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Postharvest Biology and Technology 57 (2010) 52–60
Contents lists available at ScienceDirect
Postharvest Biology and Technology
journal homepage: www.elsevier.com/locate/postharvbio
Effects of calcium ascorbate treatments and storage atmosphere on antioxidant
activity and quality of fresh-cut apple slices
Encarna Aguayoa,∗, Cecilia Requejo-Jackmanb, Roger Stanleyb, Allan Woolfb
aPostharvest and Refrigeration Group, Department of Food Engineering. Technical University of Cartagena, P◦Alfonso XIII, 48, 30203 Cartagena, Murcia, Spain
bThe New Zealand Institute for Plant & Food Research Ltd, Private Bag 92169 Auckland, New Zealand
article info
Article history:
Received 17 July 2009
Accepted 1 March 2010
Keywords:
Minimally processed
‘Braeburn’ slices
AOS
Phenolic compounds
Vitamin C
Ascorbic acid
Low oxygen
FRAP
DPPH
abstract
Fresh-cut ‘Braeburn’ apple slices were dipped in calcium ascorbate (CaAsc; 0, 2, 6, 12 and 20%, w/w) and
stored in air or under modified atmosphere (MA) conditions for up to 28 d at 4◦C. Changes in antioxidant
levels were measured using free radical scavenging activity (DPPH), reducing activity (FRAP), ascorbic
acid content (AA) and polyphenolic content (by HPLC). Changes in browning, sensory quality and micro-
bial counts were measured to indicate eating quality. CaAsc dips increased the initial levels of AA from
0.19 g kg−1in the untreated control to 3.8 g kg−1for the 20% CaAsc treatment. Ascorbic acid content of
treated slices during storage decreased by more than 50% in CaAsc concentrations of 6, 12 and 20%. Sim-
ilar patterns were observed for FRAP and DPPH activities. Untreated or 2% CaAsc treated slices stored in
air or MA showed browning, microbial deterioration and poor sensory quality, thus resulting in a short
shelf life (<7 d). However, apples dipped in 6 or 12% CaAsc and stored in MA packaging, or dipped in 20%
CaAsc and packaged in air or MA had a shelf life of 21–28 d. Total antioxidant activity in these treatments
was provided by both exogenous ascorbic acid and endogenous phenolic compounds; the latter varied in
composition, but were relatively stable during storage compared with ascorbate in higher CaAsc concen-
tration treatments. Thus, the antioxidant levels (as measured by FRAP and DPPH) were related to shelf life
and it appears that an antioxidant activity remaining above 2 gkg−1(DPPH or FRAP) may be a minimum
level to achieve long shelf life in ‘Braeburn’ apple slices.
© 2010 Elsevier B.V. All rights reserved.
1. Introduction
Sales of fresh-cut product continue to grow through consumers’
increasing willingness to pay for prepared, ready-to-eat or ready-
to-use fresh produce (Rico et al., 2007). A range of treatments have
been applied to extend the shelf life of fresh-cut apples includ-
ing use of natural browning inhibitors (Luo and Barbosa-Canovas,
1996; Buta et al., 1999; Rojas-Grau et al., 2006), salt and chemi-
cal treatments (Gil et al., 1998; Zuo and Lee, 2004; Varela et al.,
2007), coating agents and reduced oxygen atmospheres (Anese et
al., 1997; Rocculi et al., 2004; Pérez-Gago et al., 2006).
A key approach used to avoid browning in apples has been the
use of reducing agents, often with the addition of calcium chloride
(CaCl2),in combination with modified (reduced O2) atmospheres
and low temperature storage (Sapers et al., 1990; Luo and Barbosa-
Canovas, 1996). Tortoe et al. (2007) observed moderate browning
on ‘Golden Delicious’ apple slices using ascorbic acid (AA, 0.5 M
with or without sodium chloride) stored at 4 ◦Cupto14d.Son et
al. (2001) also reported the effect of AA on browning of fresh-cut
∗Corresponding author. Tel.: +34 968 325750; fax: +34 968 325435.
E-mail address: encarna.aguayo@upct.es (E. Aguayo).
apples. Selected combinations of treatments have been shown to
be effective in the prevention of browning of apple slices for up to
eight weeks at 0.2 ◦C(Anese et al., 1997). While flesh browning may
be minimised during extended storage, other organoleptic factors
such as texture and flavour may not be acceptable. An understand-
ing of the physiology behind such changes and the development of
mechanisms to prevent them is required to improve the shelf life
of fresh-cut products.
The role of oxidative-related senescence in postharvest quality
loss is well recognised (Hodges, 2003), but quality loss due to accel-
erated postharvest senescence is not consistently associated with
loss of antioxidant concentration or activity (Hodges, 2003). Active
oxygen species (AOS) have been associated with induced or natural
senescence processes (Hodges and Forney, 2000). Changes in lipid
membranes as a result of oxidative stresses can trigger lipoxyge-
nase degradation and the generation of biological signals that result
in apoptosis and necrosis (Spiteller, 2003).
Levels of AOS are regulated by their relative rates of genera-
tion and degradation which includes scavenging by enzymatic and
non-enzymatic antioxidants (Hodges, 2003). In vivo, the intrinsic
antioxidant content is compartmentalised in different organelles
and may have variable impacts on senescence and programmed
cell death. Calcium plays a pivotal role in cell signals related to
0925-5214/$ – see front matter © 2010 Elsevier B.V. All rights reserved.
doi:10.1016/j.postharvbio.2010.03.001
E. Aguayo et al. / Postharvest Biology and Technology 57 (2010) 52–60 53
AOS (Bhattacharjee, 2005). Calcium dips have been implicated in
enhancing membrane stability, slowing senescence, and improving
the retention of membrane integrity (Picchioni et al., 1996).
