Postharvest Biology and Technology 110 (2015) 158–165

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Postharvest Biology and Technology

journal homepage: www.elsevier.com/locate/postharvbio

Hot water treatment in combination with calcium ascorbate dips

increases bioactive compounds and helps to maintain fresh-cut apple
quality
Encarna Aguayoa,* , Cecilia Requejo-Jackmanb , Roger Stanleyc , Allan Woolfb
a
Postharvest and Refrigeration Group. Department of Food Engineering, Technical University of Cartagena. Paseo Alfonso XIII 48, 30203 Cartagena, Murcia,
Spain
b
The New Zealand Institute for Plant & Food Research Ltd, Private Bag 92169, Auckland, New Zealand
c
Centre for Food Innovation, University of Tasmania, Locked Bag 1370, Launceston, TAS 7250, Australia

A R T I C L E I N F O A B S T R A C T

Article history: Fresh-cut ‘Braeburn’ apple slices were dipped into cold water (4  C for 2 min) or hot water (HWT, 48  C or
Received 23 October 2014 55  C for 2 min) followed by dips into 0 or 6% w/v aqueous calcium ascorbate (CaAsc, 2 min, 0  C) and
Received in revised form 1 July 2015 stored in air up to 28 d at 4  C. Microbial counts, changes in browning and sensory acceptance were
Accepted 3 July 2015
determined to indicate changes in quality. Changes in antioxidant levels were measured using free radical
Available online 15 August 2015
scavenging activity (DPPH), reducing activity (FRAP), ascorbic acid content (AA) and polyphenolic
content (by HPLC). CaAsc dips had a strong impact reducing the browning through increasing the flesh
Keywords:
luminosity and hue angle. 6% CaAsc in fresh-cut apples extended the overall acceptability from less than 7
Minimally processed
Apple slices
d to 14 d. Immediately after CaAsc treatment, AA content was 5 fold higher (0.25–1.25 g kg 1) than those
Heat treatment not dipped into CaAsc. However, the combination of HWT treatments and CaAsc dips led to seven fold
Shelf life increased levels of AA inside the apple tissue (0.25–1.85 g kg 1) and consequently increased the
Phenolic compounds antioxidant activity. HWT did not increase the AA content when not combined with CaAsc dips. The HWT
Vitamin C CaAsc dip extended the overall acceptability to 21 d compared to 14 d for samples not heated but dipped
Ascorbic acid into CaAsc. Shelf life was ultimately limited by sensory quality. At day 28, total plate counts were reduced
Antioxidant from 5.3 log cfu/g (untreated slices) to 4.6 log cfu/g in the 6% CaAsc dips and further to 3.9 log cfu/g with
the combination of HWT and CaAsc dip. Changes in the content of phenolic compounds with time, HWT
and CaAsc dip were generally not significant except for slightly increased quercetin and phloridzin levels
and decreased p-coumaric and procyanidins over time. The combination of HWT at 48  C for 2 min
followed by 6% CaAsc dip would be best for preserving the eating quality of apple slices.
ã 2015 Elsevier B.V. All rights reserved.

1. Introduction oxidase (PPO) that exists in particularly high amounts in apple

(Whitaker, 1972). The resulting browning makes the product
Apples are one of the most widely consumed fruits (FAOSTAT, unsuitable to the consumer. Ranges of treatments have been
2011) and are a good source of phytochemicals (Boyer and Liu, applied to extend the shelf life of fresh-cut apples, mainly dipping
2004). Epidemiological studies have linked the consumption of in solutions of a wide range of anti-browning agents. Ascorbic acid,
apples with reduced risk of some cancers, cardiovascular disease, oxalic acid, oxalacetic acid, kojic acid, erythorbic acid, citric acid,
asthma, and diabetes (Boyer and Liu, 2004). One way to increase and/or calcium, cysteine, 4-hexylresorcinol have all been exam-
fresh fruit consumption is processing fruit into fresh-cut product to ined at different concentrations (Son et al., 2001; Rojas-Grau et al.,
be sold as convenient single servings. However, fresh-cut 2006; Tortoe et al., 2007). Among the aforementioned methods,
processing results in major tissue disruption of surface cells and the use of calcium ascorbate (CaAsc) has been found to be the most
injury stress of underlying tissues (Toivonen, 2004). The main effective anti-browning agent and can be marketed as a minimal
problem for fresh-cut apple is oxidation caused by polyphenol chemical input. Its application increases the antioxidant status and
extends shelf life of apple slices (Aguayo et al., 2010). On the other
hand, several methods, including heat shock, have been used to
* Corresponding author.
inhibit polyphenol oxidase activity since this reduces the rate of
E-mail address: [email protected] (E. Aguayo). the polymerisation step of browning reactions (Kwak and Lim,

http://dx.doi.org/10.1016/j.postharvbio.2015.07.001
0925-5214/ ã 2015 Elsevier B.V. All rights reserved.
 E. Aguayo et al. / Postharvest Biology and Technology 110 (2015) 158–165 159

