40% after one day and even more there onwards (Fig. 1A). Accordingly, the activity of ADH in mature green fruit stored in 2% and 21% O2 at 20 C remained low throughout the entire storage period at 20 C, but in those fruit exposed to 0.5% O2 at 20 C, the activity signicantly increased 14 times by day 4 and 18 times by day 7, respectively (Fig. 1B).

2.7. Measurement of respiration rate and ethylene production Rates of respiration and ethylene production were determined after various storage times. Two fruit were sealed inside a 1 L jar for 1 h at 20 C. Respiration rate was obtained by measuring CO2 concentration. Yanaco model G80 GC was used to qualify and quantify CO2 concentration. The GC was equipped with a TCD and a stainless steel column (3 mm 1.0 m) containing 80/100 mesh Porapak Q. The ow rate of the carrier helium gas was 40 mL min1 . The oven was set at 60 C and the injector was kept at 125 C. Ethylene concentrations were measured using a GC (GC-8A, Shimadzu) equipped with FID and a glass column (3 mm 0.5 m) containing 80/100 mesh activated alumina. The ow rate of the carrier N2 gas was 40 mL min1 . The oven was set at 40 C and the injector was kept at 140 C.

3.2. Changes in external appearance, surface color, rmness, SSC, eating quality, respiration rate and ethylene production during post-storage ripening Until ripening was triggering, all fruit remained green with an L*b*/a* value of 22, regardless of O2 concentration in storage at 20 C. There was no visible injury or decay on any fruit. However, weak off-avors were detected in fruit stored in 0.5% O2 at 20 C. During post-storage ripening at 20 C, control fruit degreened rapidly as shown in increases of L*b*/a* values from 22 at the beginning of ripening to 7 after 4 days (Fig. 2A). In contrast, degreening of the fruit stored in 0.5 or 2% O2 at 20 C was slower,

2.8. Statistical analysis Data for the analytical determinations were subjected to analysis of variance (ANOVA) using Statcel 2 (OMS, Saitama, Japan). Sources of variation were time of storage, low O2 level and duration of treatments. Mean separations were performed using HSD in Tukeys test to examine if differences between treatments and storage time were signicant at P = 0.05.

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15 10 5 Suface L*b*/a* value 0

A
a a ab b Ripning index

8 7 6 5 4

B
a

b b b b

b -5 -10 c -15 -20 -25 4 7 1 Days at 20C after treatment a a a

3 2 a a a

1
0

4 7 1 Days at 20C after treatment 30

120

C
a

D
a

100 b c 80 Firmness (N) a 60 b 40 b a a a

SSC(%)

25

a
b 20 b

b b

15

10 a a a

20

0 1 4 7 Days at 20C after treatment

0 4 7 1 Days at 20C after treatment

Fig. 2. Surface L*b*/a* value (A), ripening index (B), rmness (C) and soluble solids content in pulp (D) of bananas during post-storage ripening at 20 C. Fruits were stored with 21% O2 (white bars), 2% O2 (shaded bars) or 0.5% O2 (black bars) for 7 days at 20 C before triggering off ripening. Data represent means S.E. from three replicate samples. Values labeled with the same letter are not different at the 5% level.

especially for the fruit stored in 0.5% O2 at 20 C (Fig. 2A). It took 7 days for those fruit stored in low O2 turn to yellow (Fig. 2A). The ripening index reected the changes in L*b*/a* values and revealed that the fruit stored in 0.5 or 2% O2 at 20 C ripened slowly (Fig. 2B). The pretreatment with low oxygen had a signicant effect on banana appearance and was efcient in delaying ripening. During post-storage ripening at 20 C, control fruit softened rapidly as shown in the decreases of values from 80 N at the beginning of ripening to 45 N after 4 days (Fig. 2C). In contrast, the decreases of the fruit stored in 0.5 or 2% O2 at 20 C were less, especially for the fruit stored in 0.5% O2 at 20 C (Fig. 2C). SSC in the fresh pulp was very low before ripening was initiated (Fig. 3). However, four days after initiation of ripening at 20 C, SSC rapidly increased from about 5 to over 20%. SSC continually increased slowly afterwards (Fig. 2D). Low O2 storage at 20 C slowed the increase by a small margin (Fig. 2D). After initiation of ripening with ethylene and transfer to air at 20 C, scores for avor, sweetness and softness of the control fruit were higher than in the low O2 stored fruit at 20 C (Table 1).