Exogenous antioxidant treatments should be able to interfere
with senescence-related oxidation reactions but there is little direct
evidence. Antioxidant dips for fresh-cut apples reduce the brown-
ing and presumably also reduce the levels of AOS. Ideally, there
should be an efficient level of antioxidant which would decrease or
even neutralise the AOS, resulting in prevention of induced senes-
cence and thus, extension of shelf life. Apple fruit contain up to
0.25 g kg−1ascorbic acid antioxidant. In our opinion, this could
be supplemented by fresh-cut dips with exogenous ascorbate or
other antioxidants. Apples also contain large amounts of endoge-
nous phenolic antioxidant compounds (Kondo et al., 2002) that
react with oxidisers in damaged tissues to form enzymatic brown-
ing products. The fate of the added antioxidants and the influence
of the resulting levels of antioxidants on the quality of the resulting
fresh-cut produce are not known.
Since calcium ascorbate (CaAsc) is becoming the commercial
“industry standard” anti-browning treatment for fresh-cut apples,
this study sought to investigate the influence of the concentration
of CaAsc dips, subsequent storage time and modified atmosphere
conditions on antioxidant status and shelf life of fresh-cut apple
slices.
2. Materials and methods
2.1. Raw material
New Zealand grown ‘Mariri Red’ apples (Malus domestica Borkh.,
a sport of ‘Braeburn’) were sourced from coolstores of a commercial
supermarket and transported to the laboratory and stored at 0 ◦C
for 12 h. This apple variety was chosen due to high level of ascor-
bic acid (Laing et al., personal communication) and is an important
commercial cultivar. The apple boxes were opened in a food-grade
processing room (10 ◦C) and the fruit sorted to remove those dam-
aged or with significant variation in background colour. Whole
apple surfaces were sterilised by dipping in cold water (4 ◦C) with
5mgL
−1chlorine dioxide (OxineTM, Australasia Marketing Pty Ltd,
Sydney, NSW, Australia) for 15 min. The apples were then manu-
ally cored and cut into eight slices (using a handheld slicer). This
wedge cut allowed maximum use of the apple. Slices were dipped
into cold water (0 ◦C) with 2 mg L−1chlorine dioxide for 2 min, and
the slices drained. CaAsc solutions were made up using water at 0 ◦C
pre-treated with 2 mg L−1chlorine dioxide then made up to the dif-
ferent concentrations using CaAsc (99.9% purity, Wolf Canyon Asia
Pacific Ltd) at 0 (control), 2, 6, 12 and 20% (w/w). Slices were then
dipped in these CaAsc solutions for 2 min and drained. Apple skins
were not removed prior to treatment, as apple slices are currently
marketed with skin intact.
2.2. Packaging of apple slices and storage
Apple slices were randomised across packages of 15 apple slices
(350 ±20 g) per aluminium bag (25 cm ×18 cm, 80 m thickness,
Caspak, New Zealand). To maintain near-ambient oxygen con-
centration in the air treatment bags, a 5-mm hole was punched
through both sides of each bag. For the modified atmosphere (MA)
treatment, most of the air inside the bags was extracted by a vac-
uum machine and the bags were heat-sealed (vacuum impulse
seal, Model ME-4510VG, Mercier Corporation, MHsing-Chuan City,
Tapei Hsien, Taiwan).
Three replicate bags per treatment were stored at 4 ◦C per stor-
age duration: 7, 14, 21 and 28 d. Measures of antioxidant activity
were also measured on day 0 (immediately after treatment).
2.3. Parameter evaluations
2.3.1. Gas measurements
Gas composition (O2and CO2) within the bags was monitored
weekly until the end of storage using a GC (using an infra-red CO2
transducer Servomex 1505, Servomex Ltd., Sussex, UK) and oxy-
gen sensor (CiTicel oxygen cell, Model C/2, City Technology Ltd.,
London). For analysis, 0.5 mL gas samples were taken from each
bag using a plastic syringe through a silicone septum. At the same
time, the C2H4levels were determined by injection of 1 mL gas
samples into a GC (Philips PU4500; Pye Unicam, Cambridge. U.K.)
equipped with an active alumina column and a flame ionisation
detector. Three replicates were made for each treatment and eval-
uation period. Laboratory conditions for temperature were 25 ◦C
and 1 atm (≈101 kPa).
2.3.2. Colour measurement
The surface colour of the apple flesh was determined
on three equidistant points in each apple slice cut surface
with a Minolta chromameter (Model CR-300 Minolta; Ram-
sey, NY). The results were expressed as CIELAB (L*a*b*) colour
space. L* defines the lightness and a* and b* define the red-
greenness and blue-yellowness, respectively. The flesh colour
was also measured and expressed as hue angle (h◦= arctangent
[(b*)(a*)−1]), chroma (C*=[(a*)2+(b*)2]1/2) and whiteness index
(WI = 100 −[(100 −L*)2+(a*)2+(b*)2]1/2) according to Bolin and
Huxsoll (1991). The more representative colour parameters (h◦and
WI) are reported in the results. Fifteen slices per treatment (five
slices per replicate) were measured.
2.3.3. Sensory evaluation
A panel of five people was trained to recognise and score the
quality attributes of the treated apple slices. All assessments were
compared to slices freshly cut from whole air-stored apples of
the same variety and purchase data. Appearance, taste and tex-
ture were scored on a nine-point scale where 1 = complete lacking
or soft, to 9 = fully characteristic of fresh. A similar scale, where
1 = inedible, 3 = poor, 5 = fair (limit of marketability), 7 = good and
9 = excellent was used for the evaluation of the overall acceptability.
Only appearance and overall acceptability data are presented.
2.3.4. Microbial analyses
Microbial growth on the slices was determined after 28 d stor-
age by a certified laboratory (AgriQuality, Auckland, New Zealand).
From each of five slices, 10 g samples were blended with 90 mL
of sterile peptone buffered water (Merck Darmstadt, Germany) for
1 min in a sterile stomacher bag (Model 400 Bags 6141, London,
UK) using a Masticator (Colwort Stomacher 400 Lab, Seward Medi-
cal, London, UK). Appropriate dilutions were prepared. Plate Count
Agar medium (PCA, Merck) was used for TPC and Rose Bengal agar
medium (Merck) for yeast counts. Incubation conditions were 30 ◦C
for 48 h for TPC, and 22◦C for 5 d for yeasts, respectively. Microbial
counts were reported as log10 colony forming units per gram of
sample (log cfu g−1).
