Postharvest Biology and Technology 78 (2013) 1623

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Postharvest Biology and Technology

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Effect of nitric oxide (NO) and associated control treatments on the metabolism of fresh-cut apple slices in relation to development of surface browning
Roksana Huque a , R.B.H. Wills a, , Penta Pristijono a,b , J.B. Golding a,b
a b

School of Environmental and Life Sciences, University of Newcastle, Ourimbah, NSW 2258, Australia NSW Department of Primary Industries, Ourimbah, NSW 2258, Australia

a r t i c l e

i n f o

a b s t r a c t
Surface browning is an important cause of deterioration of fresh-cut apples during postharvest handling. Granny Smith apple slices treated with NO gas (10 L/L) and the NO donor compound 2,2 -(hydroxynitrosohydrazino)-bisethanamine (diethylenetriamine nitric oxide (DETANO) (10 mg/L) dissolved in phosphate buffer (pH 6.5) showed delayed development of surface browning during storage at 5 C and also resulted in a lower level of total phenols, inhibition of PPO activity, reduced ion leakage and reduced rate of respiration but had no signicant effect on ethylene production or lipid peroxide level as measured by malondialdehyde (MDA) and hydrogen peroxide levels. The two control treatments of phosphate buffer (pH 6.5) and water dips also had signicant effects compared to untreated slices. The relative effectiveness of treatments in extending postharvest life and reducing total phenols, PPO activity, ion leakage and respiration was DETANO > NO gas > phosphate buffer > water > untreated. Apple slices dipped in chlorogenic acid dissolved in water showed surface browning soon after application but dipping in DETANO solution negated the effect of chlorogenic acid whether applied before or after dipping in chlorogenic acid solution while the buffer and NO gas were also effective. It is suggested that an increase in phenols occurs on the apple surface soon after cutting, possibly as a defensive mechanism of the apple to limit damage to surface cells. The effectiveness of the applied treatments to inhibit development of surface browning may relate to their ability to minimize the level of phenols active on the cut surface possibly in conjunction with a reduced PPO activity. 2012 Elsevier B.V. All rights reserved.

Article history: Received 28 October 2012 Accepted 15 December 2012 Keywords: Apple (Malus x domestica Borkh) Fresh-cut slices Surface browning Nitric oxide Phenols

1. Introduction Apple slices are a popular component of the value-added consumer-ready market for minimally processed fresh fruit and vegetables. However, they are more perishable than intact produce due to adverse impacts arising from physical stress imposed during preparation, with a major limitation to postharvest life being the appearance of browning on the cut surface (Abbott et al., 2004). Browning of horticultural produce can be initiated by both enzymic and non-enzymic pathways. Enzymic browning is considered to occur through to the oxidation of ortho-phenols to quinones by the action of enzyme systems such as polyphenolic oxidase (PPO), and which then polymerize to brown pigments (Milani and Hamedi, 2005). PPO is generally associated with the plastid, and phenolic substrates are located in the vacuole but cellular and intracellular disruption allows the substrates to mix and hence react to produce browning (Landrigan et al., 1996). An association of PPO activity in apples with browning has been reported by

Corresponding author. Tel.: +61 2 94994437; fax: +61 2 94994437. E-mail address: Ron.Wills@newcastle.edu.au (R.B.H. Wills). 0925-5214/$ see front matter 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.postharvbio.2012.12.006

many authors (e.g. Nicolas et al., 1994; Hu et al., 2007; Toivonen and Brummell, 2008). Non-enzymic browning can occur through various sequences including through metal ion interaction with phenols (Vmos-Vigyz, 1981; Robards et al., 1999). Less work has been reported on a role for non-enzymic reactions in apple browning, although Nicoli et al. (2000) studied the interaction of a catechin model system with apple derivatives and found both enzymic and chemical oxidation of catechin was associated with the development of browning. A range of chemical agents are able to inhibit browning. Sulphites are very effective anti-browning agents but are banned in most countries on fresh fruit and vegetables due to potential harmful health effects (Iyengar and McEvily, 1992). Ascorbic acid, as a reducing agent, and citric acid, as an acidulant, alone or in combination dips have been widely reported as anti-browning agents for fresh-cut fruit and vegetables including apples (Vamos-Vigyazo, 1995; Son et al., 2001). Nitric oxide (NO) is a small, highly diffusible free radical that initially attracted attention as an environmental pollutant but has been shown to affect numerous biological processes in animals. Postharvest studies with NO have found short term fumigation with NO gas extended the storage life of a range of horticultural produce by inhibiting ripening or senescence (Leshem et al., 1998;