The respiration rate of the control fruit was higher than for the low O2 stored fruit at 20 C until they reached the climacteric peak on day 3 of post-storage ripening at 20 C (Fig. 3A). Fruit stored in low O2 at 20 C had lower respiration rates and reached the climacteric peak one day later, with lower peak values in comparison with the control fruit (Fig. 3A). The respiration rate then declined, to a

Table 1 Eating quality determined using a scale of 15 for each parameter in banana fruit during post-storage ripening. Parameters Days at 20 C after treatment Day 4 21%O2 Flavor Sweetness Softness 3.2a 3.4a 4.0a 2%O2 1.8b 2.2b 2.6b 0.5%O2 1.6b 1.6b 1.4c Day 7 21%O2 4.6a 4.6a 4.8a 2%O2 3.8a 4.4a 3.8a 0.5%O2 4.0a 3.6b 3.6a

Values labeled with the same letter are not different at the 5% level.

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200

250

a
150 CO2(mgkg-1hr-1)

200

Ethanol (g100g-1 FW)

100

b
150

50

21%O2 2%O2 0.5%O2

c
100

0 0 1 2 3 4 5 6 Days at 20 C after treatment 7 8

50

a a
21%O 2 2%O2 0.5%O2

a a a

b
1 4 7

C2H4(Lkg-1hr-1)

Days at 20C after treatment

150

a a a

ADH (molNADHmin-1mg-1protein)

b
100

0 0 1 2 3 4 5 6 Days at 20 C after treatment 7 8

Fig. 3. Changes in respiration rate (A) and ethylene production (B) in banana fruit during post-storage ripening. Fruits were stored in 21% O2 ( ), 2% O2 ( ), or 0.5% O2 ( ) for 7 days at 20 C prior to triggering off ripening by ethylene. Data represent means S.E. from three replicate samples.

a
50

lower level in the control fruit than in the low O2 stored fruit at 20 C (Fig. 3A). The ethylene production of the control fruit reached the climacteric peak on day 3 of post-storage ripening at 20 C (Fig. 3B). Fruit stored in low O2 at 20 C had lower production rates and reached the climacteric peak with lower peak values in comparison with the control fruit (Fig. 3B).

0 1 4 7

Days at 20C after treatment

Fig. 4. Changes in ethanol content (A) and alcohol dehydrogenase activity (B) in banana fruits during post-storage ripening. Fruits were pretreated with 21% O2 (white bars), 2% O2 (shaded bars) or 0.5% O2 (black bars) for 7 days at 20 C prior to triggering off ripening by ethylene. Data represent means S.E. from three replicate samples. Values labeled with the same letter are not different at the 5% level.

3.3. Ethanol contents and ADH activity during post-storage ripening After initiation of ripening with ethylene and transfer to air at 20 C, the ethanol concentrations increased rapidly (Fig. 4A). In spite of higher ethanol concentrations in the fruit stored in low O2 atmospheres at 20 C in green bananas, the build up during poststorage ripening at 20 C was much slower than in control fruit, which reached 210 L L1 on day 7 of ripening, from trace levels on day 0 to 30 L L1 on day 4 at 20 C (Fig. 4A). Similarly, there was an increase in ADH activity with time during the post-storage ripening, but the increased levels of ADH activity persisted in banana fruit from all treatments on return to air at 20 C (Fig. 4B). In contrast to low O2 treatments at 20 C of green banana fruit, the activity was lower in low O2 pretreated fruit than in air-stored fruit, although differences were signicant only for fruit treated under 0.5% O2 at 20 C (Fig. 4B).