2.3.5. Chemical measurements
Fruit pieces were flash frozen in liquid nitrogen and stored at
−80 ◦C for a maximum of two months. To ensure uniformity, frozen
samples (200 g) were either homogenised in 100 mL of distilled
water in a commercial blender (Sunbeam Model PB7600, Type 504,
230–240 V, Auckland, New Zealand) to produce a juice extract (for
the antioxidant activity analysis), or 150 g was ground to a fine pow-
der in a Cryomill in liquid nitrogen (for ascorbic acid content (AA)
analysis).
Antioxidant activity. Two assays were used to measure the
antioxidant activity; 2,2-diphenyl-1-picrylhydrazyl (DPPH) and
54 E. Aguayo et al. / Postharvest Biology and Technology 57 (2010) 52–60
ferric reducing antioxidant power (FRAP) assays. In both cases, to
obtain an accurate absorbance reading, the juice (1, 0.5 or 0.1 mL)
was diluted in a volume of methanol (0, 0.5 or 0.9 mL) according
to the concentration of the CaAsc treatment (0, 2 and 6% or 12
and 20% of CaAsc, respectively). The results were multiplied for the
dilution factor (1, 2 or 10, respectively). The DPPH analysis, which
determines the free radical scavenging activity, was conducted
according to Brand-Williams et al. (1995) using DPPH reagent. The
decrease in absorbance at 515 nm was measured by spectropho-
tometer (Spectromax Plus 384, Molecular Devices, Sunnyvale, CA,
USA) after 20 min incubation at room temperature.
The reducing activity was measured using the FRAP assay
according to Benzie and Strain (1996) with some modifications.
FRAP reagent contained 25 mL of 300 mM acetate buffer (pH 3.6),
2.5 mL of 10 mM TPTZ (2,4,6-tris 2-pyridyl-s-triazine) solution in
40 mM HCl and 2.5 mL of 20 mM ferric chloride (FeCl3·6H2O). This
reagent was freshly made up each day. The assay was performed
using 198 L of warmed (37 ◦C for 2 h) FRAP reagent mixed with
6L of the corresponding juice homogenate and incubated for
20minat20
◦C. The ferric reducing ability of apple extracts was
measured by the increase of absorbance at 593 nm.
All the antioxidant assays were carried out in triplicate.
Calibration curves were made for each assay using Trolox (6-
hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid) and AA
as standards. The antioxidant activity (DPPH, FRAP assay) was
expressed as grams of Trolox and AA equivalent antioxidant activity
per kilogram fresh weight of apple tissue.
Ascorbic acid evaluation. The method was adapted from Rassam
and Laing (2005). A 0.2 g sample of powdered apple tissue was
suspended in 1 mL of 6% metaphosphoric acid, 2 mM EDTA and
1% PVPP containing 4 mM TCEP (tris[2-carboxyethyl] phosphine
hydrochloride). The slurry was vortexed for 20 s, and incubated
in a heating block for 2 h at 40 ◦C to ensure full reduction of any
dehydroascorbate. The extract was clarified by centrifugation at
4◦C for 10 min, and 20 L of the supernatant was injected into
a 7.8 mm ×300 mm Aminex HPX-87H HPLC column (Bqsio-Rad,
Merck, Darmstadt, Germany). The column was run with 2.8 mM
H2SO4as the mobile phase, at a flow rate of 0.6 mL min−1. The
amount of AA was detected using a Waters 996 photodiode-array
detector set at 245.5 nm for absorbance using AA (Sigma, St. Louis,
MO) as a standard. The peak was authenticated as AA by showing
that it was completely degraded by ascorbate oxidase at pH 5.5.
Phenolic compounds evaluation. One millilitre of apple juice
extract was combined with 0.25 mL of methanol and HCl (90:10,
v/v) and vortexed for 20 s. A clear extract was made by cen-
trifugation at 10,000 rpm for 10 min and the supernatant was
injected into the HPLC following the method of Kammerer et
al. (2007). Polyphenol analyses were carried out using a Shi-
madzu HPLC system (Kyoto, Japan). The separation was performed
with a Phenomenex (Torrance, CA, USA) C18 Synergi Hydro-RP
(250 mm ×4.6 mm i.d., 4 m particle size), with a C18 ODS guard
column (4.0 mm ×3.0 mm i.d.), operated at 35 ◦C. The mobile phase
consisted of 0.1% formic acid in acetonitrile (v/v) (eluent A) and 0.1%
formic acid in water and acetonitrile (95:5, v/v; eluent B) using
a gradient programme following Kammerer et al. (2007). Chro-
matograms were recorded at 280 and 340 nm at a flow rate of
0.017 mL s−1. Peak assignment was performed by comparison of the
retention times and UV spectra with those of reference compounds
and by mass spectrometric analyses. The phenolic compounds are
expressed as gram per kilogram fresh weight of apple tissue. LC–MS
employed an LCQ Deca ion trap mass spectrometer fitted with an
ESI interface (ThermoQuest, Finnigan, San José, CA, USA) coupled to
a SurveyorTM HPLC and PDA detector. Phenolic compound separa-
tion was achieved using a Prodigy 5ODS(3) 100 Å (Phenomenex,
Torrance, CA, USA), 150 mm×2 mm analytical column maintained
at 35 ◦C. Solvents were (A) acetonitrile + 0.1% formic acid and (B)
water + 0.1% formic acid and the flow rate was 3.3 Ls
−1. The ini-
tial mobile phase, 5% A/95% B, was held for 5 min then ramped
linearly to 10% A at 10 min, 17% A at 25 min, 23% A at 30 min,
30% A at 40 min and 97% A between 48 and 53 min before reset-
ting to the original conditions. Sample injection volume was 10 L.