2005; Barbagallo et al., 2012). Hot water treatment (HWT) is an 2.2.2. Sensory evaluation
effective physical treatment, free of chemical residues, and readily A panel of five people was trained to recognise and score the
applicable in the fresh-cut industry during the washing process quality attributes of the treated apple slices. All assessments were
(Kim et al., 1993; Abreu et al., 2003). Previous studies have shown compared to freshly cut slices. Appearance, taste and texture were
that HWT is sufficiently effective to maintain product quality for scored on a nine-point scale where 1 = complete lacking or soft, to
fresh-cut products such as lettuce (Murata et al., 2004; Moreira 9 = fully characteristic of fresh. A similar scale, where 1 = inedible,
et al., 2006), rocket (Koukounaras et al., 2009), spinach (Gómez 3 = poor, 5 = fair (limit of marketability), 7 = good and 9 = excellent
et al., 2008; Glowacz et al., 2013), eggplants (Barbagallo et al., was used for the evaluation of the overall acceptability.
2012), and onions (Siddiq et al., 2013).
The use of non-chemical technologies that can improve product 2.2.3. Microbial analyses
quality response by additional interactions could improve the After 28 d storage, microbial growth on the slices was
effectiveness of CaAsc in the fresh-cut apple industry. This study determined by a certified food analysis laboratory (AgriQuality,
sought to determine whether HWT could be recommended as a Auckland, New Zealand). Only samples dipped into 6% CaAsc and
method in combination with CaAsc dips to help to maintain the both controls (0 and 6% CaAsc) from the HWT and control were
sensory quality of fresh-cut apples. analyzed. From each of five slices, 10 g samples were blended with
90 mL of sterile peptone buffered water (Merck Darmstadt,
2. Materials and methods Germany) for 1 min in a sterile stomacher bag (Model 400 Bags
6141, London, UK) using a Masticator (Colwort Stomacher 400 Lab,
2.1. Raw material Seward Medical, London, UK). Appropriate dilutions were pre-
pared. Plate Count Agar medium (PCA, Merck) was used for TPC
New Zealand grown ‘Mahana Red’ apples (Malus domestica and Rose Bengal agar medium (Merck) for the yeast and mould
Borkh., a sport of ‘Braeburn’) were sourced from the refrigerated counts. Incubation conditions were 30  C for 48 h for TPC, and 22  C
storage of a commercial supermarket. The apples had been stored for 5 d for yeasts and moulds, respectively. Microbial counts were
for up to 6 months under controlled atmosphere (2 kPa O2 plus reported as log 10 colony forming units per gram of sample
1 kPa CO2) at 0  C. Apples boxes were transported to the laboratory (log cfu g 1).
and stored at 0  C for 12 h. The boxes were opened in a food-grade
processing room (10  C) and the fruit sorted to remove those 2.2.4. Chemical measurements
damaged or with significant variation in background colour. Whole Fruit pieces were flash frozen in liquid nitrogen and stored at
apple surfaces were washed by dipping in cold water (4  C) with 80  C for a maximum of 2 months. To ensure uniformity, frozen
5 mg L 1 chlorine dioxide (OxineTM, Australasia Marketing Pty Ltd. samples (200 g) were either homogenised in 100 mL of distilled
Sydney, NSW, Australia) for 10 min. The apples were then manually water in a commercial blender (Sunbeam Model PB7600, Type 504,
cored with a metal corer and cut into 8 wedge slices using a 230–240 V, Sydney, Australia) to produce a juice extract for
handheld knife. All the slices were dipped into cold water (4  C) antioxidant activity analysis, or 150 g was ground to a fine powder
