Postharvest Biology and Technology 78 (2013) 16–23

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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 significant 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 significant 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 (Vámos-Vigyázó, 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;

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. For each apple slice. Ethylene was determined with a gas sample (1 mL) injected into a flame ionization gas chromatograph (Varian Star CX-3400. Osaka) to measure the L value (lightness)..d. 2. 2008). 2.C. Each factor was assessed on a different batch of fruit. Zhang et al. 2006). filtered and the solution diluted with distilled water. 2003. Bowyer. 2. 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. Total phenol content Total phenols was determined according to the Folin–Ciocalteu (FC) method (Singleton and Rossi. The mean postharvest life of all six slices in a treatment unit was expressed as the postharvest life for the treatment unit. respectively in the dark at 20◦ C at −18 ◦ C. ion leakage and lipid peroxidation which are factors that have been linked to either browning development or NO metabolism in plants. five apple slices from five different apples were combined to provide a treatment unit. / Postharvest Biology and Technology 78 (2013) 16–23 17 Sozzi et al. Materials and methods 2. Walnut Creek. 2007. 1999). 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. Produce ‘Granny Smith’ apples (Malus x domestica Borkh) of similar size. The desired concentration of SNP and Piloty’s acid were obtained by dissolving in water. 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. solution and its interaction with DETANO. each from a different apple with a total weight of 100 ± 10 g. Each container contained six slices. Bowyer et al. 2.) packed with Porapak Q (80–100 mesh) (Altech.2 mm i.R. A . Small studies were also made of the effect on browning development of (i) dipping in chlorogenic acid. University of Newcastle as a powder) was dissolved in 0.. Flores et al. PPO activity. shape and color were harvested in three seasons from commercial orchards in Orange. Zhu et al. After draining. The concentration of carbon dioxide in the gas sample was determined by thermal conductivity gas chromatography (Gow-Mac 580. Hou et al.3.1. transported to the laboratory and stored in air 0 ◦ C for up to 6 months. 2. a gas sample (1 mL) was collected in a syringe. 2008. 1993. 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. 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. 2006.. and this comprised a treatment unit.2 mm o. × 2. Sydney).01 M phosphate buffer at pH 6. The frozen tissue was homogenized at 4 ◦ C with cold methanol. One apple slice was removed from each container at various times up to 8 days of storage to measure a range of biochemical factors. They found the optimal treatment to delay browning was dipping slices in 10 mg/L DETANO in pH 6. Apple slices were analysed for respiration. DETANO) increased the vase life of carnation flowers while dipping in sodium nitroprusside (SNP) inhibited internal browning in intact longan and plum fruit (Duan et al. The lid of the container had an open port and a beaker of water as for NO gas. 1/2. 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.5 (1. centrifuged. Ann Arbor. Sydney). 2. apples were periodically selected for inclusion in a series of experiments. Zaharah and Singh. ethylene production. A treated unit of produce was placed into a sealed 800 mL plastic container at various days after treatment. Respiration was calculated as mg CO2 /kg h.. NJ) with a stainless steel column (60 cm × 1 mm i. 2008).5. After 4 h in the sealed container.2 -(hydroxynitrosohydrazino)-bisethanamine (diethylenetriamine/nitric oxide...5.5 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. Biochemical and physical assessments For biochemical assessment. Treatment with NO A unit of fresh cut apple slices was sealed in a 4 L container and NO gas (BOC Gases. NSW. Six readings were taken from cortex tissue of each apple slice.1. total phenol content. Apple slices were treated as for DETANO... NO can also be utilized in biological systems with donor compounds that degrade quantitatively under controlled conditions to release NO (Hrabie et al. Bridgewater. and 2/3 from the calyx.. Treated units of five apple slices were stored in a 4 L container at 5 ◦ C. The time taken for the L value of each apple slice to decline to 75.. 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. All treated and control containers were then stored at 5◦ C. 1965).d.6 was taken as the postharvest life (Pristijono et al. (2003) reported postharvest dipping in a solution of 2. The gas chromatograph response was calibrated with a standard gas mixture containing 5% CO2 in nitrogen (BOC Gases.d.