UV–vis detection was by absorbance at 200–600 nm. MS data were
acquired in the negative mode using a data-dependent LC–MS3
method with dynamic exclusion enabled and a repeat count of
2. The ESI voltage, capillary temperature, sheath gas pressure and
auxiliary gas were set at −47 V, 300 ◦C, 345 kPa, and 690 kPa, respec-
tively.
2.4. Statistical analysis
A randomised design with three replicates per treatment was
used where each bag constituted a replicate. To determine the
effect of storage time, CaAsc concentration and type of packag-
ing on each dependent variable, a three-way analysis of variance
(ANOVA, P< 0.05) was carried out (Statgraphic Plus, (version 5.1,
2001), Manugistic Inc, Rockville, MD, USA). Mean values were com-
pared by LSD (multiple range least significant difference test) to
identify significant differences among treatments and significant
interactions between factors.
3. Results
3.1. Storage atmospheres
The control/air bags (with punched holes) resulted in an atmo-
sphere close to ambient (<0.5 kPa CO2and >20 kPa O2, data not
shown). In the MA bags, the O2concentration of 20.8 kPa at day 0
reduced to ∼
=3–5 kPa after 7d, and then to ∼
=1 kPa by day 28 (Fig. 1).
Over the storage period, CO2increased from ambient to 16–20 kPa
after 7 d and to ∼
=30–35 kPa after 28 d. There was no consistent
effect of CaAsc concentration on the resulting atmospheres in the
bags.
Ethylene levels increased in all treatments over two weeks
of storage to 124–180 LL
−1, and after this decreased to
80–100 LL
−1(data not shown). Apple slices treated with 20%
CaAsc were the only treatment that had lower C2H4production
during storage than the control. This difference was greater ini-
tially (∼
=30% of the other treatments at day 7, 80% by day 14, and
not significantly different by day 28).
Fig. 1. Changes in O2and CO2levels of fresh-cut apple dipped into 0, 2, 6, 12 or 20%
calcium ascorbate solution, packaged in modified atmosphere and stored for 28 d at
4◦C. Means of three replicates ±S.E. Solid mark: kPa of O2. Clear mark: kPa of CO2.
Laboratory conditions for temperature were 25 ◦C and 1atm (≈101 kPa).
E. Aguayo et al. / Postharvest Biology and Technology 57 (2010) 52–60 55
Fig. 2. ◦Hue of fresh-cut apple dipped into 0, 2, 6, 12 or 20% of calcium ascor-
bate solution, packaged in air or modified atmosphere and stored for 28 d at
4◦C. Solid mark and continuous line: apples packaged in air. Clear mark and
dashed line: apples packaged in MA. Analysis of variance showed as significant all
simple factors (time of storage, CaAsc dips and kind of packaging) as the inter-
actions (time ×CaAsc dips ×kind of packaging). LSDs (5%) of significant effect:
LSDtime×CaAsc dips ×packaged = 1.84.
3.2. Colour
Immediately after CaAsc treatment, the hue angle (h◦) of apple
slices was higher (indicating slightly more green colour) for treated
than for control fruit (Fig. 2). Over time in storage, control slices
stored in air showed a consistent decrease in h◦as tissue browned.
Storing control (untreated) slices under MA conditions resulted in
no significant decrease in h◦over 28 d. For slices dipped in CaAsc,
there was a slight linear decrease in h◦over the storage time.
There was a significant effect of MA conditions on h◦during storage
(P< 0.05).
CaAsc dips also increased the whiteness colour in the slices
(Fig. 3). CaAsc treatments of 2 and 6% resulted in whiter pulp tissue
than the 12 or 20% treatment. Whiteness levels were similar after
28 d with only minor differences between treatment slices stored
in air or MA.
Fig. 3. Whiteness index of fresh-cut apple dipped into 0, 2, 6, 12 or 20% of cal-
cium ascorbate solution, packaged in air or modified atmosphere and stored for
28dat4
◦C. Solid mark and continuous line: apples packaged in air. Clear mark
and dashed line: apples packaged in MA. Analysis of variance showed as signifi-
cant simple factors: time of storage, CaAsc dips and kind of packaging. Significant
interactions: time ×CaAsc dips and CaAsc dips ×packaged. LSDs (5%) of significant
effects: LSDtime×CaAsc dips = 0.58; LSDCaAsc dips ×packaged = 0.37.
Fig. 4. Appearance of fresh-cut apple dipped into 0, 2, 6, 12 or 20% of calcium
ascorbate solution, packaged in air or modified atmosphere and stored for 28 d
at 4 ◦C. Data based on hedonic scale where 1 =unusable, 3 =poor, 5 =fair (limit of
marketability), 7 =good and 9= excellent. Solid mark and continuous line: apples
packaged in air. Clear mark and dashed line: apples packaged in MA. Analysis of
variance showed as significant simple factors: time of storage, CaAsc dips and kind
of packaging. Significant interactions: time ×packaged and CaAsc dips×packaged.
LSDs (5%) of significant effects: LSDtime×packaged = 0.68; LSDCaAsc dips ×packaged = 0.68.
3.3. Sensory results
The appearance of slices was improved by dipping in CaAsc solu-
tions. Immediately after treatment, the average appearance rating
increased from 6 points for the untreated control to 7.7–8 points for
CaAsc treatments of 6, 12 and 20% (Fig. 4). Slices stored under MA
had a better appearance than slices packaged in air. This effect was
most pronounced for CaAsc dip solutions of 2 and 6% where the 2%
treatment packaged in air scored under the limit of marketability
after only one week of storage. After 28 d, slices treated with 6, 12
and 20% CaAsc packed under MA were marketable, as were 12 and
20% packed in air, while 6% CaAsc dips packed in air scored just
under the limit of marketability.
In general, slices maintained good texture during the whole stor-
age period and only the control slices packaged in air scored under
the limit of marketability (data not shown). Modified atmosphere
packaging delayed loss of firmness compared with the control slices
(0% CaAsc) and calcium from CaAsc dips resulted in firmer tissue
than the control slices (0% CaAsc) following 28 d of cool storage
(data not shown).
