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Postharvest Biology and Technology 78 (2013) 16–23

Contents lists available at SciVerse ScienceDirect

Postharvest Biology and Technology

journa l h o me pa g e: www.elsev ier .com/ locate /postharvbio

ffect of nitric oxide (NO) and associated control treatments on the metabolismf fresh-cut apple slices in relation to development of surface browning

oksana Huquea, R.B.H. Willsa,∗, Penta Pristijonoa,b, J.B. Goldinga,b

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

r t i c l e i n f o

rticle history:eceived 28 October 2012ccepted 15 December 2012

eywords:pple (Malus x domestica Borkh)resh-cut slicesurface browningitric oxidehenols

a b s t r a c t

Surface browning is an important cause of deterioration of fresh-cut apples during postharvesthandling. ‘Granny Smith’ apple slices treated with NO gas (10 �L/L) and the NO donor compound2,2′-(hydroxynitrosohydrazino)-bisethanamine (diethylenetriamine nitric oxide (DETANO) (10 mg/L)dissolved in phosphate buffer (pH 6.5) showed delayed development of surface browning during storageat 5 ◦C and also resulted in a lower level of total phenols, inhibition of PPO activity, reduced ion leakageand reduced rate of respiration but had no significant effect on ethylene production or lipid peroxidelevel as measured by malondialdehyde (MDA) and hydrogen peroxide levels. The two control treatmentsof 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, PPOactivity, ion leakage and respiration was DETANO > NO gas > phosphate buffer > water > untreated. Appleslices 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 dip-ping in chlorogenic acid solution while the buffer and NO gas were also effective. It is suggested that anincrease in phenols occurs on the apple surface soon after cutting, possibly as a defensive mechanism ofthe apple to limit damage to surface cells. The effectiveness of the applied treatments to inhibit devel-opment of surface browning may relate to their ability to minimize the level of phenols active on the cutsurface possibly in conjunction with a reduced PPO activity.

. Introduction

Apple slices are a popular component of the value-addedonsumer-ready market for minimally processed fresh fruit andegetables. However, they are more perishable than intact pro-uce due to adverse impacts arising from physical stress imposeduring preparation, with a major limitation to postharvest lifeeing the appearance of browning on the cut surface (Abbott et al.,004). Browning of horticultural produce can be initiated by bothnzymic and non-enzymic pathways. Enzymic browning is consid-red to occur through to the oxidation of ortho-phenols to quinonesy the action of enzyme systems such as polyphenolic oxidasePPO), and which then polymerize to brown pigments (Milani andamedi, 2005). PPO is generally associated with the plastid, andhenolic substrates are located in the vacuole but cellular and

ntracellular disruption allows the substrates to mix and henceeact to produce browning (Landrigan et al., 1996). An associa-ion 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).

925-5214/$ – see front matter © 2012 Elsevier B.V. All rights reserved.ttp://dx.doi.org/10.1016/j.postharvbio.2012.12.006

© 2012 Elsevier B.V. All rights reserved.

many authors (e.g. Nicolas et al., 1994; Hu et al., 2007; Toivonenand Brummell, 2008). Non-enzymic browning can occur throughvarious sequences including through metal ion interaction withphenols (Vámos-Vigyázó, 1981; Robards et al., 1999). Less workhas been reported on a role for non-enzymic reactions in applebrowning, although Nicoli et al. (2000) studied the interaction ofa catechin model system with apple derivatives and found bothenzymic and chemical oxidation of catechin was associated withthe development of browning. A range of chemical agents are ableto inhibit browning. Sulphites are very effective anti-browningagents but are banned in most countries on fresh fruit and vegeta-bles due to potential harmful health effects (Iyengar and McEvily,1992). Ascorbic acid, as a reducing agent, and citric acid, as an acidu-lant, alone or in combination dips have been widely reported asanti-browning agents for fresh-cut fruit and vegetables includingapples (Vamos-Vigyazo, 1995; Son et al., 2001).