3.4. Ester biosynthesis during ripening of bananas after storage under low O2 atmospheres Until the end of the storage, ester production was almost not detectable regardless of O2 levels in the storage atmosphere at 20 C (Fig. 5). Four days after initiating ripening, ester production rapidly increased and the increase continued (Fig. 5). However, production of esters in fruit stored in low O2 atmospheres at 20 C increased slowly in the rst four days and speeded up later (Fig. 5). Ester production and activity of related enzymes during ripening differed according to storage conditions at 20 C. Fruit stored under low

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25

A
a

12

a ab

Isoamyl acetate (g100g-1 FW)

20

b 15

Isobutyl acetate (g100g-1 FW)

10

b
6

10 a a

5 a a a

2 a

bb a a
1 4 7 Days at 20C after treatment

1 4 7 Days at 20C after treatment

35 30

C
a

50

a b b

40
Ethyl acetate (g100g-1 FW)
25 20
15 b 10 5 a

AAT (unitsmg-1protein)

30

20 a a 10 b 0 4 7 1 Days at 20C after treatment bb c

b
0

a a

b b

1 4 7 Days at 20C after treatment

Fig. 5. Changes in isoamyl acetate (A), isobutyl acetate (B) and ethyl acetate (C) contents, and alcohol acetyltransferase activity (D) in banana fruits during post-storage ripening. Fruits were stored in 21% O2 (white bars), 2% O2 (shaded bars) or 0.5% O2 (black bars) for 7 days at 20 C prior to triggering off ripening by ethylene. Data represent means S.E. from three replicate samples. Values labeled with the same letter are not different at the 5% level.

O2 conditions at 20 C showed signicantly lower ester production during ripening (Fig. 5). AAT activity increased gradually with storage time in all treatments, being signicantly lower in the fruit exposed to low O2 pretreatments at 20 C (Fig. 5D). The AAT activity was low before ripening and increased as ripening progressed. 4. Discussion 4.1. Low O2 directly or through ethanol accumulation inhibits triggering of ripening in mature green bananas Wills et al. (1982) showed that exposure of mature green Williams banana fruit to 0.11.0% O2 for 23 days, prior to storage in air, extended the time required for the fruit to ripen. Similarly, Pesis et al. (2001) found that application of 3% O2 to mature green banana fruit for 48 h effectively delayed ripening. It is wellknown that storage in various CA treatments results in a general retardation of metabolism of many fruit (Wills et al., 1982). The retardation of ripening during storage in low O2 treatment at 20 C can be attributed directly to an effect of low O2 itself which reduces respiration rates and ethylene production, and slows ripening metabolism. However, it is also possible that the induced

production of ethanol metabolites delays ripening. Both storage under relatively low O2 conditions or exogenous application of ethanol vapor in mature green banana fruit has been shown to delay ripening and decay (Yi et al., 2006). Therefore, it could be hypothesized that the retardation of fruit ripening by low O2 treatment at 20 C was related to levels of ethanol fermentative metabolism in the mature green banana fruit. Imahori et al. (1998) showed that when storing ethylene treated pre-climacteric bananas in low O2 atmospheres, ethanol accumulated in the fruit and ripening was delayed. Pesis et al. (2001) also showed that compared to 3% O2 , mature green bananas exposed to 1.8% accumulated levels of ethanol, and the fruit maintained a better appearance and a lower decay incidence. Exogenous application of ethanol to fruit inhibits the ripening and extends the storage life of many climacteric and nonclimacteric fruit (Pesis, 2005). Kelly and Saltveit (1988) reported that ethanol application inhibited color development, lycopene production and ripening in tomato fruit. Bai et al. (2011) observed that ethanol vapor treatment inhibited total anthocyanin accumulation and softening of non-climacteric sweet cherry fruit. Bai et al. (2004) applied ethanol to intact apples and Plotto et al. (2006) applied it to mangoes showing that treating the intact fruit with