Sensory analysis detected a mouldy aroma after 14 d for the
control treatment (no CaAsc) and for the 2% CaAsc packaged in air
(data not shown). After 28 d storage in air, this effect was also found
in slices dipped in 6% CaAsc. In addition, a slight ‘off-flavour’ was
reported in slices dipped in 12 and 20% CaAsc and packaged in air
storage (data not shown).
Taking into account the combination of sensory parameters such
as appearance, texture, aroma, taste, and off-flavour, the overall
acceptability score was found to be the highest for 6, 12 and 20%
CaAsc treatments stored under MA, and after 28 d there was little
difference between these three treatments (Fig. 5). A high score was
also found for the 20% CaAsc treated slices packaged in air.
3.4. Microbial analyses
At the end of storage, only samples that scored above the limit
of marketability for appearance were analysed for microbial count.
The 12 and 20% CaAsc dips resulted in decreased microbial counts
when stored in MA packaging (Table 1). After 28 d storage, the
56 E. Aguayo et al. / Postharvest Biology and Technology 57 (2010) 52–60
Fig. 5. Overall quality of fresh-cut apple dipped into 0, 2, 6, 12 or 20% of calcium
ascorbate solution, packaged in air or modified atmosphere and stored for 28 d
at 4 ◦C. Data based on hedonic scale where 1=unusable, 3 =poor, 5 =fair (limit of
marketability), 7 =good and 9= excellent. Solid mark and continuous line: apples
packaged in air. Clear mark and dashed line: apples packaged in MA. Analysis of
variance shows as significant simple factors: time of storage, CaAsc dips and kind of
packaging. Significant interactions: time ×packaged. LSDs (5%) of significant effects:
LSDCaAsc dips = 0.61; LSDtime×packaged = 0.86.
microbial counts for these treatments remained relatively low for
a fresh-cut food.
3.5. Antioxidant activity
Antioxidant activity was measured using the free radical scav-
enging activity (DPPH) and the ferric reducing ability (FRAP). In
both cases, the values calculated as AA equivalents were very simi-
lar to those found for Trolox equivalents (data not shown) and thus,
results are only presented as AA equivalents (Figs. 6 and 7).
The DPPH assay of antioxidant activity of untreated apples slices
on day 0 was 0.4 g kg−1. This level increased proportionally as the
concentration of CaAsc in the dip solution increased (Fig. 6). Slices
dipped in 20% of CaAsc had 16 times more antioxidant activity
than control (0% CaAsc) on day 0 of storage. DPPH decreased more
markedly for apple slices packaged in air than those in MA (low
O2/high CO2), and this difference was more marked the higher the
CaAsc concentration (Fig. 6). However, neither storage time nor
packaging affected the relatively low antioxidant activity levels of
0 or 2% CaAsc treated slices. In order to maintain an antioxidant
activity greater than the level in the original apple slice (control), a
treatment of at least 6% of CaAsc was required.
The FRAP measure of antioxidant followed similar patterns to
that of the DPPH assay including the effect of storage time and
atmosphere. The FRAP assay for control slices (0% CaAsc) at day 0
Table 1
Microbial counts (mean log cfug−1) of fresh-cut apple dipped into 6, 12 or 20% cal-
cium ascorbate solution then stored in air or modified atmosphere conditions for
28dat4◦C.
Packaging atmosphere CaAsc treatment 28 d at 4◦C
Total plate count Yeast
Air 12% >5.4a>3.2
20% >5.4 >3.2
MA 6% >5.4 3.4
12% 5.2 <2
20% 4.4 <2
aData are the means of three replicates.
Fig. 6. Antioxidant activity (DDPH as ascorbic acid) of fresh-cut apple dipped into 0,
2, 6, 12 or 20% of calcium ascorbate solution, packaged in air or modified atmosphere
and stored for 28 d at 4◦C. Solid mark and continuous line: apples packaged in air.
Clear mark and dashed line: apples packaged in MA. Analysis of variance showed as
significant all simple factors and their interactions. LSDs (5%) of one of the significant
effects: LSDtime×CaAsc dips ×packaged = 0.58. Result expressed as fresh weight.
showed an activity of 1.05 gkg−1(Fig. 7) which was 2.5-fold higher
than the value obtained by the DPPH assay (0.41 g kg−1;Fig. 6).
Similarly, treatment with 2% CaAsc resulted in higher activity as
measured by FRAP as that of DPPH.
3.6. Ascorbic acid determination
The initial AA content of untreated apple slices was of
0.19 g kg−1. Immediately after treatment, AA content was 2.5-, 5-,
13.3- and 20-fold higher for 2, 6, 12 and 20% CaAsc, respectively,
than for the control (Fig. 8). The pattern of decrease with storage
time and atmosphere followed the same trends as those FRAP and
DPPH activities, indicated that these changes were the result of loss
of ascorbic acid content.
3.7. Phenolic compounds
The main phenolic compounds in the untreated (control) apple
slices were procyanidins (B1, B2 and C1) followed by phloridzin,
Fig. 7. Antioxidant activity (FRAP as ascorbic acid) of fresh-cut apple dipped into 0,
2, 6, 12 or 20% of calcium ascorbate solution, packaged in air or modified atmosphere
and stored for 28 d at 4◦C. Solid mark and continuous line: apples packaged in air.
Clear mark and dashed line: apples packaged in MA. Analysis of variance showed as
significant all simple factors and their interactions. LSDs (5%) of one of the significant
effects: LSDtime×CaAsc dips ×packaged = 0.27. Result expressed as fresh weight.
E. Aguayo et al. / Postharvest Biology and Technology 57 (2010) 52–60 57
Table 2
Main phenolic compounds (mg kg−1) of fresh-cut apple slices dipped in 0 (control), 2, 6, 12 or 20% calcium ascorbate solution. Slices were then packaged in air or modified
atmosphere conditions and stored for up to 28 d at 4◦C.