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17

Sozzi et al., 2003; Flores et al., 2008; Zhu et al., 2009; Zaharah and Singh, 2011). NO can also be utilized in biological systems with donor compounds that degrade quantitatively under controlled conditions to release NO (Hrabie et al., 1993; Hou et al., 1999). Bowyer et al. (2003) reported postharvest dipping in a solution of 2,2 -(hydroxynitrosohydrazino)-bisethanamine (diethylenetriamine/nitric oxide, DETANO) increased the vase life of carnation owers while dipping in sodium nitroprusside (SNP) inhibited internal browning in intact longan and plum fruit (Duan et al., 2007; Zhang et al., 2008). NO applied as a gas and as a DETANO dip has also been shown delay the browning of fresh-cut apple slices (Pristijono et al., 2006, 2008). They found the optimal treatment to delay browning was dipping slices in 10 mg/L DETANO in pH 6.5 phosphate buffer but the buffer solution itself also gave a delay in browning over slices dipped in water which in turn showed delayed browning over untreated slices. Fumigation with 10 L/L NO gas showed delayed browning over its control treatment of untreated but was not as effective as the DETANO treatment. Thus both NO treatments and the three control treatments showed differing responses to the development of surface browning. In order to better understand how NO inhibited development of surface browning, this study examined the metabolism of Granny Smith apple slices that had been exposed to NO gas and DETANO solution before storage at 5 C. Apple slices were analysed for respiration, ethylene production, total phenol content, PPO activity, ion leakage and lipid peroxidation which are factors that have been linked to either browning development or NO metabolism in plants. Small studies were also made of the effect on browning development of (i) dipping in chlorogenic acid, the major phenol in apples (Lee et al., 2003), solution and its interaction with DETANO, (ii) other forms of NO, namely sodium nitroprusside (SNP) and Pilotys acid, and (iii) preparing apple slices with a metal and a ceramic knife. 2. Materials and methods 2.1. Produce Granny Smith apples (Malus x domestica Borkh) of similar size, shape and color were harvested in three seasons from commercial orchards in Orange, NSW, transported to the laboratory and stored in air 0 C for up to 6 months. During this period, apples were periodically selected for inclusion in a series of experiments. The required number of apples in each experiment were removed to 20 C and after 2 h each apple was hand-cut longitudinally into six unpeeled slices using a sharp stainless steel knife and slices were placed into 4 L containers with a sealable lid. Each container contained six slices, each from a different apple with a total weight of 100 10 g, and this comprised a treatment unit. Each experiment was repeated at least three times on different occasions and there were three treatment units in each replicate. 2.2. Treatment with NO A unit of fresh cut apple slices was sealed in a 4 L container and NO gas (BOC Gases, Sydney) was applied from a syringe through an injection port in the lid of the container to give a concentration of 10 L/L NO. After application of NO gas for 2 h, the lids of the containers were opened to atmospheric air and the lid replaced but an injection port (4 mm diameter) in the lid was opened to prevent carbon dioxide accumulation inside the container. A beaker with water was provided in each container to maintain a high humidity. All treated and control containers were then stored at 5 C. DETANO (supplied by Dr. M.C. Bowyer, University of Newcastle as a powder) was dissolved in 0.01 M phosphate buffer at pH