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. Each experiment was repeated at least three times on different occasions and there were three treatment units in each replicate. A beaker with water was provided in each container to maintain a high humidity.4. SNP (Na2 [Fe(CN)5 NO]·2H2 O) (Ajax Chemicals. the major phenol in apples (Lee et al. 2011). Assessment of postharvest life The browning of apple slices was assessed on the color of the cut surface using a colorimeter (Minolta CR-300. namely sodium nitroprusside (SNP) and Piloty’s acid. The ethylene production rate was calculated as L C2 H4 /kg h. (ii) other forms of NO. During this period. M.4 g sodium dihydrogen phosphate was dissolved in approximately 900 mL distilled water. Victoria) and Piloty’s acid (N-hydroxybenzenesulfonamide) (Cayman Chemical. Thus both NO treatments and the three control treatments showed differing responses to the development of surface browning. and (iii) preparing apple slices with a metal and a ceramic knife.) of Haysep N (80–100 mesh) (Altech. 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. In order to better understand how NO inhibited development of surface browning. The colorimeter was calibrated with a white tile plate (Calibration Plate CR-A43). 2009.2. DETANO (supplied by Dr. CA) fitted with a stainless steel column (2 m × 3. After application of NO gas for 2 h. 8. the slices were allowed to dry for 2–5 min before placing in a 4 L container and storied at 5 ◦ C. Sydney). Respiration and ethylene measurement Fresh cut slices of ‘Granny Smith’ apples were weighed before treatment. Huque et al. 2003). MI) were stored.

2007). 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. 6 months and the Season 2 experiments. 3. 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.1. Gallic acid was the calibration standard and the data was expressed as mg/L gallic acid equivalents. 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.5 cm thick section of fresh apple flesh (2 g) was placed in a beaker containing de-ionized water and incubated for 2 h at 25 ◦ C. The percentage of the ion leakage was calculated. Treated slices were stored in a 4 L container at 5 ◦ C as previously stated. The absorbance of the supernatant was determined at 560 and 600 nm and using the MDA molar extinction coefficient (155 mM/cm). the order was reversed with apple slices first dipped into chlorogenic acid solution. Sydney) dissolved in water for 10 s. 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. The main trial in both Season 1 and Season 2 over 8 days therefore only examined respiration. A subsequent experiment conducted on slices that had been stored for 6 months and including a phosphate buffer dip showed significant differences between treatments with the order of effectiveness in extending postharvest life being DETANO > NO gas = phosphate buffer > water = untreated.. 2.7. Jenway. (2008). Ion leakage Ion leakage from cells was measured using the method described by Song et al. MDA was measured by the method described by Heath and Packer (1968). H2 O2 was calculated using an extinction coefficient 0.18 R. 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. Fruit obtained in Season 2 were evaluated after 3 months storage and each treatment had a similar postharvest life as in the previous season. Comparison of the postharvest life obtained by DETANO and NO gas in the preliminary study showed a similar extension in postharvest life.1. but a phosphate buffer control was also included. Changes in total phenols as determined by the Folin–Ciocalteu (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. 2. IL). Physiological and biochemical parameters 3.6. 2. Statistical analysis Statistical procedures were performed using SPSS for Microsoft version 18. Apple slices were dipped in DETANO solution. The homogenate was centrifuged at 17. Zheng et al. . the content of MDA ( mole) was calculated from (A560 − A600 )/155.. 2.05 was used. 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.5. which also showed a significant difference between all treatments. Table 1 also shows the mean values for the seven replicates evaluated in the Season 1.01 unit change in absorbance per min at 410 nm at 25 ◦ C. There was a significant 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 first analytical time of 2 days.0 software package (SPSS Chicago. H2 O2 was determined by the method of Sun et al.5. 2009). 3. 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 2.5. NO gas and DETANO reduced respiration but ethylene showed no significant effect of NO treatment (data not given).000 × g for 15 min at 4 ◦ C and extracts were filtered through three layers of cheese cloth. Staffordshire) as the initial reading and again after boiling for 15 min and cooling at room temperature. in phosphate buffer only or in water for 5 min and allowed to drain.3. 2007). (2010). Results 3.0) and 0.2. Supernatant (1 mL) combined with sodium phosphate buffer and pyrocatechol and one unit of PPO activity was measured spectrophotometrically as a 0. 