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ethanol inhibited respiration rates and slowed the deterioration of fresh-cut products. However, external application of ethanol to extend the green life of bananas was not an effective treatment, as ethanol might fail to penetrate the fruit when the banana remained intact (Hewage et al., 1995). In this research, ethanol accumulation occurred only when storage O2 was as low as 0.5%, however, there was no difference to the control when O2 concentration was 2% (Fig. 1). Trends of ADH activities paralleled the ethanol concentration in the pulp of bananas (Fig. 1). Further research is required to distinguish if the low O2 inhibits triggering of ripening caused by ethanol accumulation under anaerobic conditions. 4.2. Triggering of ripening and consequent basic physiological and quality changes in bananas after storage in low O2 atmospheres Yi et al. (2006) showed that exposure of banana fruit to exogenous ethylene could overcome the inhibitory effect of a shorter anoxia treatment on fruit ripening, and suggested that the effect of a reduced O2 atmosphere was only transient. Our results showed that 2% did not affect ADH activity and ethanol content in pulp during storage of the mature green fruit, and the residual effect was revealed after triggering ripening: the climacteric peak and yellowing of surface color were delayed by 23 days in comparison with the control (Figs. 2 and 3). However, sugar content, i.e. starch hydrolyzation, was inhibited only slightly by the low O2 (Fig. 2), indicating differential ripening processes in quality attributes: sweetening (starch hydrolyzation), yellowing of the surface, rmness, respiratory and ethylene production (Figs. 2 and 3). Lower O2 concentrations resulted in stronger inhibition of the ripening on-set (Figs. 2 and 3). During banana ripening, starch degradation and sugar accumulation are the main changes occurring in fruit composition (Li et al., 2009). Jayanty et al. (2002) observed that the carbon conversion peaks at about the same time that respiration reaches its maximum, agreeing with our observations (Figs. 2 and 3). However, there was little difference in SSC between 0.5 and 2% low O2 storage (Fig. 2). Once the ripening of banana fruit is initiated by ethylene application, carbohydrate metabolism of fruit is stimulated, and as a result, the pulp starch is degraded rapidly and sugars accumulate. Similarly, treatment with reduced O2 for short periods before storage retarded the pre-climacteric ripening of bananas by delaying the climacteric peak (Wills et al., 1982; Pesis et al., 2001). Although post-treatment inhibition of ripening was attained after 2 or 3 days of storage in low O2 , presumably, this period was required to effect the suppression of ethylene synthesis (Wills et al., 1982). It was shown earlier that reducing or absorbing ethylene after ethylene treatment could prolong postharvest life of ethylene-treated bananas (Pesis et al., 2005). In various other fruit such as many subtropical fruit, low O2 pretreatment also delays fruit ripening. In cherimoya fruit, as low O2 pretreated fruit ripened normally after being transferred to air storage at 20 C, the time needed to reach an edible condition differed with O2 level and was inversely proportional to O2 concentration during storage (Palma et al., 1993). In mango fruit, low O2 treatment prior to storage slowed fruit softening and sugar accumulation (Burdon et al., 1994). It has been shown that storage of bell pepper fruit held for 5 days at 20 C in 1.5% O2 resulted in post-storage respiratory suppression of CO2 production after transfer to air and a marked reduction in the oxidative capacity of isolated mitochondria (Rahman et al., 1995). 4.3. Ethanol and ester metabolism after ethylene application to initiate ripening It is also suggested that in banana ripening, ethanol increases as part of the processes of ripening and aroma production, and