Nitric oxide (NO) is a small, highly diffusible free radical thatinitially 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 fumigationwith NO gas extended the storage life of a range of horticulturalproduce by inhibiting ripening or senescence (Leshem et al., 1998;

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ozzi et al., 2003; Flores et al., 2008; Zhu et al., 2009; Zaharah andingh, 2011). NO can also be utilized in biological systems withonor compounds that degrade quantitatively under controlledonditions to release NO (Hrabie et al., 1993; Hou et al., 1999).owyer et al. (2003) reported postharvest dipping in a solutionf 2,2′-(hydroxynitrosohydrazino)-bisethanamine (diethylenetri-mine/nitric oxide, DETANO) increased the vase life of carnationowers while dipping in sodium nitroprusside (SNP) inhibited

nternal browning in intact longan and plum fruit (Duan et al.,007; Zhang et al., 2008). NO applied as a gas and as a DETANOip has also been shown delay the browning of fresh-cut applelices (Pristijono et al., 2006, 2008). They found the optimal treat-ent to delay browning was dipping slices in 10 mg/L DETANO in

H 6.5 phosphate buffer but the buffer solution itself also gave aelay in browning over slices dipped in water which in turn showedelayed browning over untreated slices. Fumigation with 10 �L/LO gas showed delayed browning over its control treatment ofntreated but was not as effective as the DETANO treatment. Thusoth NO treatments and the three control treatments showed dif-ering responses to the development of surface browning.

In order to better understand how NO inhibited development ofurface browning, this study examined the metabolism of ‘Grannymith’ apple slices that had been exposed to NO gas and DETANOolution before storage at 5 ◦C. Apple slices were analysed for res-iration, ethylene production, total phenol content, PPO activity,

on leakage and lipid peroxidation which are factors that have beeninked to either browning development or NO metabolism in plants.mall studies were also made of the effect on browning develop-ent of (i) dipping in chlorogenic acid, the major phenol in apples

Lee et al., 2003), solution and its interaction with DETANO, (ii)ther forms of NO, namely sodium nitroprusside (SNP) and Piloty’scid, and (iii) preparing apple slices with a metal and a ceramicnife.

. Materials and methods

.1. Produce

‘Granny Smith’ apples (Malus x domestica Borkh) of similar size,hape and color were harvested in three seasons from commer-ial orchards in Orange, NSW, transported to the laboratory andtored in air 0 ◦C for up to 6 months. During this period, applesere periodically selected for inclusion in a series of experiments.

he required number of apples in each experiment were removedo 20 ◦C and after 2 h each apple was hand-cut longitudinally intoix unpeeled slices using a sharp stainless steel knife and slices werelaced into 4 L containers with a sealable lid. Each container con-ained six slices, each from a different apple with a total weight of00 ± 10 g, and this comprised a treatment unit. Each experimentas repeated at least three times on different occasions and thereere three treatment units in each replicate.

.2. Treatment with NO

A unit of fresh cut apple slices was sealed in a 4 L container andO gas (BOC Gases, Sydney) was applied from a syringe throughn injection port in the lid of the container to give a concentrationf 10 �L/L NO. After application of NO gas for 2 h, the lids of theontainers were opened to atmospheric air and the lid replaced butn injection port (4 mm diameter) in the lid was opened to preventarbon dioxide accumulation inside the container. A beaker with

ater was provided in each container to maintain a high humidity.ll treated and control containers were then stored at 5◦C.

DETANO (supplied by Dr. M.C. Bowyer, University of Newcas-le as a powder) was dissolved in 0.01 M phosphate buffer at pH

d Technology 78 (2013) 16–23 17

6.5 (1.4 g sodium dihydrogen phosphate was dissolved in approx-imately 900 mL distilled water, 8.05 g sodium chloride was addedand the volume made up to 1 L with distilled water) to give a con-centration of 10 mg/L DETANO. A unit of fresh-cut slices was placedin a stainless steel mesh strainer and dipped into the DETANO solu-tion at 20 ◦C for 5 min. After draining, the slices were allowed to dryfor 2–5 min before placing in a 4 L container and storied at 5 ◦C. Thelid of the container had an open port and a beaker of water as forNO gas.

SNP (Na2[Fe(CN)5NO]·2H2O) (Ajax Chemicals, Victoria) andPiloty’s 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 Piloty’s acid wereobtained by dissolving in water. Apple slices were treated as forDETANO.