6.5 (1.4 g sodium dihydrogen phosphate was dissolved in approximately 900 mL distilled water, 8.05 g sodium chloride was added and the volume made up to 1 L with distilled water) to give a concentration of 10 mg/L DETANO. A unit of fresh-cut slices was placed in a stainless steel mesh strainer and dipped into the DETANO solution at 20 C for 5 min. After draining, the slices were allowed to dry for 25 min before placing in a 4 L container and storied at 5 C. The lid of the container had an open port and a beaker of water as for NO gas. SNP (Na2 [Fe(CN)5 NO]2H2 O) (Ajax Chemicals, Victoria) and Pilotys acid (N-hydroxybenzenesulfonamide) (Cayman Chemical, Ann Arbor, MI) were stored, respectively in the dark at 20 C at 18 C. The desired concentration of SNP and Pilotys acid were obtained by dissolving in water. Apple slices were treated as for DETANO. 2.3. Assessment of postharvest life The browning of apple slices was assessed on the color of the cut surface using a colorimeter (Minolta CR-300, Osaka) to measure the L value (lightness). Six readings were taken from cortex tissue of each apple slice, three from each side of a slice with the measuring head placed along the longitudinal axis on the midpoint between the core and skin at 1/3, 1/2, and 2/3 from the calyx. The colorimeter was calibrated with a white tile plate (Calibration Plate CR-A43). The time taken for the L value of each apple slice to decline to 75.6 was taken as the postharvest life (Pristijono et al., 2006). The mean postharvest life of all six slices in a treatment unit was expressed as the postharvest life for the treatment unit. 2.4. Respiration and ethylene measurement Fresh cut slices of Granny Smith apples were weighed before treatment. A treated unit of produce was placed into a sealed 800 mL plastic container at various days after treatment. After 4 h in the sealed container, a gas sample (1 mL) was collected in a syringe. The concentration of carbon dioxide in the gas sample was determined by thermal conductivity gas chromatography (Gow-Mac 580, Bridgewater, NJ) with a stainless steel column (60 cm 1 mm i.d.) of Haysep N (80100 mesh) (Altech, Sydney). The gas chromatograph response was calibrated with a standard gas mixture containing 5% CO2 in nitrogen (BOC Gases, Sydney). Respiration was calculated as mg CO2 /kg h. Ethylene was determined with a gas sample (1 mL) injected into a ame ionization gas chromatograph (Varian Star CX-3400, Walnut Creek, CA) tted with a stainless steel column (2 m 3.2 mm o.d. 2.2 mm i.d.) packed with Porapak Q (80100 mesh) (Altech, Sydney). The ethylene production rate was calculated as L C2 H4 /kg h. 2.5. Biochemical and physical assessments For biochemical assessment, ve apple slices from ve different apples were combined to provide a treatment unit. Treated units of ve apple slices were stored in a 4 L container at 5 C. One apple slice was removed from each container at various times up to 8 days of storage to measure a range of biochemical factors. Each factor was assessed on a different batch of fruit. 2.5.1. Total phenol content Total phenols was determined according to the FolinCiocalteu (FC) method (Singleton and Rossi, 1965). For each apple slice, a section of tissue (1 g) was cut from the outside of the core area to just below the skin at a point halfway from the calyx to core and the sample was immediately placed at 18 C for 30 min. The frozen tissue was homogenized at 4 C with cold methanol, centrifuged, ltered and the solution diluted with distilled water. A

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R. Huque et al. / Postharvest Biology and Technology 78 (2013) 1623