Storage time Storage
atmosphere
CaAsc
treatment
Chlorogenic Quercetin
derivatives
Epicatechin Coumaric
acid
Procyanidins
(B1, B2, C1)
Phloridzin Total
phenol
0d 0% 89.0a10.5 57.5 112.5 192.6 166.9 628.9
2% 86.9 12.3 51.5 115.6 194.6 173.1 633.9
6% 82.0 15.6 54.7 109.8 205.6 178.1 645.7
12% 78.4 14.7 36.5 99.9 170.5 168.9 568.9
20% 80.0 11.2 37.6 94.2 160.8 175.6 567.6
7 d Air 0% 84.7 10.8 46.3 135.5 176.3 156.6 607.3
2% 82.8 11.0 48.9 125.6 184.8 159.1 610.2
6% 80.0 11.0 48.5 109.6 195.1 159.1 603.3
12% 85.7 9.5 47.3 121.0 172.2 150.0 583.8
20% 89.8 10.0 43.4 131.4 185.6 163.7 622.0
MA 0% 79.1 11.2 46.8 133.9 168.9 182.1 625.0
2% 78.9 10.6 41.1 115.9 163.0 164.2 573.7
6% 85.4 11.0 40.6 112.6 156.9 166.7 573.1
12% 88.0 10.9 44.6 115.4 162.6 175.8 597.3
20% 78.4 10.7 37.8 107.6 150.8 157.1 544.4
14 d Air 0% 89.3 12.2 46.7 144.3 173.5 154.0 622.9
2% 92.6 12.3 49.7 145.8 186.7 177.5 664.7
6% 88.7 12.2 52.2 137.3 191.8 158.3 637.7
12% 94.7 12.0 47.7 115.6 205.5 136.2 611.6
20% 90.6 13.8 42.3 112.3 200.2 143.8 602.9
MA 0% 75.8 10.1 46.8 121.7 154.3 158.9 565.4
2% 89.0 10.2 51.3 128.8 177.2 177.6 634.2
6% 82.5 10.8 44.3 130.0 159.4 166.6 593.6
12% 83.6 8.9 39.6 116.1 170.2 154.5 573.0
20% 93.4 7.8 37.1 125.2 159.0 153.2 575.7
21 d Air 0% 94.5 13.7 49.8 149.5 173.8 161.0 642.3
2% 90.9 11.3 48.9 135.8 179.7 145.9 612.6
6% 94.3 15.9 48.5 134.3 189.2 150.5 632.6
12% 89.5 12.3 45.0 118.4 212.1 141.7 619.0
20% 82.2 12.1 42.2 121.7 188.7 141.0 588.0
MA 0% 83.5 9.8 51.5 134.4 182.3 166.1 626.4
2% 92.1 11.7 48.4 140.7 188.3 178.3 662.5
6% 85.9 8.3 46.9 131.1 185.0 150.7 607.8
12% 82.2 9.0 46.6 129.4 191.4 149.4 608.1
20% 78.6 9.2 41.2 122.4 165.2 142.7 559.4
28 d Air 0% 90.3 10.7 47.3 130.6 172.6 160.6 612.1
2% 93.9 11.3 45.2 114.5 168.2 158.4 591.6
6% 96.3 13.1 46.0 109.5 175.4 155.3 595.2
12% 98.0 12.7 41.6 107.2 181.3 144.4 585.5
20% 88.0 10.0 44.9 109.3 189.2 149.9 591.2
MA 0% 86.9 12.1 50.2 130.5 184.5 168.2 632.5
2% 82.5 11.1 51.1 126.1 182.3 170.8 623.9
6% 82.5 10.8 47.4 118.0 177.4 161.5 597.5
12% 82.1 8.8 43.4 106.8 172.8 154.3 572.8
20% 83.8 7.3 40.7 135.6 164.6 149.0 580.9
Time (4.0) (1.3) NS (6.1) NS (7.7) NS
Packaging (2.5) (0.8) (1.2) NS (3.9) (4.9) NS
% CaAsc NS (1.3) (1.9) (6.1) (6.2) (7.7) (20.3)
Time ×packaging NS (1.8) NS NS NS NS NS
Time ×CaAsc NS (2.8) NS NS NS NS NS
Packaging ×CaAsc NS NS NS NS 8.8 NS NS
Time ×packaging ×CaAsc NS (4.0) NS NS NS NS NS
aValues are means (n= 3); NS, not significant. LSD values are in brackets at P<0.05. %CaAsc: percentage of calcium ascorbate in the dips solution. Results expressed as
fresh weight.
coumaric acid, chlorogenic, epicatechin and a small proportion of
quercetin derivatives (3-O-galactoside, 3-O-rhamnoside and 3-O-
glucoside) (Table 2). In the procyanidins group, B1 was 68% and B2
was 28% of the total with procyanidin C1 at just 6%. In the quercetins
derivatives, 3-O-galactoside was 52%, 3-O-rhamnoside was 38% and
quercetin-3-O-glucoside was 10% of the total.
The total phenolic content in control (untreated) slices prior to
storage was 629 mg kg−1and treatment with high concentrations
of CaAsc (12 and 20%) tended to reduce total phenolics (Table 2).
Over storage, phenolics ranged from 540 to 660 mgkg−1, although
only CaAsc treatment had a significant effect while storage time
and atmosphere had none (Table 2).
Although there were some statistically significance changes
in the phenolic content of apple slices (Table 2), the changes
were overall relatively small (≤15%). With increasing storage time,
the concentrations of coumaric acid, chlorogenic and phloridzin
decreased, while coumaric acid and chlorogenic increased slightly.
Phloridzin levels were retained better under MA conditions but
chlorogenic and epicatechin trended to decrease under that con-
dition.