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. Enzyme activity in the supernatant was determined according to the method described by Yingsanga et al. NO was similarly effective on freshly harvested fruit and fruit stored for 6 months. Thus. 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. A phosphate buffer dip was not included in the experiment. but with a significant difference between all treatments for DETANO > NO gas > phosphate buffer > water > untreated. (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). cooled to room temperature and centrifuged.2. 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.2. Five minutes after completion of the treatment. 2002. To determine significant difference between treatments least significant difference (LSD) at P = 0.25 g polyvinylpolypyrrolidone (10 mL). A 0.4. The conductivity of the solution was measured with a conductivity meter (Model 4071. slices were dipped into different concentrations of chlorogenic acid (Sigma–Aldrich.2.5 mL) was added FC reagent and sodium carbonate solution and the absorbance at 765 nm was determined. Huque et al. Respiration also significantly (P < 0. (2006). The precipitate was dissolved in H2 SO4 and the absorbance at 415 nm recorded. Postharvest life A preliminary experiment conducted soon after the Season 1 harvest confirmed the findings obtained by Pristijono et al. 2.28 mol/cm (Hung and Kao. / Postharvest Biology and Technology 78 (2013) 16–23 sample (0. In another experiment.001) increased in all treatments during storage. Linear regression equations were calculated to determine the relationship between postharvest life and an applied treatment.2.

2 6 31.5d 55.6 47.8 1.8 49.2 142. Ion leakage Ion leakage showed a significant difference (P < 0.6 181.8 149. Ion leakage of buffer and water-treated slices were not significantly different but both were significantly lower than untreated slices. Analysis of the data for the other four treatments over both seasons showed that PPO activity was significantly different (P < 0.2 17.05 and LSD values are at P = 0.7 56.2.77 4 3.1d 8. .001).001) between treatments (Table 2). Storage period had a significant effect (P < 0.053 – 0. Storage period also had a significant effect (P < 0.038 0.5 53.R.1 139. but the difference between DETANO and NO gas was not significant at P = 0.62 7 19 Meanb Each replicate contained 3 treatment units. Huque et al.7 59.6 29.1b 5.0 Water 139.8 2.061 0.2 25. increasing during storage. MDA was used as the biomarker for lipid peroxidation but the data showed no significant difference between treatments with slices exhibiting about 2.9 5.5 4.051 0. of replicates Postharvest life (days)a Season 1 0 months 4.0 176.55 3 6 months 3. The total phenols were significantly different (P < 0.3.056 0.5 6.6 31.05.2 6.047a 0.065 0.6e 0.2 151. with total phenol content of all treatments increasing during storage.0 NO gas 121.6 0.041 0.2d 154.6 159.8c 7.060 – 0. Treatment Amount during storage 2 Respiration rate (mg CO2 /kg h) Untreated Water Buffer NO gas DETANO LSD 24.2 51.0 23.3 158.8 22.0 51.24 4 27.3 177.7bc 51.8 14. a Postharvest life was the time taken for the HunterLab L value to fall to 75.8 22.072 0. 3.6 1.3 62.05.6. Lipid peroxidation For the fruit in Season 1.4a 4.2 54.4 151.062d 0.051b 0.64 3 Season 2 3 months 3.001) on PPO activity in all treatments.002 58.9 8.8 132.0 8 days 34.17 54. b Mean values are of data from Season 1. 3.8b 136.7 49.043 0.0d 24.4e 27.6 57.8 6.054 0.1 7.5 3.8a 7.5 Buffer 128.0 Mean 29.5 6.5 54. Examination of the effect of treatments on PPO activity was incomplete as the phosphate buffer was not included in Season 1.001) between treatments with DETANO < NO gas < water < untreated.0 1.057c – 0.32 LSD PPO activity ( Abs/min) Untreated Water Buffer NO gas DETANO LSD Ion leakage (%) Untreated Water Buffer NO gas DETANO LSD 171.2 28.0 44.2 8.8a 2.047 – 0. mean values with different superscript letters are significantly different at P = 0.4 4. In this and all subsequent tables.04 0. PPO activity and ion leakage of ‘Granny Smith’ apple slices during storage at 5 ◦ C.051 0.4.4c 22.71 Values are the mean of 6 replicates with 3 treatment units in each replicate.2 25.6 51.6 55.3ab 48. / Postharvest Biology and Technology 78 (2013) 16–23 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.7c 143.1 168.8 58.6 18.067 – 0.4 191.4 DETANO 16. A significantly 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.5 9.0 26.00 Total phenols (gallic acid equivalent in mg/L) 161.8 49.0 mol MDA/g (data not Table 2 Effect of NO treatments on respiration rate.048 0.058 0.6 51. 6 months and Season 2.2 Untreated 149.0b 19.4 26.2.001) between treatments with the mean levels throughout storage in slices treated with DETANO < NO gas < phosphate buffer < water < untreated.003 0.0 21.6 20.1 158.3e 163.7c 53.6 58.4 20.7 168. total phenols.