with 2 mg L 1 chlorine dioxide for 2 min, and the slices drained. in a Cryomill in liquid nitrogen for ascorbic acid content (AA)
Slices for HWT were dipped in hot water (48  C or 55  C) for 2 min. analysis.
A stainless water-bath (Grant 40 L, with a Grant temperature
controller units (0.1  C, 1.4 kW heater model GRAVF, U.K.) with 2.2.4.1. Antioxidant activity. Two assays were used to measure the
continuous hot water recirculation and stirring was used to antioxidant activity; 2,2-diphenyl-1-picrylhydrazyl (DPPH) and
maintain the relevant temperature. All apple slices (heated or not) ferric reducing antioxidant power (FRAP) assays. All the material,
were then dipped for 2 min into 0% (control) or 6% CaAsc solution method and equipment has been previously reported (Aguayo
(w/w; 99.9% purity, Wolf Canyon Asia Pacific Ltd) and drained. This et al., 2010). All the antioxidant assays were carried out in
solution was made using water at 0  C that had been pre-treated triplicate. Calibration curves were made for each assay using Trolox
with 2 mg L 1 chlorine dioxide. Apple skins were not removed (6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid) and
prior to treatment, as apple slices are currently marketed with skin AA as standards. The antioxidant activity (DPPH, FRAP assay)
intact. For each treatment, apple slices were randomised across was expressed as Trolox and AA equivalent antioxidant activity per
packages of 15 apple slices (350  20 g) per aluminium bag kg fresh weight of apple tissue.
(25 cm  18 cm, 80 mm thickness, Caspak, New Zealand). To
maintain nearly ambient oxygen concentration in the bags, two 2.2.4.2. Ascorbic acid evaluation. The method was adapted from
5-mm holes were punched through both sides of each bag. Three Rassam and Laing (2005). A 0.2 g sample of powdered apple tissue
replicate bags per treatment were stored at 4  C per storage was suspended in 1 mL of 6% metaphosphoric acid, 2 mM EDTA and
duration: 7, 14, 21 and 28 d. Measures of antioxidant activity were 1% PVPP containing 4 mM TCEP (tris[2-carboxyethyl] phosphine
also measured on day 0 (immediately after treatment). 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
2.2. Parameter evaluations 4  C for 10 min, and 20 mL of the supernatant was injected into a
7.8  300 mm Aminex HPX-87H HPLC column (Bio-Rad, Merck,
2.2.1. Colour measurement Darmstadt, Germany). The column was run with 2.8 mM H2SO4 as
The surface colour of the apple flesh was determined on three the mobile phase, at a flow rate of 0.01 mL s 1. The amount of AA
equidistant points in each apple slice cut surface with a Minolta was detected using a Waters 996 photodiode-array detector
chromameter (D65 light source, Minolta Camera Co., Osaka, Japan). (Milford, MA, USA) set at 245.5 nm for absorbance using AA (Sigma,
The results were expressed as CIELAB (L*a*b*) colour space. L* St. Louis, MO) as a standard. The peak was authenticated as AA by
defines the lightness and a* and b* define the red-greenness and showing that it was completely degraded by ascorbate oxidase at
blue-yellowness, respectively. The flesh colour was also measured pH 5.5.
and expressed as hue angle (h = arctangent [(b*) (a*) 1]) and
chroma (C* = [(a*)2 + (b*)2]1/2). Fifteen slices per treatment (5 slices 2.2.4.3. Phenolic compounds evaluation. One mL of apple juice
per replicate) were measured. extract was combined with 0.25 mL of methanol and HCl
160 E. Aguayo et al. / Postharvest Biology and Technology 110 (2015) 158–165