CaAsc treatment resulted in a decrease in coumaric acid, epicat-
echin and phloridzin, while the changes in procyanidins showed an
interaction between packaging and CaAsc treatments. Slices dipped
at 0, 2, 6 or 12% CaAsc packaged in MA, or dips at 6% CaAsc packaged
58 E. Aguayo et al. / Postharvest Biology and Technology 57 (2010) 52–60
Fig. 8. Ascorbic acid content of fresh-cut apple dipped into 0, 2, 6, 12 or 20% of
calcium ascorbate solution, packaged in air or modified atmosphere and stored for
28 d at 4 ◦C. Solid mark and continuous line: apples packaged in air. Clear mark
and dashed line: apples packaged in MA. Analysis of variance showed as significant
all simple factors and their interactions. LSDs (5%) of one of the significant effects:
LSDtime×CaAsc dips ×packaged = 0.29. Result expressed as fresh weight.
in air retained the procyanidins content. However, if the slices were
dipped into 20% CaAsc and stored in air or MA, the procyanidins
concentration tended to decrease.
4. Discussion
We have confirmed the strong anti-browning potential of cal-
cium ascorbate in fresh-cut apples, and the concentrations required
for maintaining antioxidant levels in tissue after treatment and sen-
sory during long storage periods. A treatment of over 6% exogenous
CaAsc was needed to maintain the quality for 28 d at 4 ◦C when
packaged in air, while for slices packed in MA 6% CaAsc was enough.
The retention of overall quality appears to be correlated with the
retention of antioxidant levels (as measured by FRAP) of at least
twice the endogenous level in the apple slices in addition, levels
of antioxidant decreased faster when slices were stored in air as
opposed to when they were packed in MA.
Browning and MA treatments. Various approaches to control the
surface browning of fresh-cut apples have been investigated. Mod-
ified atmospheres (elevated CO2and low O2) and low temperature
storage (4 ◦C) alone have been found to be effective in preventing
browning of fresh-cut apples and pears (Anese et al., 1997), as we
found here in apple. Gorny (1997) concluded that a reduction of
O2levels to near 0 kPa is required to inhibit polyphenol oxidase
(PPO) mediated browning of many fresh-cut fruit products. In our
study, O2concentrations in the MA treatment were low by the end
of the storage time (∼
=1 kPa), and is likely to have been low enough
to effectively delay browning (most likely the action of PPO). How-
ever, we found that regardless of the storage atmosphere, treating
the slices with 6, 12 or 20% CaAsc improved the ◦Hue of the slices
and provided a whiter pulp tissue than non-treated slices. The MA
atmosphere generated in this work was somewhat extreme in that
the CO2levels were high (>30 kPa at the end of storage), although
the low O2(∼
=1 kPa) did avoid the anaerobic limit (since no alco-
hol off-flavours were detected by panellists). Thus, clearly there
is warrant in examining a range of atmospheres and their effect
on slice quality and antioxidant status, preferably under a flow-
through system where CO2and O2concentration can be varied
independently.
Browning and CaAsc treatments. Although MA is an important
tool, the most potent way to reduce, or even eliminate brown-
ing is the use of antioxidant dips, particularly AA, although there
are significant differences in the efficacy of the antioxidant com-
pound (Son et al., 2001). Others have used AA to reduce browning
in apples with good results (Sapers et al., 1990; Luo and Barbosa-
Canovas, 1996). Tortoe et al. (2007) observed moderate browning
on ‘Golden Delicious’ apple slices using AA (0.5 M) with or without
sodium chloride after 14 d at 4 ◦C. Son et al. (2001) also reported the
effect of AA on browning of fresh-cut apples. However, they found
the reducing power of a 1% AA dip was depleted within 20 min,
and that the browning developed rapidly thereafter. Our work has
demonstrated that lower concentrations of CaAsc are less effective,
particularly if a longer shelf life is sought. Thus, to achieve durations
of up to 21 d, a concentration of 6% or more may be required to pre-
vent enzymatic browning. That is, levels of AA of 0.9 g kg−1or higher
were required to directly inhibit the PPO and/or to reduce the oxi-
dation of the o-diphenols at the wounded surfaces to o-quinones,
which polymerise to produce brown pigments (melanin) enzymat-
ically formed (Vamos-Vigyazo, 1981). In addition to the benefit of
CaAsc in reducing browning, the slower softening observed here
may have been related to the increased calcium level in fruit tis-
sue (Fallahi et al., 1997). Stabilization of membrane systems and
formation of calcium pectates increased the rigidity of the middle
lamella and cell walls and retards polygalacturonase (PG) activity
(Poovaiah, 1986).
Microbial counts. Similarly, many authors have shown applica-
tion of calcium to fresh-cut fruit also reduces microbial growth, due
to calcium increasing the rigidity of the cell wall and resistance to
fungal enzymes (Demarty et al., 1984). For example, Sharples and
Johnson (1977) delayed Alternaria growth in apples by dipping in 4%
CaCl2solution and Luna-Guzmán and Barret (2000) and Aguayo et
al. (2008) found that 2.5 or 0.5% CaCl2reduced bacterial microbial
growth in fresh-cut melon. Here we found that CaAsc treatment,
particularly in combination with MA packaging, very efficiently
reduced TPC and the yeast population. For example, while control or
low CaAsc concentrations fell under the limit of marketability after
only one week, concentrations of 6, 12 and 20% stored in MA pack-
aging (or even in air for 20%) resulted in the best sensory parameters
scores with low microbial counts after even 28 d.
Antioxidant status. The difference in results between the antiox-
idant assays used in this work reflects the relative sensitivity to
ascorbic acid compared to polyphenolic compounds. The FRAP
assay reflects total antioxidant power involving the single electron
transfer reaction, whereas DPPH is based on free radical scaveng-
ing activity. Brand-Williams et al. (1995) reported that phenols are
poor antiradical compounds measured by DPPH assay. However,
on slices dipped into CaAsc solution of 6, 12 and 20% the relation
between FRAP and DPPH was highly correlated, thus supporting the
hypothesis that the increase in antioxidant activity and concomi-
tant extension of shelf life was primarily due to AA from the CaAsc
solution.