2.3. Assessment of postharvest life

The browning of apple slices was assessed on the color of the cutsurface using a colorimeter (Minolta CR-300, Osaka) to measure theL value (lightness). Six readings were taken from cortex tissue ofeach apple slice, three from each side of a slice with the measuringhead placed along the longitudinal axis on the midpoint betweenthe core and skin at 1/3, 1/2, and 2/3 from the calyx. The colorimeterwas calibrated with a white tile plate (Calibration Plate CR-A43).The time taken for the L value of each apple slice to decline to 75.6was taken as the postharvest life (Pristijono et al., 2006). The meanpostharvest life of all six slices in a treatment unit was expressedas the postharvest life for the treatment unit.

2.4. Respiration and ethylene measurement

Fresh cut slices of ‘Granny Smith’ apples were weighed beforetreatment. A treated unit of produce was placed into a sealed800 mL plastic container at various days after treatment. After 4 h inthe sealed container, a gas sample (1 mL) was collected in a syringe.The concentration of carbon dioxide in the gas sample was deter-mined by thermal conductivity gas chromatography (Gow-Mac580, Bridgewater, NJ) with a stainless steel column (60 cm × 1 mmi.d.) of Haysep N (80–100 mesh) (Altech, Sydney). The gas chro-matograph response was calibrated with a standard gas mixturecontaining 5% CO2 in nitrogen (BOC Gases, Sydney). Respirationwas calculated as mg CO2/kg h. Ethylene was determined with a gassample (1 mL) injected into a flame ionization gas chromatograph(Varian Star CX-3400, Walnut Creek, CA) fitted with a stainless steelcolumn (2 m × 3.2 mm o.d. × 2.2 mm i.d.) packed with Porapak Q(80–100 mesh) (Altech, Sydney). The ethylene production rate wascalculated as �L C2H4/kg h.

2.5. Biochemical and physical assessments

For biochemical assessment, five apple slices from five differentapples were combined to provide a treatment unit. Treated units offive apple slices were stored in a 4 L container at 5 ◦C. One apple slicewas removed from each container at various times up to 8 days ofstorage to measure a range of biochemical factors. Each factor wasassessed on a different batch of fruit.

2.5.1. Total phenol contentTotal phenols was determined according to the Folin–Ciocalteu

(FC) method (Singleton and Rossi, 1965). For each apple slice, asection 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 coreand the sample was immediately placed at −18 ◦C for 30 min. Thefrozen tissue was homogenized at 4 ◦C with cold methanol, cen-trifuged, filtered and the solution diluted with distilled water. A

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ample (0.5 mL) was added FC reagent and sodium carbonate solu-ion and the absorbance at 765 nm was determined. Gallic acid washe calibration standard and the data was expressed as mg/L galliccid equivalents.

.5.2. PPO activityFrozen apple tissue (1 g) obtained as described above was

omogenized in a mortar and pestle with a cold solution (4 ◦C)ontaining 100 mM sodium phosphate buffer (pH 7.0) and 0.25 golyvinylpolypyrrolidone (10 mL). The homogenate was cen-rifuged at 17,000 × g for 15 min at 4 ◦C and extracts were filteredhrough three layers of cheese cloth. Enzyme activity in the super-atant was determined according to the method described byingsanga et al. (2008). Supernatant (1 mL) combined with sodiumhosphate buffer and pyrocatechol and one unit of PPO activityas measured spectrophotometrically as a 0.01 unit change in

bsorbance per min at 410 nm at 25 ◦C.

.5.3. Lipid peroxidationMalondialdehyde (MDA) was considered to be a suitable

iomarker for lipid peroxidation caused by reactive oxygen speciesROS) which are purported as a major cause of membrane deterio-ation in plant tissues (Mittler, 2002; Zheng et al., 2007). MDA waseasured by the method described by Heath and Packer (1968).

resh apple tissue (1 g) was homogenized in a mortar and pestleith a solution containing thiobarbituric acid and trichloroacetic

cid then incubated at 90 ◦C, cooled to room temperature and cen-rifuged. The absorbance of the supernatant was determined at60 and 600 nm and using the MDA molar extinction coefficient155 mM/cm), the content of MDA (�mole) was calculated fromA560 − A600)/155.