(90:10 v/v) and vortexed for 20 s. As mentioned, all the details have Table 2
Effect of CaAsc dip concentration (0 or 6%) on L*,  h, texture and taste of apple slices
previously been reported (Aguayo et al., 2010). Chromatograms
(P = 0.05). Slices were packaged in air and stored up to 28 d at 4  C.
were recorded at 280 and 340 nm at a flow rate of 0.02 mL s 1. Peak

assignment was performed by comparison of the retention times % CaAs L* h Texture Taste
and UV spectra with those of reference compounds and by mass 0 73.0 93.0 6.0 5.0
spectrometric analyses. Sample injection volume was 10 mL. UV– 6 76.2 100.7 7.0 6.2
vis detection was by absorbance at 200–600 nm.

2.3. 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, HWT and CaAsc 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 compared by LSD (least significant
difference test) when significant differences among treatments
and interactions between factors was found.

3. Results

Table 1 shows ANOVA results of time of storage, hot water

treatment and CaAsc dips (0 or 6%) factors and their interaction on
apple slices.

3.1. Colour
Fig. 1. Chroma of apple slices that were cold water (4  C, 2 min) or hot water treated
(48  C or 55  C, 2 min), then either dipped in 6% CaAsc or water. Slices were
CaAsc dips had a strong immediate impact increasing L* and packaged in bags at ambient atmosphere, and stored up to 28 d at 4  C. Solid
hue angle ( h) (Table 2) and reducing the chroma of apple slices symbols and lines indicate slices were treated with 6% CaAsc, while empty symbols
(Fig. 1). This indicated a lighter, slightly greener colour with less and dashed lines indicated slices dipped in water only (0% CaAsc). LSDs of
saturation in the apple flesh. In contrast, the HWT reduced the significant effect (P = 0.05): LSDTimeHWTCaAsc dips = 1.23.
luminosity of apple slices (Table 3) and slices that were not heated
showed the higher levels of L* and  h and decreased chroma. After
Table 3
28 d of storage  h was slightly reduced from 97 to 96 without Effect of hot water treatment on luminosity (L*) and  h of apple slices that were cold
significant changes in the L* parameter. water (4  C, 2 min) or plus hot water treated (48  C or 55  C, 2 min). Slices were
packaged in air and stored up to 28 d at 4  C. Letters differentiate values compared
by the least significant difference (LSD) at P = 0.05.
3.2. Sensory changes

Treatment L* h
The appearance of slices was improved by dipping in CaAsc 4  C/2 min 76.2 a 97.3 a
solutions. Immediately after treatment, the average appearance 48  C/2 min 73.7 b 97.2 a
rating increased from 6 points for the non CaAsc treatment to 7.8–8 55  C/2 min 73.8 b 96.1 b
points for CaAsc treatments (Fig. 2). After 7 d, HWT significantly
improved the retention of appearance of apple slices treated with
CaAsc. The appearance decreased with storage time in all limit of marketability except the slices heated at 55  C and not
treatments. Slices not treated in CaAsc decreased in appearance dipped in CaAsc, which fell below marketability at 28 d (data not
to below marketability limit before 7 d of storage. The CaAsc also shown). At day 21, the taste from slices not treated with CaAsc fell
improved the texture and the taste of the apple slices (Table 2) and down under the limit of marketability. At the end of the 28 d
the time of storage reduced both parameters and aroma (Table 4). storage period, the taste and a mouldy aroma from all treatments
During storage time, all treatments kept texture levels above the qualified the apple slices as not commercially acceptable (Table 4).

Table 1
Statistical analysis of parameters studied: time of storage (T, 28 d packaged in air at 4  C), hot water treatment (HWTs, 48  C or 55  C for 2 min or not heated) and CaAsc dips (0
or 6%) and their interaction for apple slices through analysis of variance.

T HWT CaAsc T x HWT T x CaAsc HWT x CaAsc T x HWT x CaAsc

L* – *** *** – – – –

h ** ** *** – – – –
Chroma – *** *** ** * – *
Appearance *** – *** – *** ** –
Texture *** – *** – – – –
Taste *** – *** – – – –
Aroma *** – – – – – –
Overall acceptability *** – *** * *** – –
DPPH *** *** *** * *** *** *
FRAP *** *** *** *** *** *** ***
Ascorbic acid *** *** *** – *** *** –

Dash represents non-significance difference at P > 0.05 and *, **, *** significant difference at P < 0.05; 0.01 and 0.001, respectively.
 E. Aguayo et al. / Postharvest Biology and Technology 110 (2015) 158–165 161

Taking into account the combination of sensory parameters

such as appearance, texture, taste and aroma, the overall
acceptability score was found to be the highest for CaAsc
treatments extending the shelf life up to 14 d (Fig. 3). In contrast,
slices not treated with CaAsc had a very short shelf life (<7 d). From
14 to 28 d of storage, the use of HWT before CaAsc dips was a
significant factor in the overall acceptability changes. The use of
this combination helped to extend the shelf life from 14 d (slices
not heated) up to 21 d (slices heated).