sample (0.5 mL) was added FC reagent and sodium carbonate solution and the absorbance at 765 nm was determined. Gallic acid was the calibration standard and the data was expressed as mg/L gallic acid equivalents. 2.5.2. PPO activity Frozen apple tissue (1 g) obtained as described above was homogenized in a mortar and pestle with a cold solution (4 C) containing 100 mM sodium phosphate buffer (pH 7.0) and 0.25 g polyvinylpolypyrrolidone (10 mL). The homogenate was centrifuged at 17,000 g for 15 min at 4 C and extracts were ltered through three layers of cheese cloth. Enzyme activity in the supernatant was determined according to the method described by Yingsanga et al. (2008). Supernatant (1 mL) combined with sodium phosphate buffer and pyrocatechol and one unit of PPO activity was measured spectrophotometrically as a 0.01 unit change in absorbance per min at 410 nm at 25 C. 2.5.3. Lipid peroxidation Malondialdehyde (MDA) was considered to be a suitable biomarker for lipid peroxidation caused by reactive oxygen species (ROS) which are purported as a major cause of membrane deterioration in plant tissues (Mittler, 2002; Zheng et al., 2007). MDA was measured by the method described by Heath and Packer (1968). Fresh apple tissue (1 g) was homogenized in a mortar and pestle with a solution containing thiobarbituric acid and trichloroacetic acid then incubated at 90 C, cooled to room temperature and centrifuged. The absorbance of the supernatant was determined at 560 and 600 nm and using the MDA molar extinction coefcient (155 mM/cm), the content of MDA ( mole) was calculated from (A560 A600 )/155. Hydrogen peroxide (H2 O2 ) was used as an alternative biomarker for oxidative stress generated by reactive oxygen species (ROS) in plant tissues during normal metabolism (Lu et al., 2009). H2 O2 was determined by the method of Sun et al. (2010). A section of fresh apple tissue (2 g) was homogenized with cold acetone then centrifuged and an extract (1 mL) was mixed with titanium dioxide and ammonia solutions and centrifuged. The precipitate was dissolved in H2 SO4 and the absorbance at 415 nm recorded. H2 O2 was calculated using an extinction coefcient 0.28 mol/cm (Hung and Kao, 2007). 2.5.4. Ion leakage Ion leakage from cells was measured using the method described by Song et al. (2006). A 0.5 cm thick section of fresh apple esh (2 g) was placed in a beaker containing de-ionized water and incubated for 2 h at 25 C. The conductivity of the solution was measured with a conductivity meter (Model 4071, Jenway, Staffordshire) as the initial reading and again after boiling for 15 min and cooling at room temperature. The percentage of the ion leakage was calculated. 2.6. Treatment with chlorogenic acid The effect of chlorogenic acid on the development of browning of fresh-cut apple slices was examined in conjunction with dipping in DETANO solution. Apple slices were dipped in DETANO solution, in phosphate buffer only or in water for 5 min and allowed to drain. Five minutes after completion of the treatment, slices were dipped into different concentrations of chlorogenic acid (SigmaAldrich, Sydney) dissolved in water for 10 s. In another experiment, the order was reversed with apple slices rst dipped into chlorogenic acid solution. Treated slices were stored in a 4 L container at 5 C as previously stated. The L value of each slice was measured daily and

the mean postharvest life of all six slices in a treatment unit was determined as previously stated. 2.7. Statistical analysis Statistical procedures were performed using SPSS for Microsoft version 18.0 software package (SPSS Chicago, IL). To determine signicant difference between treatments least signicant difference (LSD) at P = 0.05 was used. Linear regression equations were calculated to determine the relationship between postharvest life and an applied treatment. 3. Results 3.1. Postharvest life A preliminary experiment conducted soon after the Season 1 harvest conrmed the ndings obtained by Pristijono et al. (2008) that DETANO was the most effective treatment in inhibiting browning while apples fumigated with NO gas had a longer postharvest life than those dipped in water and untreated (Table 1). A phosphate buffer dip was not included in the experiment. A subsequent experiment conducted on slices that had been stored for 6 months and including a phosphate buffer dip showed signicant differences between treatments with the order of effectiveness in extending postharvest life being DETANO > NO gas = phosphate buffer > water = untreated. Comparison of the postharvest life obtained by DETANO and NO gas in the preliminary study showed a similar extension in postharvest life. Thus, NO was similarly effective on freshly harvested fruit and fruit stored for 6 months. Fruit obtained in Season 2 were evaluated after 3 months storage and each treatment had a similar postharvest life as in the previous season, but with a signicant difference between all treatments for DETANO > NO gas > phosphate buffer > water > untreated. Table 1 also shows the mean values for the seven replicates evaluated in the Season 1, 6 months and the Season 2 experiments, which also showed a signicant difference between all treatments. 3.2. Physiological and biochemical parameters 3.2.1. Respiration and ethylene production A preliminary study in Season 1 examined changes in respiration and ethylene over 4 days but without inclusion of a phosphate buffer treatment. NO gas and DETANO reduced respiration but ethylene showed no signicant effect of NO treatment (data not given). The main trial in both Season 1 and Season 2 over 8 days therefore only examined respiration, but a phosphate buffer control was also included. The changes due to the applied treatments and over the storage period were consistent in both seasons so the combined data are presented in Table 2. There was a signicant difference (P < 0.001) in respiration between treatments with DETANO < NO gas < phosphate buffer < water < untreated with the effect of the treatments evident from the rst analytical time of 2 days. Respiration also signicantly (P < 0.001) increased in all treatments during storage. 3.2.2. Total phenol content and PPO activity Changes in each biochemical factor examined was assessed on a different batch of apple slices with measurements taken on fruit from Season 1 and Season 2. Changes in total phenols as determined by the FolinCiocalteu (FC) method and in PPO activity due to applied treatments were consistent in fruit from both seasons and hence the combined data for both seasons are presented in Table 2.