Hydrogen peroxide (H2O2) was used as an alternative biomarkeror oxidative stress generated by reactive oxygen species (ROS) inlant tissues during normal metabolism (Lu et al., 2009). H2O2 wasetermined by the method of Sun et al. (2010). A section of freshpple tissue (2 g) was homogenized with cold acetone then cen-rifuged and an extract (1 mL) was mixed with titanium dioxidend ammonia solutions and centrifuged. The precipitate was dis-olved in H2SO4 and the absorbance at 415 nm recorded. H2O2 wasalculated using an extinction coefficient 0.28 �mol/cm (Hung andao, 2007).

.5.4. Ion leakageIon leakage from cells was measured using the method

escribed by Song et al. (2006). A 0.5 cm thick section of freshpple flesh (2 g) was placed in a beaker containing de-ionized waternd incubated for 2 h at 25 ◦C. The conductivity of the solutionas measured with a conductivity meter (Model 4071, Jenway,

taffordshire) as the initial reading and again after boiling for5 min and cooling at room temperature. The percentage of the

on leakage was calculated.

.6. Treatment with chlorogenic acid

The effect of chlorogenic acid on the development of browningf fresh-cut apple slices was examined in conjunction with dippingn DETANO solution. Apple slices were dipped in DETANO solution,n phosphate buffer only or in water for 5 min and allowed to drain.ive minutes after completion of the treatment, slices were dippednto different concentrations of chlorogenic acid (Sigma–Aldrich,

ydney) dissolved in water for 10 s. In another experiment, therder was reversed with apple slices first dipped into chlorogeniccid solution. Treated slices were stored in a 4 L container at 5 ◦C asreviously stated. The L value of each slice was measured daily and

d Technology 78 (2013) 16–23

the mean postharvest life of all six slices in a treatment unit wasdetermined as previously stated.

2.7. Statistical analysis

Statistical procedures were performed using SPSS for Microsoftversion 18.0 software package (SPSS Chicago, IL). To determine sig-nificant difference between treatments least significant difference(LSD) at P = 0.05 was used. Linear regression equations were calcu-lated to determine the relationship between postharvest life andan applied treatment.

3. Results

3.1. Postharvest life

A preliminary experiment conducted soon after the Season 1harvest confirmed the findings obtained by Pristijono et al. (2008)that DETANO was the most effective treatment in inhibiting brown-ing while apples fumigated with NO gas had a longer postharvestlife than those dipped in water and untreated (Table 1). A phosphatebuffer dip was not included in the experiment.

A subsequent experiment conducted on slices that had beenstored for 6 months and including a phosphate buffer dip showedsignificant differences between treatments with the order ofeffectiveness in extending postharvest life being DETANO > NOgas = phosphate buffer > water = untreated. Comparison of thepostharvest life obtained by DETANO and NO gas in the prelimi-nary study showed a similar extension in postharvest life. Thus, NOwas similarly effective on freshly harvested fruit and fruit storedfor 6 months.

Fruit obtained in Season 2 were evaluated after 3 months stor-age and each treatment had a similar postharvest life as in theprevious season, but with a significant difference between all treat-ments for DETANO > NO gas > phosphate buffer > water > untreated.Table 1 also shows the mean values for the seven replicates evalu-ated in the Season 1, 6 months and the Season 2 experiments, whichalso showed a significant difference between all treatments.

3.2. Physiological and biochemical parameters

3.2.1. Respiration and ethylene productionA preliminary study in Season 1 examined changes in respira-

tion and ethylene over 4 days but without inclusion of a phosphatebuffer treatment. NO gas and DETANO reduced respiration butethylene showed no significant effect of NO treatment (data notgiven). The main trial in both Season 1 and Season 2 over 8 daystherefore only examined respiration, but a phosphate buffer con-trol was also included. The changes due to the applied treatmentsand over the storage period were consistent in both seasons sothe combined data are presented in Table 2. There was a signifi-cant difference (P < 0.001) in respiration between treatments withDETANO < NO gas < phosphate buffer < water < untreated with theeffect of the treatments evident from the first analytical time of 2days. Respiration also significantly (P < 0.001) increased in all treat-ments during storage.

3.2.2. Total phenol content and PPO activityChanges in each biochemical factor examined was assessed on

a different batch of apple slices with measurements taken on fruitfrom Season 1 and Season 2. Changes in total phenols as deter-

mined by the Folin–Ciocalteu (FC) method and in PPO activity dueto applied treatments were consistent in fruit from both seasonsand hence the combined data for both seasons are presented inTable 2.