normal ethanol levels can reach 800 L L1 (Pesis et al., 2001). Increases of alcohols, such as ethanol during maturation and ripening, were associated with increases in other volatiles, as they are one of the precursors of natural aroma compounds (Yang et al., 2011). Additionally, the increase in ethanol appeared in the postclimacteric phase and led to the production of ethyl acetate, which increased dramatically when the banana fruit were over-ripe (Pesis et al., 2001). These compounds could be very important for the aroma prole in banana, although they are usually regarded as the components that contribute of off-avor developing in many fruit when the concentration is high (Kader, 1986; Imahori et al., 2002). The production of total volatile esters in apple fruit was shown to increase dramatically by low O2 treatment, because of induction of high concentrations of acetaldehyde and ethanol during hypoxic conditions (Pesis, 2005). In a previous study, low O2 pretreatments delayed ripening of banana fruit, as shown by a lag in appearance of the CO2 production peak, and by the levels of ethanol (Pesis et al., 2001). These results are in agreement with those in our study. In this study, it was also suggested that low O2 pretreatment delayed the on-set of ripening, as shown by a lag in the appearance of the climacteric peak (Fig. 3) and by the low levels of ethanol (Fig. 4A). This suggests that the respiratory substrates were non-limiting, which may have some bearing on the composition of the aroma volatiles produced by the fruit (Golding et al., 1998). By the time aroma volatiles synthesis was initialized, conversion of starch to sugars would have been 50% complete or more (Jayanty et al., 2002). In banana fruit during ripening, there is a dramatic increase in ethanol levels and ADH activity which contributes to the development of aroma volatiles (Pesis, 2005). ADH is involved in stress tolerance during plant development and fruit storage, but also plays a role in aroma compound biosynthesis during fruit ripening. ADH genes were expressed in a developmentally regulated manner during fruit ripening (Yang et al., 2011). It was reported that fruit-specic expression of two ADH genes is likely to play a key role in the regulation of aroma production in cantaloupe melons. Maximum transcript levels of both genes coincided with the peak of the respiratory climacteric (Whitaker, 2008). Therefore, ADH is one of the regulation points of aroma compound production during fruit ripening and has an impact on avor proles by enhancing alcohol supply for ester production during ripening (Yang et al., 2011). When banana fruit ripen, esters become an important fraction of their aroma prole. Banana fruit mainly produce volatile esters, such as isoamyl acetate and isobutyl acetate, which contribute to the characteristic aroma of the fruit (Macku and Jennings, 1987; Bai et al., 1990; Wendakoon et al., 2006). In general, the amounts of individual volatiles increased continuously to the onset of peel browning, after which they either plateaued or decreased (Macku and Jennings, 1987). Production of isoamyl acetate, and isobutyl acetate increased gradually with storage time in all treatments, being signicantly lower in the fruit with the 0.5% O2 pretreatment at 20 C (Fig. 5A and B). However, 2% O2 pretreatment at 20 C did not reduce this production. The levels of both esters decreased with reducing O2 concentration in pretreatment atmospheres. The processes responsible for generating esters may be affected at control points of volatile biosynthesis pathways, such as due to enhanced respiration. The production of most volatile compounds was significantly increased by ethylene treatment. The factor most limiting ester production during this developmental stage was not the availability of the alcohol precursors, but may be the availability of the precursor to the acid portion of the ester molecule (Jayanty et al., 2002). Production of ethyl acetate increased gradually with storage time in all treatments, being signicantly lower in the fruit exposed to low O2 pretreatments (Fig. 5C). This may reect the abundance of ethanol and acetyl CoA and not larger acid moieties coupled with ethanol. A dramatic increase in ethanol, which may have competed