1% chlorogenic acid.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.3a 2 0. (g/100 g) 0. 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. Relationship between fruit parameters and postharvest life From Table 2 it can be seen that the effect of the treatments on respiration. There was also no significant difference whether the treatments were applied before or after dipping in chlorogenic acid solution.5 <1 ha <1 ha 2.0 5.3 7. However.4a 1. (mg/L) Postharvest life (days) SNP Untreated Water 10 50 100 500 750 1000 LSD 3.75 0. a Not assessed due to flesh softening. PPO activity and ion leakage was evident at the first analysis time of 2 days after treatment and the magnitude of differences between treatments did not markedly change on further storage.7a 7.01) and ion leakage (P < 0. Prior application of a DETANO dip negated the effect of chlorogenic acid as evidenced by a postharvest life of 4.1 3 0.9 5.001).001 8. total phenols.5 days (Experiment 4. 3.5 4. Effect of SNP and Piloty’s acid on postharvest life ‘Granny Smith’ apple slices dipped in SNP dissolved in water showed a significant 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. 3.38 Values in each experiment are the mean of 3 replicates with 3 treatments units in each replicate.3.15 1. Thus. Table 3). Table 3).8 4. Effect of added chlorogenic acid on postharvest life 2. In case the lack of an effect was specific to MDA.8 5.5 6. 1 Chlorogenic acid conc. 3. total phenols (P < 0.0b 6. PPO activity (P < 0. Application of 0. apple slices dipped in Piloty’s acid dissolved in water also showed a significantly longer postharvest life with dipping in 100 mg/L 4.4.001% chlorogenic acid resulted in the postharvest life of slices being about 1.3 given).5 <1 ha <1 ha 2. / Postharvest Biology and Technology 78 (2013) 16–23 Table 3 Postharvest life at 5 ◦ C of ‘Granny Smith’ apple slices dipped in DETANO.4b 1. The relationships between the mean values for respiration.2ab 4. Huque et al.9 a Piloty’s acid 3.8 3. phosphate buffer and water before or after dipping in chlorogenic acid solution.0 6. dipping in DETANO solution and the buffer negated much of the effect of chlorogenic acid with no significant difference with water and untreated slices.05). However.1 7.4 4.31 4. phosphate buffer and water and after 5 min were then dipped into 0.9 1. All further experiments were conducted with the chlorogenic acid dip applied as the second dip. Dipping slices in buffer also negated most of the effect of chlorogenic acid but the postharvest life was significantly lower than for the DETANO dip.05) than the combined treatment. The data in Table 3 show that in both experiments.20 R. total phenol content.001).01% chlorogenic acid resulted in a slightly longer postharvest life of about 2 h for chlorogenic acid-treated slices (Experiment 3. .7 mol H2 O2 /g (data not given). apple slices were dipped in DETANO.5b 4 0. The data in Fig.3 a 0. 1 show that there was a significant inverse linear relationship with a greater postharvest life associated with a lower rate of respiration (P < 0.1% chlorogenic acid solution while in the second study.9b ∼2 ha ∼2 ha 3. Application of 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. 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.7c 7. Table 4 Postharvest life at 5 ◦ C of ‘Granny Smith’ apple slices dipped in SNP and Piloty’s acid in water for 5 min at 20 ◦ C. hydrogen peroxide was used in Season 2 as the biomarker for lipid peroxidation. a Treatment was not included in statistical analysis. the dipping order was reversed.4 days which was not significantly different to water-dipped and untreated slices which had no added chlorogenic acid.3b 1.5.95 Values are the mean of 3 replicates with 3 treatments units in each replicate. Two sets of experiments were conducted with 0. Dip conc. but again no significant difference between treatments was found with slices showing about 0. the buffer dip fully negated the effect of the added chlorogenic acid with both treatments not significantly different. The addition of a DETANO dip largely negated the effect of chlorogenic acid although the postharvest life of the DETANO-only dip was still significantly greater (P < 0. In the first experiment. the mean values for each treatment given in Table 2 can represent the comparative level of each factor.8 2.4 5. Expt.2 7.5 6.89 4.