R. Huque et al. / Postharvest Biology and Technology 78 (2013) 16–23 19

Table 1Effect of NO treatments on the postharvest life at 5 ◦C of ‘Granny Smith’ apple slices.

Treatment Postharvest life (days)a

Season Season 1 Season 2 Meanb

Storage time 0 months 6 months 3 months

Untreated 4.5 3.5 3.4 3.4aWater dip 4.8 3.9 4.2 4.1bPhosphate buffer dip 5.5 6.1 5.8c10 �L/L NO gas 6.5 6.9 7.2 7.1d10 mg/L DETANO dip 9.0 8.6 8.6 8.6eLSD 1.55 1.64 0.77 0.62No. of replicates 3 3 4 7

Each replicate contained 3 treatment units.In this and all subsequent tables, mean values with different superscript letters are significantly 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.

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b Mean values are of data from Season 1, 6 months and Season 2.

The total phenols were significantly different (P < 0.001)etween treatments with the mean levels throughout stor-ge in slices treated with DETANO < NO gas < phosphateuffer < water < untreated. Storage period also had a signifi-ant effect (P < 0.001), with total phenol content of all treatmentsncreasing during storage.

Examination of the effect of treatments on PPO activity wasncomplete as the phosphate buffer was not included in Season. Analysis of the data for the other four treatments over both sea-

ons showed that PPO activity was significantly different (P < 0.001)etween treatments with DETANO < NO gas < water < untreated.torage period had a significant effect (P < 0.001) on PPO activityn all treatments, increasing during storage.

able 2ffect of NO treatments on respiration rate, total phenols, PPO activity and ion leakage of

Treatment Amount during storage

2 4

Respiration rate (mg CO2/kg h)Untreated 24.8 27.2

Water 22.4 25.8

Buffer 20.2 22.6

NO gas 17.8 20.6

DETANO 14.8 18.2

LSD 2.24

Total phenols (gallic acid equivalent in mg/L)Untreated 161.2 171.1

Water 149.0 158.4

Buffer 139.5 151.1

NO gas 128.0 139.8

DETANO 121.4 132.6

LSD 16.32

PPO activity (�Abs/min)Untreated 0.051 0.058

Water 0.047 0.053

Buffer – –

NO gas 0.041 0.048

DETANO 0.038 0.043

LSD 0.003

Ion leakage (%)Untreated 58.6 54.0

Water 55.5 51.7

Buffer 53.6 49.6

NO gas 51.8 47.0

DETANO 49.5 44.6

LSD 6.17

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

3.2.3. Ion leakageIon leakage showed a significant difference (P < 0.001) between

treatments (Table 2). A significantly lower ion leakage was foundin NO gas and DETANO-treated apple slices compared to those ofthe respective control slices of buffer and water, but the differencebetween DETANO and NO gas was not significant at P = 0.05. Ionleakage of buffer and water-treated slices were not significantlydifferent but both were significantly lower than untreated slices.

3.2.4. Lipid peroxidationFor the fruit in Season 1, MDA was used as the biomarker for lipid

peroxidation but the data showed no significant difference betweentreatments with slices exhibiting about 2.0 �mol MDA/g (data not

‘Granny Smith’ apple slices during storage at 5 ◦C.

6 8 days Mean

31.2 34.6 29.4e28.4 31.6 27.0d26.0 29.0 24.4c23.0 26.2 22.0b21.0 25.0 19.8

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181.7 191.3 176.3e168.6 177.1 163.2d159.8 168.3 154.7c149.2 158.2 143.8b142.4 151.0 136.8a

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58.7 62.7 58.5d56.2 59.6 55.7c54.2 57.5 53.7bc51.8 54.6 51.3ab49.3 51.8 48.8a

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Table 3Postharvest life at 5 ◦C of ‘Granny Smith’ apple slices dipped in DETANO, phosphatebuffer and water before or after dipping in chlorogenic acid solution.