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Y. Imahori et al. / Postharvest Biology and Technology 77 (2013) 1927 Chervin, C., Speirs, J., Loveys, B., Patterson, B.D., 2000. Inuence of low oxygen storage on aroma compounds of whole pears and crushed pear esh. Postharvest Biology and Technology 19, 279285. Fellman, J.K., Mattinson, D.S., Bostick, B.C., Mattheis, J.P., Patterson, M.E., 1993. Ester biosynthesis in Rome apples subjected to low-oxygen atmospheres. Postharvest Biology and Technology 3, 201214. Golding, J.B., Shearer, D., Wyllie, S.G., McGlasson, W.B., 1998. Application of 1-MCP and propylene to identify ethylene-dependent ripening processes in mature banana fruit. Postharvest Biology and Technology 14, 8798. Hewage, K.S., Wainwright, H., Luo, Y., 1995. Effect of ethanol and acetaldehyde on banana ripening. The Journal of Horticultural Science 70, 5155. Imahori, Y., Kishioka, K., Uemura, K., Yoshioka, H., Ueda, Y., Ishimaru, M., Chachin, K., 2002. Physiological responses and quality attributes of Japanese pear Kosui frujit kept in low oxygen atmospheres. The Journal of Horticultural Science and Biotechnology 77, 677682. Imahori, Y., Kota, M., Ueda, Y., Chachin, K., 1998. Effects of low-oxygen atmospheres on quality and ethanol and acetaldehyde formation of ethylene-treated bananas. Nippon Shokuhin Kagaku Kogaku Kaishi 45, 572576. Imahori, Y., Suzuki, Y., Uemura, K., Kishioka, I., Fujiwara, H., Ueda, Y., Chachin, K., 2004. Physiological and quality responses of Chinese chive leaves to low oxygen atmospheres. Postharvest Biology and Technology 31, 295303. Jayanty, S., Song, J., Rubinstein, N.M., Chong, A., Beaudry, R.M., 2002. Temporal relationship between ester biosynthesis and ripening events in bananas. Journal of the American Society for Horticultural Science 127, 9981005. Kader, A.A., 1986. Biochemical and physiological basis for effects of controlled and modied atmospheres on fruit and vegetables. Food Technology 40, 99104. Kanellis, A.K., Solomos, T., Mattoo, A.K., 1989. Changes in sugars, enzymic activities and acid phosphatase isoenzyme proles of bananas ripened in air or stored in 2.5% O2 with and without ethylene. Plant Physiology 90, 251258. Ke, D., Zhou, L., Kader, A.A., 1994. Mode of oxygen and carbon dioxide action on strawberry ester biosynthesis. Journal of the American Society for Horticultural Science 119, 971975. Kelly, M.O., Saltveit, M.E., 1988. Effect of endogenously synthesized and exogenously applied ethanol on tomato fruit ripening. Plant Physiol. 88, 143147. Klieber, A., Bagnato, N., Barrett, R., Sedgley, M., 2002. Effect of post-ripening nitrogen atmosphere storage on banana shelf life, visual appearance and aroma. Postharvest Biology and Technology 25, 1524. Lara, I., Mir, R.M., Fuentes, T., Sayez, G., Graell, J., Lpez, M.L., 2003. Biosynthesis of volatile aroma compounds in pear fruit stored under long-term controlled-atmosphere conditions. Postharvest Biology and Technology 29, 2939. Li, W., Shao, Y., Chen, W., Jia, W., 2009. The effects of harvest maturity on storage quality and sucrose-metabolizimg enzymes during banana ripening. Food and Bioprocess Technology 4, 12731280. Macku, C., Jennings, W.G., 1987. Production of volatiles by ripening bananas. Journal of Agricultural and Food Chemistry 35, 845848. Palma, T., Stanley, D.W., Aguilera, J.M., Zoffoli, J.P., 1993. Respiratory behavior of cherimoya (Annona cherimola Mill.) under controlled atmospheres. HortScience 28, 647549. Palomer, X., Roig-Villanova, I., Grima-Calvo, D., Vendrell, M., 2005. Effects of nitrous oxide (N2 O) treatment on the postharvest ripening of banana fruit. Postharvest Biology and Technology 36, 167175. Pesis, E., 2005. The role of the anaerobic metabolites, acetaldehyde and ethanol, in fruit ripening, enhancement of fruit quality and fruit deterioration. Postharvest Biology and Technology 37, 119. Pesis, E., Copel, A., Ben-Arie, R., Feygenberg, O., Aharoni, Y., 2001. Low-oxygen treatment for inhibition of decay and ripening in organic bananas. The Journal of Horticultural Science and Biotechnology 76, 648652. Pesis, E., Arie, R.B., Feygenberg, O., Villamizar, F., 2005. Ripening of ethylenepretreated bananas is retarded using modied atmosphere and vacuum packaging. HortScience 40, 726731. Plotto, A., Bai, J., Narciso, J.A., Brecht, J.K., Baldwin, E.A., 2006. Ethanol vapor prior to processing extends fresh-cut mango shelf-life by decreasing spoilage, but does not always delay ripening. Postharvest Biology and Technology 39, 134145. Rahman, A.S.A., Huber, D.J., Brecht, J.K., 1995. Low-O2 -induced post-storage suppression of bell pepper fruit respiration and mitochondrial oxidative activity. Journal of the American Society for Horticultural Science 120, 10451049. Sugiura, A., Tomana, T., 1983. Relationship of ethanol production by seeds of different types of Japanese persimmons and their tannin content. HortScience 18, 319321. Thompson, A.K., 1998. Banana, Musa, Controlled Atmosphere Storage of Fruits and Vegetables. CAB International, Bedford. Wendakoon, S.K., Ueda, Y., Imahori, Y., Ishimaru, M., 2006. Effect of short-term anaerobic conditions on the production of volatiles, activity of alcohol acetyltransferase and other quality traits of ripened bananas. Journal of the Science of Food and Agriculture 86, 14751480. Whitaker, B.D., 2008. Postharvest avor deployment and degradation in fruits and vegetables. In: Brckner, B., Wyllie, S.G. (Eds.), Fruit and Vegetable Flavour Recent Advances and Future Prospects. Woodhead Publishing, Cambridge, pp. 103131. Wills, R.B.H., Pitakserikul, S., Scott, K.J., 1982. Effects of pre-storage in low oxygen or high carbon dioxide concentrations on delaying the ripening of bananas. Australian Journal of Agricultural Research 33, 10291036.