It had been reported that antioxidant activity of fruit pulp did
not correlate with the AA content (Wang et al., 1996). Our results
are in disagreement with Wang et al. (1996) since we found a
strong correlation of antioxidant activity (as measured by DPPH
and FRAP assays) with AA content resulting from exogenous appli-
cation. The higher AA levels found in the CaAsc treated samples
was reflected in the higher antioxidant activities measured by the
FRAP and DPPH assays in comparison to the control. We found
that the high antioxidant activity in the 12 and 20% dipped slices
packed in air was much less stable than if they were under MA,
while control slices or those dipped in 2% CaAsc (which had much
lower antioxidant activity and AA levels) seemed to be more sta-
ble. Kalt et al. (1999) showed that in vegetable cells most of the
AA is located in the vacuole (a very low pH environment), together
with phenolic flavonoids. It is possible that the reducing action of
flavonoids together with the low pH prevented the rapid oxidation
of AA (Klein, 1987). As Cocci et al. (2006) reported, it is proba-
ble that exogenous AA in the dipped slices diffused into the apple
E. Aguayo et al. / Postharvest Biology and Technology 57 (2010) 52–60 59
tissue from the dipping solutions and thus, being located more
superficially, was therefore more exposed to oxygen. We note that
‘Braeburn’ apples have a naturally high AA content since 0.19 g kg−1
is significantly greater than 0.02–0.03 g kg−1found in ‘Red Deli-
cious’ apples, or the 0.03–0.04 g kg−1in ‘Fuji’ (Franke et al., 2004),
and these levels are maintained more effectively during storage
(Davey and Keulemans, 2004).
We observed that MA packaging significantly slowed the reduc-
tion in antioxidant and AA levels. However, others authors reported
that high concentrations of CO2(more than 5 kPa) had a nega-
tive effect on AA maintenance of apple slices (Cocci et al., 2006),
although this appears to be dependent on the product pH (Gil et al.,
1998).
Phenolics. The total phenolic content in the flesh of ‘Braeburn’
apple (control, day 0) was 630 mg kg−1, this being significantly
higher than reported in other cultivars such as 280 mgkg−1in ‘Red
Delicious’ (Napolitano et al., 2004), or 380 mg kg−1in ‘Golden Deli-
cious’ (Chinnici et al., 2004). Several authors have reported the level
of individual phenolics in apples (Lee et al., 2003; Chinnici et al.,
2004; Franke et al., 2004; Napolitano et al., 2004) but they do not
reach a consensus on the main phenolic compound since it is influ-
enced by cultivar, cultivation (organic or integrated), and time in
storage. Here we found that procyanidins and total hydroxycin-
namic acids (coumaric acid and chlorogenic acid) were the most
important phenolic components.
Exogenous CaAsc dips had small but significant effects on the
polyphenolic content of the apple slices but overall, phenolic
compounds were relatively stable with storage duration and atmo-
sphere. The lack of changes in the phenolic content relative to the
total antioxidant content suggests that the level of phenolics is
controlled by mechanisms other than ascorbic acid.
Correlation of antioxidant and shelf life. A significant observa-
tion in this work was that the antioxidant status of the fruit may
provide a useful indicator of the resulting overall slice quality.
When the postharvest oxidative stress exceeds the natural antiox-
idant system’s capacity, protection against AOS declines, resulting
in AOS-induced injury translated into disorders such as browning,
microbial contamination and poor sensory quality. These effects
were found on treatments with a low initial antioxidant activity
(DPPH = 0.94 or FRAP = 1.2 g kg−1) which had a relatively short shelf
life (7 d at 4 ◦C. However, if the antioxidant activity was maintained
at ≥2gkg
−1(DPPH or FRAP) apple slice shelf life was still accept-
able after 28 d at 4 ◦C. The validity of this proposal is supported
particularly by the 6% CaAsc treatment where quality was unac-
ceptable in only the air-stored treatment, while the MA-stored fruit
maintained a level of FRAP/DPPH that was >2 g kg−1and resulted
in acceptable quality.
It is important to point out that ‘Braeburn’ is a cultivar that
has particularly high levels of vitamin C and phenolic compounds
(Laing et al., unpublished data), and they have an intrinsic capa-
bility to withstand the stress. However, it is probable that in other
fruit, the antioxidant activity level may be due to other antioxidant
compounds such as organic acids, or even high levels of pheno-
lic compounds. Also, polyphenols may offer an indirect protection
by activating endogenous defence systems (Masella et al., 2005). It
could be useful to know the minimum level of endogenous or even
exogenous antioxidant in each fruit that is required to keep the AOS
under a level that is correlated with overall commercial product
acceptability and therefore to know the limit of storage life. How-
ever, we are in agreement with Toivone (2003) that these changes
may not be consistent between commodities or even within a com-
modity.
It is also worth noting that this experiment was carried out
in laboratory conditions. Despite all intent to replicate techniques
that could be used in industry, it is possible some variations to the
processes may be required to fully optimise industrial application.
5. Conclusion
We have confirmed that CaAsc is an effective anti-browning
agent in fresh-cut ‘Braeburn’ apples which would otherwise have a
short shelf life. The use of MA storage results in significant improve-
ment in quality as measured by sensory, chemical and visual
properties. Application of exogenous CaAsc significantly increased
the antioxidant status of the slices, and although the antioxidant
level decreased with time of storage, it could be maintained by the
use of MA conditions. The antioxidant levels were related to shelf
life and maintaining an antioxidant activity level of over 2 g kg−1
equivalent (for both FRAP and DPPH), obtained by dipping slices
in 6% of CaAsc and packaged in MA, appeared to be required to
maintain acceptable storage quality. Using CaAsc dips at 20%, the
vitamin C content of sliced apples can be raised to at least 3.8 g kg−1
of fruit without having a deleterious effect on the sensory quality,
and adding a significant nutritional benefit of vitamin C to regular
diets.
Acknowledgements
Encarna Aguayo thanks the Technical University of Cartagena
for supporting her nine months’ sabbatical stay at the New Zealand
Institute for Plant & Food Research (Auckland). Thanks to Reginald
Wibisono for his valuable assistance in the laboratory, and David
Stevenson and Janine Cooney for their knowledge on the phenolic
compound identification.
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