(2002) who reported that NO affects the function of mitochondria in plant cells and reduces cell respiration by inhibiting the cytochrome pathway. respectively) had a significantly longer postharvest life (P < 0. / Postharvest Biology and Technology 78 (2013) 16–23 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.02 60 2 4 6 8 10 Ion leakage (%) 55 50 y = 62. Apple slices treated with 750 mg/L SNP and 1000 mg/L Piloty’s acid caused damage to slices by rapidly softening the flesh to an unacceptable level.6 -1. of ‘Granny Smith’ apple slices with NO and the associated control treatments as shown by Pristijono et al. It is possible that this decreases the release of browning precursors such as phenols from cells to the surface of the cut fruit.6c 9.6. 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. There was. respectively).4 days. (2006.001) than those not dipped in water (3.7x 45 40 2 4 6 8 10 Postharvest life (days) Fig. Treatment Water for 5 min 10 L/L NO gas for 1 h 100 mg/L Piloty’s acid for 5 min 500 mg/L SNP for 5 min 10 mg/L DETANO for 5 min LSD Postharvest life (days) 4. 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. Relationship between postharvest life and mean respiration.1d 1. however.2x 120 100 2 4 6 8 10 0.23 15 10 y = 17. NO has previously been . no significant difference in postharvest life between the metal and ceramic knife-cut apple slices whether dipped or not dipped in water. 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 lack of effect of NO is probably due to the apples being post-climacteric and thus having a substantial production of ethylene.08 PPO activity (ΔAbs/min) 0. The lack of a significant 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. resulting in the longest postharvest life. The lack of a significant 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).0.06 .04 y = 0. 2005). PPO activity and ion leakage of Granny Smith apple slices during storage at 5 ◦ C. The postharvest life of apple slices cut by the metal and ceramic knives then dipped in water (4. The inhibition of browning. It is generally considered that PPO catalyses the oxidation of phenolic compounds to quinones which then condense to form brown polymers (Milani and Hamedi.8 and 5.3b 7.4 . 1.1b 6. 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 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 significantly more effective than SNP.0.R.002x 0. 4. The postharvest life of apple slices fumigated with 10 L/L NO gas and dipped in 100 mg/L Piloty’s acid solution were not significantly different. This is consistent with Millar and Day (1996) and Zottini et al. Each point is the mean of 6 replicates with 3 units per replicate. 2000). 2008) has been shown to be quantitatively associated with a decrease in respiration rate.0 days. Comparison was made of the effect of the optimum concentration of SNP and Piloty’s acid dissolved in water with DETANO in phosphate buffer and fumigation with NO gas to inhibit browning. phenol content. as reflected in extension of postharvest life.06 0. 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. PPO activity and ion leakage but was not correlated with ethylene production or lipid peroxidation. Nevertheless. Discussion 180 160 140 y = 192 -7. ppm) 3. Huque et al. 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.9x 5 0 200 Values are the mean of 3 replicates with 3 treatments units in each replicate.3 and 3.3a 6. total phenols. 2 4 6 8 10 Total phenols (Gallic acid.