R. Huque et al. / Postharvest Biology and Technology 78 (2013) 1623 Table 1 Effect of NO treatments on the postharvest life at 5 C of Granny Smith apple slices. Treatment Season Storage time Untreated Water dip Phosphate buffer dip 10 L/L NO gas 10 mg/L DETANO dip LSD No. of replicates Postharvest life (days)a Season 1 0 months 4.5 4.8 6.5 9.0 1.55 3 6 months 3.5 3.9 5.5 6.9 8.6 1.64 3 Season 2 3 months 3.4 4.2 6.1 7.2 8.6 0.77 4 3.4a 4.1b 5.8c 7.1d 8.6e 0.62 7

19

Meanb

Each replicate contained 3 treatment units. In this and all subsequent tables, mean values with different superscript letters are signicantly different at P = 0.05 and LSD values are at P = 0.05. a Postharvest life was the time taken for the HunterLab L value to fall to 75.6. b Mean values are of data from Season 1, 6 months and Season 2.

The total phenols were signicantly different (P < 0.001) between treatments with the mean levels throughout storage in slices treated with DETANO < NO gas < phosphate buffer < water < untreated. Storage period also had a signicant effect (P < 0.001), with total phenol content of all treatments increasing during storage. Examination of the effect of treatments on PPO activity was incomplete as the phosphate buffer was not included in Season 1. Analysis of the data for the other four treatments over both seasons showed that PPO activity was signicantly different (P < 0.001) between treatments with DETANO < NO gas < water < untreated. Storage period had a signicant effect (P < 0.001) on PPO activity in all treatments, increasing during storage.

3.2.3. Ion leakage Ion leakage showed a signicant difference (P < 0.001) between treatments (Table 2). A signicantly lower ion leakage was found in NO gas and DETANO-treated apple slices compared to those of the respective control slices of buffer and water, but the difference between DETANO and NO gas was not signicant at P = 0.05. Ion leakage of buffer and water-treated slices were not signicantly different but both were signicantly lower than untreated slices. 3.2.4. Lipid peroxidation For the fruit in Season 1, MDA was used as the biomarker for lipid peroxidation but the data showed no signicant difference between treatments with slices exhibiting about 2.0 mol MDA/g (data not

Table 2 Effect of NO treatments on respiration rate, total phenols, PPO activity and ion leakage of Granny Smith apple slices during storage at 5 C. Treatment Amount during storage 2 Respiration rate (mg CO2 /kg h) Untreated Water Buffer NO gas DETANO LSD 24.8 22.4 20.2 17.8 14.8 2.24 4 27.2 25.8 22.6 20.6 18.2 6 31.2 28.4 26.0 23.0 21.0 8 days 34.6 31.6 29.0 26.2 25.0 Mean 29.4e 27.0d 24.4c 22.0b 19.8 1.00

Total phenols (gallic acid equivalent in mg/L) 161.2 Untreated 149.0 Water 139.5 Buffer 128.0 NO gas 121.4 DETANO 16.32 LSD PPO activity ( Abs/min) Untreated Water Buffer NO gas DETANO LSD Ion leakage (%) Untreated Water Buffer NO gas DETANO LSD

171.1 158.4 151.1 139.8 132.6

181.7 168.6 159.8 149.2 142.4

191.3 177.1 168.3 158.2 151.0

176.3e 163.2d 154.7c 143.8b 136.8a 7.04

0.051 0.047 0.041 0.038 0.003

0.058 0.053 0.048 0.043

0.065 0.060 0.054 0.051

0.072 0.067 0.061 0.056

0.062d 0.057c 0.051b 0.047a 0.002

58.6 55.5 53.6 51.8 49.5 6.17

54.0 51.7 49.6 47.0 44.6

58.7 56.2 54.2 51.8 49.3

62.7 59.6 57.5 54.6 51.8

58.5d 55.7c 53.7bc 51.3ab 48.8a 2.71

Values are the mean of 6 replicates with 3 treatment units in each replicate.