3.3. Microbial counts

At day 28, non heated apple slices dipped in CaAsc had

microbial TPC counts of 4.6 log cfu/g compared to 5.3 log cfu/g for
untreated slices. However, the biggest reduction to 3.9 log cfu/g
was obtained with the combination of HWT and CaAsc dips
(Table 5). Yeast and mould growth was higher than TPC and the
reduction obtained using CaAsc alone or in combination with HWT
was lower. HWT using 48  C instead of 55  C provided a better
microbial quality.
Fig. 2. Appearance of apple slices that were cold water (4  C, 2 min) or hot water
treated (48  C or 55  C, 2 min), then either dipped in 6% CaAsc or water. Slices were
packaged in bags at ambient atmosphere, and stored up to 28 d at 4  C. Solid 3.4. Antioxidant activity
symbols and lines indicate slices were treated with 6% CaAsc, while empty symbols
and dashed lines indicated slices dipped in water only (0% CaAsc). Data based on
Antioxidant activity was measured using the free radical
hedonic scale where 1 = unusable, 3 = poor, 5 = fair (limit of marketability), 7 = good
and 9 = excellent. LSDs of significant effect (P = 0.05): LSDTimeCaAsc dips = 0.59. scavenging activity (DPPH) and the ferric reducing ability (FRAP)
LSDHWTCaAsc dips = 0.46. assays. In both cases, the values, calculated as AA equivalents, were
very similar to those found for Trolox equivalents (data not shown)
and thus, results are only presented as AA equivalents. The DPPH
Table 4 assay of antioxidant activity of untreated apples slices on day 0 was
Effect of days of storage on  h, texture, taste and aroma of apple slices stored in air
0.25 g kg 1. This level increased between 1.8 and 2.4 g kg 1 with
up to 28 d at 4  C. Letters differentiate values compared by the least significant
difference (LSD) at P = 0.05. slices dipped in CaAsc solution (Fig. 4) obtaining the highest
concentration when both HWT were used. The slices dipped in

Days of storage h Texture Taste Aroma
HWT at 55  C for 2 min and 6% of CaAsc had nearly 10 times more
0 97.3 a 7.3 a 7.7 a 6.4 a antioxidant activity than the control (0% CaAsc, not heated) on day
7 97.6 a 7.2 a 6.5 b 5.3 b
0 of storage. However, HWT did not affect the relatively low
14 96.8 ab 6.5 b 5.9 bc 5.1 b
21 96.8 ab 6.2 b 5.3 c 5.3 b
antioxidant activity levels of slices when they were not dipped into
28 95.9 b 5.2 c 2.6 d 1.9 c CaAsc. Independently of type of HWT, DPPH decreased with time of
storage and the proportional decrease was greater in apple slices
dipped in CaAsc than those not dipped (70% vs 30% lost) although
the level of antioxidant was higher in the CaAsc treated slices. The
greatest reduction was found before 7 d of storage.
The FRAP measure followed similar patterns to that of the DPPH
assay including the effect of dipping in CaAsc and storage time. The
FRAP assay for control slices at day 0 showed an antioxidant
activity of 0.99 g kg 1 (Fig. 5) which was 4-fold higher than the
value obtained by the DPPH assay (0.25 g kg 1; Fig. 4). Treatment
with 6% CaAsc resulted in higher activity as measured by FRAP as
that of DPPH and during the first 21 d, both HWT provided a similar
increase on antioxidant compared to non-heated sample. In order
to maintain an antioxidant activity greater than the level in the
original apple slice (control), a heat treatment (48 or 55  C)

Table 5
Microbial counts (mean log cfu g 1) at day 28 d of apple slices that were cold water
(4  C, 2 min) or hot water treated (HWT, 48  C or 55  C, 2 min), then either dipped in
6% CaAsc or water. Slices were packaged in bags at ambient atmosphere and stored
at 4  C. Letters differentiate values compared by the least significant difference
(LSD) at P = 0.05.

HWT % CaAsc 28 d at 4  C
Fig. 3. Overall acceptability of apple slices that were cold water (4  C, 2 min) or hot
water treated (48  C or 55  C, 2 min), then either dipped in 6% CaAsc or water. Slices Total plate count Yeasts and moulds
were packaged in bags at ambient atmosphere, and stored up to 28 d at 4  C.
4  C/2 min 0% 5.3z a 5.9 a
Solid symbols and lines indicate slices were treated with 6% CaAsc, while empty
6% 4.6 b 5.4 a
symbols and dashed lines indicated slices dipped in water only (0% CaAsc). Data
based on hedonic scale where 1 = unusable, 3 = poor, 5 = fair (limit of marketability),
48  C/2 min 6% 3.6 c 4.4 b
7 = good and 9 = excellent. LSDs of significant effect (P = 0.05): LSDTimeHWT = 0.67.
55  C/2 min 6% 3.9 c 4.7 b
LSDTimeCaAsc dips = 0.54.
z
Data are the means of 3 replicates.
162 E. Aguayo et al. / Postharvest Biology and Technology 110 (2015) 158–165

2.0
Not heated
1.8 48º C/2 min
55 ºC/2 min
1.6

Ascorbic acid content (g/kg)