with other alcohols for acetyl CoA, leaded to accumulation of ethyl acetate (Lara et al., 2003). Ethyl acetate is usually regarded as the component that contributes of off-avors developing in many fruit (Imahori et al., 2002). However, this compound increased continuously even into late senescence and could be very important for the volatile prole in banana fruit (Macku and Jennings, 1987). It was suggested that the dramatic increase in ethanol, which may have competed with other alcohols for acetyl CoA, was the major driving force for ethyl acetate accumulation (Ke et al., 1994). Such increased ethanol levels and/or ADH activity in fruit stored under low-O2 atmospheres may be retained during subsequent ripening in air, and in some cases may lead to off-avors (Lara et al., 2003). The basal expression of AAT genes was found even in unripe green banana fruit and increased as ripening proceeded (Jayanty et al., 2002). As a general rule, expression of AAT genes in climacteric fruit is ethylene-dependent, and consequently perception and responsiveness are required for volatile ester production (Whitaker, 2008). Thus, the production of most volatile compounds was signicantly increased by ethylene treatment. The nal enzymatic step of esterication of alcohols and acyl-CoA by banana AAT is an important transcriptional regulation point of ethylene for aroma production in banana fruit (Yang et al., 2011). As ripeness advanced, AAT activity, rather than substrate availability, may have exerted the greater inuence in ester formation as fruit became suitable for consumption (Jayanty et al., 2002). Low O2 pretreatments are both effective means of maintains overall quality and extending storage life, resulting in a decrease in production of esters.

5. Conclusions Storage treatment of banana fruit with low O2 atmospheres at 20 C inhibited ripening and decay in banana fruit, and extended the time required for the fruit to ripen. Our results indicate that it would be necessary to keep banana fruit at 20 C because of a lack of refrigeration in the distribution system, or in a modied atmosphere package designed to develop an optimum atmosphere during retail display, and suggest a potential for using low O2 atmospheres at higher temperatures to help maintain fruit quality. There is a high potential for low O2 treatment as a simple and inexpensive postharvest technology.

Acknowledgment This work was supported in part by a Grant-in-Aid for Scientic Research (No. 20580037) from Japanese Society for the Promotion Science.

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