. Since the differences between treatments were evident by 2 days after application.. Trends Food Sci. Zhu et al. Golding. .. whatever the metabolic sequence induced by cutting that leads to browning. Evidence for the function of the free radical gas – nitric oxide (NO·) – as an endogenous maturation and senescence regulating factor in higher plants. 2003) applied to apple slices at 0. Biophys. 417–441. Wills. Duan.. Nitric oxide inhibits the cytochrome oxidase but not the alternative oxidase of plant mitochondria. DETANO and NO gas resulted in a 50% and 100% increase in postharvest life over their respective control treatments of phosphate buffer and untreated.. Wills. 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. 2000.. Kao. were having a similar affect on inhibiting that pathway but with different levels of effectiveness. P. 2008. Flores. L.X. Technol. J. J. 125. and leaf senescence in rice. 1472–1476.. Inhibitory effects of various antibrowning agents on apple slices. Design 5. 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. as an inhibitor of browning in apple slices. Agric. Synthesis chemistry. Wills. 5. McEvily. R.. Postharvest Biol.H. 58. that.. Badiyan. / Postharvest Biology and Technology 78 (2013) 16–23 reported to reduce the MDA content of intact longan (Duan et al.Y.G.... 2006. However. R. J. 189–198. Biochem.. W. A. Kim. 1999. 2007) and kiwifruit (Zhang et al. 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. 7. Phenolic compounds and their role in oxidative processes in fruits. Klose. Goupy. The small but significant benefit of a water dip could be to remove reactive material. Pristijono. Campbell-Palmer.T. 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. S. 36.. 2221–2226.. A. Rev. L.. 1994. H. Dordrecht. L. A modified chemiluminescence method for hydrogen peroxide determination in apple fruit tissues.B. browning could be inhibited by NO affecting other some aspect of apple metabolism.. 66.001% solution resulted in browning after 2 days compared to 4–5 days for water dipped slices. Tucker..J. J.W. D. 369–373.P.. 83.A. Pristijono. M. F. Nitric Oxide in Plants: Occurrence. H. Postharvest water relationship and tissue browning of rambutan fruit. C. Moon. Saavedra. Robards. Lee. Glover.. K. Iyengar....B. M. W. Enzymatic browning reactions in apple and apples product.. Thus. Eamus. Plant Sci.1 and 0.. 1996.T. K. 1993.. 227. Cammack. Y. 2007. Jiang. Effect of nitric oxide on pericarp browning of harvested longan fruit in relation to phenolic metabolism. 449–458. while the DETANO treatment was more effective than NO gas... Food Res.. 120. Janczuk. 1427–1434. 2007. 127–140. Nicoli.. Current trends in the development of nitric oxide donors..J. Sci.H. 2007.. 73. 2001. M.. Landrigan. 1996. Curr. If lipid peroxidation is involved in the browning of apple slices. all treatments including a water-dip. 571–576. Y. 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... R. antioxidants and stress tolerance. Qu. 6516–6520.G. S. Antibrowning agents alternatives to use of sulfites in food... Food Chem. 2002. Food Chem.B. R. and where phenol content and PPO activity are both implicated in the browning sequence. M...C..’ and ‘Granny Smith’ apples. 1999. Biochem. Postharvest Biol. 51. X.. Trends Plant Sci.R. Song. J..K.L. R. R. calcium.. Leshem. Hu. Heath. Amiot. Y.’ ‘Golden Delicious. PPO activity and ion leakage with postharvest life suggests firstly. Hughes and Cammack. 16. An obvious consideration is that the metal knife used to cut the slices could have left traces of metal which catalysed some enzyme sequence. Lara Manzocco. Am.V. 155–158. L. K. A. J. M. Rossi. Calligaris.B. Martínez-Madrid.. 1999. Arch.C. Postharvest Biol. Lee. M.22 R.. Extending the postharvest life of carnations with nitric oxide—comparison of fumigation and in vivo delivery.S. 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. 1968. Hamedi. 2003. chlorogenic acid (Lee et al... Hrabie.Z.E. total phenols. D. Sci.. X. 1992. Susceptibility of five apple cultivars to enzymatic browning.T.. 405–410.. 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