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R. Huque et al. / Postharvest Biology and Technology 78 (2013) 1623 Table 3 Postharvest life at 5 C of Granny Smith apple slices dipped in DETANO, phosphate buffer and water before or after dipping in chlorogenic acid solution. Expt. 1 Chlorogenic acid conc. (g/100 g) 0.1 Treatment Untreated Water Chlorogenic acid Water + chlorogenic acid Phosphate buffer + chlorogenic acid DETANO + chlorogenic acid LSD Untreated Water Chlorogenic acid Chlorogenic acid + water Chlorogenic acid + phosphate buffer Chlorogenic acid + DETANO LSD Untreated Water Chlorogenic acid Water + chlorogenic acid Phosphate buffer + chlorogenic acid DETANO + chlorogenic acid LSD Chlorogenic acid Water + chlorogenic acid Phosphate buffer Phosphate buffer + chlorogenic acid DETANO DETANO + chlorogenic acid LSD Postharvest life (days) 3.8 4.5 <1 ha <1 ha 2.3

given). In case the lack of an effect was specic to MDA, hydrogen peroxide was used in Season 2 as the biomarker for lipid peroxidation, but again no signicant difference between treatments was found with slices showing about 0.7 mol H2 O2 /g (data not given). 3.3. Relationship between fruit parameters and postharvest life From Table 2 it can be seen that the effect of the treatments on respiration, total phenol content, PPO activity and ion leakage was evident at the rst analysis time of 2 days after treatment and the magnitude of differences between treatments did not markedly change on further storage. Thus, the mean values for each treatment given in Table 2 can represent the comparative level of each factor. The relationships between the mean values for respiration, total phenols, PPO activity and ion leakage and browning as expressed by the postharvest life (as given by the mean values in Table 1) were examined by linear regression analysis. The data in Fig. 1 show that there was a signicant inverse linear relationship with a greater postharvest life associated with a lower rate of respiration (P < 0.001), total phenols (P < 0.001), PPO activity (P < 0.01) and ion leakage (P < 0.05). 3.4. Effect of added chlorogenic acid on postharvest life

2.8 2.31 4.4 4.5 <1 ha <1 ha 2.8 3.9 1.89 4.2ab 4.9b 2 ha 2 ha 3.3a

0.1

0.01

The effect of dipping apple slices in aqueous solutions containing chlorogenic acid on the development of browning of fresh-cut apple slices was examined in conjunction with dipping in DETANO in phosphate buffer. Two sets of experiments were conducted with 0.1% chlorogenic acid. In the rst experiment, apple slices were dipped in DETANO, phosphate buffer and water and after 5 min were then dipped into 0.1% chlorogenic acid solution while in the second study, the dipping order was reversed. The data in Table 3 show that in both experiments, browning developed within 1 h in slices dipped in chlorogenic acid or in water plus chlorogenic acid compared to about 4 days for water-dipped or untreated slices. However, dipping in DETANO solution and the buffer negated much of the effect of chlorogenic acid with no signicant difference with water and untreated slices. There was also no signicant difference whether the treatments were applied before or after dipping in chlorogenic acid solution. All further experiments were conducted with the chlorogenic acid dip applied as the second dip. Application of 0.01% chlorogenic acid resulted in a slightly longer postharvest life of about 2 h for chlorogenic acid-treated slices (Experiment 3, Table 3). Prior application of a DETANO dip negated the effect of chlorogenic acid as evidenced by a postharvest life of 4.4 days which was not significantly different to water-dipped and untreated slices which had no added chlorogenic acid. Dipping slices in buffer also negated most of the effect of chlorogenic acid but the postharvest life was signicantly lower than for the DETANO dip. Application of 0.001% chlorogenic acid resulted in the postharvest life of slices being about 1.5 days (Experiment 4, Table 3). The addition of a DETANO dip largely negated the effect of chlorogenic acid although the postharvest life of the DETANO-only dip was still signicantly greater (P < 0.05) than the combined treatment. However, the buffer dip fully negated the effect of the added chlorogenic acid with both treatments not signicantly different. 3.5. Effect of SNP and Pilotys acid on postharvest life Granny Smith apple slices dipped in SNP dissolved in water showed a signicant increase in postharvest life through delayed onset of surface browning with dipping in 500 mg/L SNP resulting in the longest postharvest life (Table 4). In a separate experiment, apple slices dipped in Pilotys acid dissolved in water also showed a signicantly longer postharvest life with dipping in 100 mg/L

4.4b 1.15 1.4a 1.7a 7.0b 6.5b

0.001

8.7c 7.3b 1.38

Values in each experiment are the mean of 3 replicates with 3 treatments units in each replicate. a Treatment was not included in statistical analysis.