Expt. Chlorogenic acidconc. (g/100 g)

Treatment Postharvest life(days)

1 0.1 Untreated 3.8Water 4.5Chlorogenic acid <1 ha

Water + chlorogenicacid

<1 ha

Phosphatebuffer + chlorogenicacid

2.3

DETANO + chlorogenicacid

2.8

LSD 2.31

2 0.1 Untreated 4.4Water 4.5Chlorogenic acid <1 ha

Chlorogenicacid + water

<1 ha

Chlorogenicacid + phosphate buffer

2.8

Chlorogenicacid + DETANO

3.9

LSD 1.89

3 0.01 Untreated 4.2abWater 4.9bChlorogenic acid ∼2 ha

Water + chlorogenicacid

∼2 ha

Phosphatebuffer + chlorogenicacid

3.3a

DETANO + chlorogenicacid

4.4b

LSD 1.15

4 0.001 Chlorogenic acid 1.4aWater + chlorogenicacid

1.7a

Phosphate buffer 7.0bPhosphatebuffer + chlorogenicacid

6.5b

DETANO 8.7cDETANO + chlorogenicacid

7.3b

LSD 1.38

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

Table 4Postharvest life at 5 ◦C of ‘Granny Smith’ apple slices dipped in SNP and Piloty’s acidin water for 5 min at 20 ◦C.

Dip conc. (mg/L) Postharvest life (days)

SNP Piloty’s acid

Untreated 3.5 3.9Water 4.4 5.010 5.5 6.850 6.3 5.2100 7.1 7.5500 7.9 6.0750 a 5.3

0 R. Huque et al. / Postharvest Bio

iven). In case the lack of an effect was specific to MDA, hydrogeneroxide was used in Season 2 as the biomarker for lipid peroxi-ation, but again no significant difference between treatments wasound with slices showing about 0.7 �mol H2O2/g (data not given).

.3. Relationship between fruit parameters and postharvest life

From Table 2 it can be seen that the effect of the treatmentsn respiration, total phenol content, PPO activity and ion leakageas evident at the first analysis time of 2 days after treatment and

he magnitude of differences between treatments did not markedlyhange on further storage. Thus, the mean values for each treatmentiven in Table 2 can represent the comparative level of each factor.

The relationships between the mean values for respiration, totalhenols, PPO activity and ion leakage and browning as expressedy the postharvest life (as given by the mean values in Table 1)ere examined by linear regression analysis. The data in Fig. 1

how that there was a significant inverse linear relationship with areater postharvest life associated with a lower rate of respirationP < 0.001), total phenols (P < 0.001), PPO activity (P < 0.01) and ioneakage (P < 0.05).

.4. Effect of added chlorogenic acid on postharvest life

The effect of dipping apple slices in aqueous solutions contain-ng chlorogenic acid on the development of browning of fresh-cutpple slices was examined in conjunction with dipping in DETANOn phosphate buffer. Two sets of experiments were conducted with.1% chlorogenic acid. In the first experiment, apple slices wereipped in DETANO, phosphate buffer and water and after 5 minere then dipped into 0.1% chlorogenic acid solution while in the

econd study, the dipping order was reversed. The data in Table 3how that in both experiments, browning developed within 1 h inlices dipped in chlorogenic acid or in water plus chlorogenic acidompared to about 4 days for water-dipped or untreated slices.owever, dipping in DETANO solution and the buffer negated muchf the effect of chlorogenic acid with no significant difference withater and untreated slices. There was also no significant differencehether the treatments were applied before or after dipping in

hlorogenic acid solution.All further experiments were conducted with the chlorogenic

cid dip applied as the second dip. Application of 0.01% chloro-enic acid resulted in a slightly longer postharvest life of about 2 hor chlorogenic acid-treated slices (Experiment 3, Table 3). Priorpplication of a DETANO dip negated the effect of chlorogenic acids evidenced by a postharvest life of 4.4 days which was not signif-cantly different to water-dipped and untreated slices which hado added chlorogenic acid. Dipping slices in buffer also negatedost of the effect of chlorogenic acid but the postharvest life was

ignificantly lower than for the DETANO dip.Application of 0.001% chlorogenic acid resulted in the posthar-

est life of slices being about 1.5 days (Experiment 4, Table 3). Theddition of a DETANO dip largely negated the effect of chlorogeniccid although the postharvest life of the DETANO-only dip was stillignificantly greater (P < 0.05) than the combined treatment. How-ver, the buffer dip fully negated the effect of the added chlorogeniccid with both treatments not significantly different.

.5. Effect of SNP and Piloty’s acid on postharvest life

‘Granny Smith’ apple slices dipped in SNP dissolved in waterhowed a significant increase in postharvest life through delayed

nset of surface browning with dipping in 500 mg/L SNP resultingn the longest postharvest life (Table 4). In a separate experiment,pple slices dipped in Piloty’s acid dissolved in water also showed

significantly longer postharvest life with dipping in 100 mg/L

1000 a

LSD 0.75 0.95

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

R. Huque et al. / Postharvest Biology and Technology 78 (2013) 16–23 21

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Table 5Postharvest life at 5 ◦C of ‘Granny Smith’ apple slices treated at 20 ◦C with the opti-mum concentration of different forms of NO.

Treatment Postharvest life (days)

Water for 5 min 4.3a10 �L/L NO gas for 1 h 6.1b100 mg/L Piloty’s acid for 5 min 6.3b500 mg/L SNP for 5 min 7.6c10 mg/L DETANO for 5 min 9.1d

The lack of a significant effect of NO on MDA or hydrogen

PO activity and ion leakage of Granny Smith apple slices during storage at 5 ◦C.ach point is the mean of 6 replicates with 3 units per replicate.

esulting in the longest postharvest life. Apple slices treated with50 mg/L SNP and 1000 mg/L Piloty’s acid caused damage to slicesy rapidly softening the flesh to an unacceptable level.

Comparison was made of the effect of the optimum concentra-ion of SNP and Piloty’s acid dissolved in water with DETANO inhosphate buffer and fumigation with NO gas to inhibit brown-

ng. The results in Table 5 show that all forms of NO extended theostharvest life of apple slices over water but 10 mg/L DETANOnd 500 mg/L SNP solutions were the most effective treatments

ith DETANO significantly more effective than SNP. The posthar-

est life of apple slices fumigated with 10 �L/L NO gas and dippedn 100 mg/L Piloty’s acid solution were not significantly different.

LSD 1.23

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

3.6. Effect of cutting slices with a metal or ceramic knife onpostharvest life

A study examined the development of browning of apples slicesthat had been cut from the whole fruit with a stainless steel or aceramic knife then dipped or not dipped in water at 20 ◦C withall slices stored at 5 ◦C. The postharvest life of apple slices cutby the metal and ceramic knives then dipped in water (4.8 and5.0 days, respectively) had a significantly longer postharvest life(P < 0.001) than those not dipped in water (3.3 and 3.4 days, respec-tively). There was, however, no significant difference in postharvestlife between the metal and ceramic knife-cut apple slices whetherdipped or not dipped in water.

4. Discussion

The inhibition of browning, as reflected in extension of posthar-vest life, of ‘Granny Smith’ apple slices with NO and the associatedcontrol treatments as shown by Pristijono et al. (2006, 2008) hasbeen shown to be quantitatively associated with a decrease in res-piration rate, phenol content, PPO activity and ion leakage but wasnot correlated with ethylene production or lipid peroxidation.

The reduced the rate of respiration in apple slices suggests thatNO has an anti-senescent action which could be a general reductionin the rate of cellular metabolism. This is consistent with Millar andDay (1996) and Zottini et al. (2002) who reported that NO affectsthe function of mitochondria in plant cells and reduces cell respira-tion by inhibiting the cytochrome pathway. The lack of a significanteffect of NO on ethylene production was surprising as the mode ofaction of NO is often attributed to an antagonistic effect againstethylene (Leshem, 2000). This lack of effect of NO is probably dueto the apples being post-climacteric and thus having a substantialproduction of ethylene. Nevertheless, the data tend to suggest thatethylene is either not a direct causative factor in surface brown-ing of apples or NO can inhibit browning through other modes ofaction.