Table 4 Postharvest life at 5 C of Granny Smith apple slices dipped in SNP and Pilotys acid in water for 5 min at 20 C. Dip conc. (mg/L) Postharvest life (days) SNP Untreated Water 10 50 100 500 750 1000 LSD 3.5 4.4 5.5 6.3 7.1 7.9
a

Pilotys acid 3.9 5.0 6.8 5.2 7.5 6.0 5.3

a

0.75

0.95

Values are the mean of 3 replicates with 3 treatments units in each replicate. a Not assessed due to esh softening.

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20

21

Respiration (ml CO2 kg -1 hr -1 )

Table 5 Postharvest life at 5 C of Granny Smith apple slices treated at 20 C with the optimum concentration of different forms of NO. Treatment Water for 5 min 10 L/L NO gas for 1 h 100 mg/L Pilotys acid for 5 min 500 mg/L SNP for 5 min 10 mg/L DETANO for 5 min LSD Postharvest life (days) 4.3a 6.1b 6.3b 7.6c 9.1d 1.23

15

10

y = 17.4 - 0.9x
5

0 200

Values are the mean of 3 replicates with 3 treatments units in each replicate.
2 4 6 8 10

Total phenols (Gallic acid, ppm)

3.6. Effect of cutting slices with a metal or ceramic knife on postharvest life A study examined the development of browning of apples slices that had been cut from the whole fruit with a stainless steel or a ceramic knife then dipped or not dipped in water at 20 C with all slices stored at 5 C. The postharvest life of apple slices cut by the metal and ceramic knives then dipped in water (4.8 and 5.0 days, respectively) had a signicantly longer postharvest life (P < 0.001) than those not dipped in water (3.3 and 3.4 days, respectively). There was, however, no signicant difference in postharvest life between the metal and ceramic knife-cut apple slices whether dipped or not dipped in water. 4. Discussion

180 160 140

y = 192 -7.2x
120 100

10

0.08

PPO activity (Abs/min)

0.06

0.04

y = 0.06 - 0.002x

0.02 60

10

Ion leakage (%)

55

50

y = 62.6 -1.7x
45

40

10

Postharvest life (days)

Fig. 1. Relationship between postharvest life and mean respiration, total phenols, PPO activity and ion leakage of Granny Smith apple slices during storage at 5 C. Each point is the mean of 6 replicates with 3 units per replicate.

resulting in the longest postharvest life. Apple slices treated with 750 mg/L SNP and 1000 mg/L Pilotys acid caused damage to slices by rapidly softening the esh to an unacceptable level. Comparison was made of the effect of the optimum concentration of SNP and Pilotys acid dissolved in water with DETANO in phosphate buffer and fumigation with NO gas to inhibit browning. The results in Table 5 show that all forms of NO extended the postharvest life of apple slices over water but 10 mg/L DETANO and 500 mg/L SNP solutions were the most effective treatments with DETANO signicantly more effective than SNP. The postharvest life of apple slices fumigated with 10 L/L NO gas and dipped in 100 mg/L Pilotys acid solution were not signicantly different.

The inhibition of browning, as reected in extension of postharvest life, of Granny Smith apple slices with NO and the associated control treatments as shown by Pristijono et al. (2006, 2008) has been shown to be quantitatively associated with a decrease in respiration rate, phenol content, PPO activity and ion leakage but was not correlated with ethylene production or lipid peroxidation. The reduced the rate of respiration in apple slices suggests that NO has an anti-senescent action which could be a general reduction in the rate of cellular metabolism. This is consistent with Millar and Day (1996) and Zottini et al. (2002) who reported that NO affects the function of mitochondria in plant cells and reduces cell respiration by inhibiting the cytochrome pathway. The lack of a signicant effect of NO on ethylene production was surprising as the mode of action of NO is often attributed to an antagonistic effect against ethylene (Leshem, 2000). This lack of effect of NO is probably due to the apples being post-climacteric and thus having a substantial production of ethylene. Nevertheless, the data tend to suggest that ethylene is either not a direct causative factor in surface browning of apples or NO can inhibit browning through other modes of action. The reduction in PPO activity due to NO is consistent with the role for PPO activity in enzymic browning that has been extensively reported for intact and fresh-cut fruit and vegetables. It is generally considered that PPO catalyses the oxidation of phenolic compounds to quinones which then condense to form brown polymers (Milani and Hamedi, 2005). The concomitant reduction of total phenols suggests that inhibition of browning could be due to a lower level of phenol available to be oxidized in conjunction with reduced PPO activity. The reduced rate of ion leakage found in NO-treated slices is indicative of NO assisting in maintaining membrane integrity and thereby reducing the rate of electrolyte leakage. It is possible that this decreases the release of browning precursors such as phenols from cells to the surface of the cut fruit. The lack of a signicant effect of NO on MDA or hydrogen peroxide in apple slices would imply that NO has no effect in mitigating any oxidative damage on the surface of apple slices caused by reactive oxygen species (ROS). NO has previously been