The reduction in PPO activity due to NO is consistent with therole for PPO activity in enzymic browning that has been exten-sively reported for intact and fresh-cut fruit and vegetables. It isgenerally considered that PPO catalyses the oxidation of pheno-lic compounds to quinones which then condense to form brownpolymers (Milani and Hamedi, 2005). The concomitant reductionof total phenols suggests that inhibition of browning could be dueto a lower level of phenol available to be oxidized in conjunctionwith reduced PPO activity. The reduced rate of ion leakage foundin NO-treated slices is indicative of NO assisting in maintainingmembrane integrity and thereby reducing the rate of electrolyteleakage. It is possible that this decreases the release of brown-ing precursors such as phenols from cells to the surface of thecut fruit.

peroxide in apple slices would imply that NO has no effect inmitigating any oxidative damage on the surface of apple slicescaused by reactive oxygen species (ROS). NO has previously been

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eported to reduce the MDA content of intact longan (Duan et al.,007) and kiwifruit (Zhang et al., 2007; Zhu et al., 2008) andydrogen peroxide in intact kiwifruit (Zhang et al., 2007). If lipideroxidation is involved in the browning of apple slices, brown-

ng could be inhibited by NO affecting other some aspect of appleetabolism.The effect of the NO treatments and the three control treatments

phosphate buffer, water and untreated) in showing an inverse lin-ar relationship between respiration, total phenols, PPO activitynd ion leakage with postharvest life suggests firstly, that, whateverhe metabolic sequence induced by cutting that leads to browning,ll treatments including a water-dip, were having a similar affectn inhibiting that pathway but with different levels of effective-ess. Since the differences between treatments were evident by 2ays after application, the action of cutting would seem to haveriggered induction of the browning sequence and that this induc-ion was inhibited to varying extents by the dipping treatments andO gas fumigation. An obvious consideration is that the metal knifesed to cut the slices could have left traces of metal which catalysedome enzyme sequence, but the lack of any difference in browningetween the ceramic and metal knives suggests that this does notccur.

Considering a scenario in which surface browning is due to leak-ge of substrates from cells to the cut surface that react to producerowning, and where phenol content and PPO activity are both

mplicated in the browning sequence, an obvious question is – areither or both phenols and PPO the key metabolites involved inhe slower development of browning caused by the treatments?o assist in answering this question, the major phenolic compoundn apple, chlorogenic acid (Lee et al., 2003) applied to apple slicest 0.1 and 0.01% showed virtual instant surface browning while an.001% solution resulted in browning after 2 days compared to 4–5ays for water dipped slices. This could imply that sufficient PPOctivity is present on the cut surface at harvest to cause browningnd that phenols are the rate limiting compounds. The addition ofETANO largely negated the action of chlorogenic acid suggest-

ng that NO was able to inhibit the involvement of chlorogeniccid in browning reactions. However, the effect of the phosphateuffer alone in negating the effect of chlorogenic acid suggestshat it also inhibits the reaction of phenols with PPO although NOnd the buffer could have different modes of action. The smallut significant benefit of a water dip could be to remove reac-ive material, possibly phenols, from the cut surface. The actionf the treatments in reducing respiration could be an indication ofeduced general metabolism and hence better retention of cellularntegrity. This would lead to a reduced rate of ion leakage and hence

lower rate of release of metabolites involved in browning on theut surface.

The current findings extend the effect of NO in inhibiting brown-ng to SNP and Piloty’s acid. The relative effectiveness of the appliedreatments in absolute terms was found to be DETANO > SNP > NOas = Piloty’s acid. However, the effectiveness of the treatmentseeds to factor in the beneficial effect of a phosphate buffernd water dip on browning development. For example, while theETANO treatment was more effective than NO gas, DETANO andO gas resulted in a 50% and 100% increase in postharvest lifever their respective control treatments of phosphate buffer andntreated. Thus, NO gas had a proportionately greater effect over itsontrol than did DETANO. The various NO compounds release dif-erent forms of NO – DETANO and NO gas release the NO· free radicalhile SNP releases the NO+ cation and Piloty’s acid releases the NO−

nion (Hou et al., 1999; Hughes and Cammack, 1999; Saavedra and

eefer, 2002). It might be expected that each NO moiety has differ-nt reactivity on browning inhibition of apple slices but it wouldeem that all released NO moieties are inter-convertible to somextent.

d Technology 78 (2013) 16–23

5. Conclusions

While inhibition of browning of apples slices by NO and associ-ated control treatments was associated with reduced respiration,ion leakage, PPO activity and phenol content, the interaction ofthese treatments with added chlorogenic acid suggests that accu-mulation of phenols on the cut surface is a key step in browningdevelopment possibly in conjunction with reduced PPO activity.

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