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reported to reduce the MDA content of intact longan (Duan et al., 2007) and kiwifruit (Zhang et al., 2007; Zhu et al., 2008) and hydrogen peroxide in intact kiwifruit (Zhang et al., 2007). If lipid peroxidation is involved in the browning of apple slices, browning could be inhibited by NO affecting other some aspect of apple metabolism. The effect of the NO treatments and the three control treatments (phosphate buffer, water and untreated) in showing an inverse linear relationship between respiration, total phenols, PPO activity and ion leakage with postharvest life suggests rstly, that, whatever the metabolic sequence induced by cutting that leads to browning, all treatments including a water-dip, were having a similar affect on inhibiting that pathway but with different levels of effectiveness. Since the differences between treatments were evident by 2 days after application, the action of cutting would seem to have triggered induction of the browning sequence and that this induction was inhibited to varying extents by the dipping treatments and NO gas fumigation. An obvious consideration is that the metal knife used to cut the slices could have left traces of metal which catalysed some enzyme sequence, but the lack of any difference in browning between the ceramic and metal knives suggests that this does not occur. Considering a scenario in which surface browning is due to leakage of substrates from cells to the cut surface that react to produce browning, and where phenol content and PPO activity are both implicated in the browning sequence, an obvious question is are either or both phenols and PPO the key metabolites involved in the slower development of browning caused by the treatments? To assist in answering this question, the major phenolic compound in apple, chlorogenic acid (Lee et al., 2003) applied to apple slices at 0.1 and 0.01% showed virtual instant surface browning while an 0.001% solution resulted in browning after 2 days compared to 45 days for water dipped slices. This could imply that sufcient PPO activity is present on the cut surface at harvest to cause browning and that phenols are the rate limiting compounds. The addition of DETANO largely negated the action of chlorogenic acid suggesting that NO was able to inhibit the involvement of chlorogenic acid in browning reactions. However, the effect of the phosphate buffer alone in negating the effect of chlorogenic acid suggests that it also inhibits the reaction of phenols with PPO although NO and the buffer could have different modes of action. The small but signicant benet of a water dip could be to remove reactive material, possibly phenols, from the cut surface. The action of the treatments in reducing respiration could be an indication of reduced general metabolism and hence better retention of cellular integrity. This would lead to a reduced rate of ion leakage and hence a lower rate of release of metabolites involved in browning on the cut surface. The current ndings extend the effect of NO in inhibiting browning to SNP and Pilotys acid. The relative effectiveness of the applied treatments in absolute terms was found to be DETANO > SNP > NO gas = Pilotys acid. However, the effectiveness of the treatments needs to factor in the benecial effect of a phosphate buffer and water dip on browning development. For example, while the DETANO treatment was more effective than NO gas, DETANO and NO gas resulted in a 50% and 100% increase in postharvest life over their respective control treatments of phosphate buffer and untreated. Thus, NO gas had a proportionately greater effect over its control than did DETANO. The various NO compounds release different forms of NO DETANO and NO gas release the NO free radical while SNP releases the NO+ cation and Pilotys acid releases the NO anion (Hou et al., 1999; Hughes and Cammack, 1999; Saavedra and Keefer, 2002). It might be expected that each NO moiety has different reactivity on browning inhibition of apple slices but it would seem that all released NO moieties are inter-convertible to some extent.

5. Conclusions While inhibition of browning of apples slices by NO and associated control treatments was associated with reduced respiration, ion leakage, PPO activity and phenol content, the interaction of these treatments with added chlorogenic acid suggests that accumulation of phenols on the cut surface is a key step in browning development possibly in conjunction with reduced PPO activity.

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