1.4

1.2

1.0

0.8 LSD Heat treatment x CaAsc

0.6
LSD Time x CaAsc
0.4

0.2

0.0
0 7 14 21 28

Storage du ration (days at 4 ºC)

Fig. 4. Antioxidant activity (DDPH as ascorbic acid) of apple slices that were cold Fig. 6. Ascorbic acid content of apple slices that were cold water (4  C, 2 min) or hot
water (4  C, 2 min) or hot water treated (48  C or 55  C, 2 min), then either dipped in water treated (48  C or 55  C, 2 min), then either dipped in 6% CaAsc or water. Slices
6% CaAsc or water. Slices were packaged in bags at ambient atmosphere, and stored were packaged in bags at ambient atmosphere, and stored up to 28 d at 4  C. Solid
up to 28 d at 4  C. Solid symbols and lines indicate slices were treated with 6% CaAsc, symbols and lines indicate slices were treated with 6% CaAsc, while empty symbols
while empty symbols and dashed lines indicated slices dipped in water only (0% and dashed lines indicated slices dipped in water only (0% CaAsc). LSDs of
CaAsc). LSDs of significant effect (P = 0.05): LSDTimeHWTCaAsc dips = 0.16. significant effect (P = 0.05): LSDTimeCaAsc dips = 0.18. LSDHWTCaAsc dips = 0.14.

3.6. Phenolic compounds

The total phenolic content in control (untreated) slices prior to

storage was 0.61 g kg 1 (Table 6). During storage the total
phenolics decreased about 20%, and ranged after 28 d from 449
to 516 mg kg 1. Interaction of time, heat treatment and CaAsc had a
significant effect on the total phenolic changes although the
response varied with the type of individual phenolic (Table 6).
Chlorogenic and epicatechin levels only were significantly affected
by time of storage. Chlorogenic decreased from day 14 and
epicatechin suffered a sharp decline at day 7 remaining stable until
the end of storage. Quercetin concentration increased using CaAsc.
During the first 21 d, the use of HWT also increased quercetin levels
although no differences were found at the end of storage when
HWT was used. A treatment of 55  C for 2 min increased the
phloridzin levels. In contrast HWT decreased p-coumaric and
procyanidins (B1, B2, C1) levels. Dipping into CaAsc further
Fig. 5. Antioxidant activity (FRAP as ascorbic acid) of apple slices that were cold reduced coumaric content although the procyanidins levels only
water (4  C, 2 min) or hot water treated (48  C or 55  C, 2 min), then either dipped in reduced in apple slices not heated and the phloridzin content
6% CaAsc or water. Slices were packaged in bags at ambient atmosphere, and stored decreased in slices not heated or heated 48  C for 2 min.
up to 28 d at 4  C. Solid symbols and lines indicate slices were treated with 6% CaAsc,
while empty symbols and dashed lines indicated slices dipped in water only (0%
CaAsc). LSDs of significant effect (P = 0.05): LSDTimeHWTCaAsc dips = 0.21.
4. Discussion
combined with dipping in 6% CaAsc was required. This treatment
provided a remaining antioxidant activity of about 1.5 g kg 1 at day 4.1. Shelf life
21.
This research has shown that use of a 6% calcium ascorbate dip
3.5. Ascorbic acid in fresh-cut apples extended the shelf life from <7 d in untreated
slices to 14 d when packaged in air and stored at 4  C. The factor
The initial AA content of untreated apple slices was of that ultimately limited the shelf life was the sensory quality. The
0.25 g kg 1. Immediately after CaAsc treatment, AA content was mechanism for the increased shelf life related to use of HWT
5–7-fold higher (1.3–1.9 g kg 1) than those not dipped into CaAsc treatments (48  C or 55  C, 2 min) before CaAsc dips which lead to
(Fig. 6). HWT helped to increase the ascorbic acid absorption from increased absorption of ascorbic acid into the apple tissue,
CaAsc dips. These increases were an average of 34% using 48  C and enhancing the ascorbic acid content and consequently the
about 25% with 55  C. The antioxidant activity measurement antioxidant activity as measured by DPPH and FRAP values. The
showed that HWT did not increase the retention of vitamin C when HWT enabled further extension of the shelf life (appearance) from
the treatment was not combined with CaAsc dips. The pattern of 14 d (samples not heated dipped in CaAsc) to 21 d. In general, there
decrease with storage time followed the same trends as those of was no significant difference between HWT at 48  C and 55  C and
the FRAP and DPPH activities, indicating that these changes were therefore a HWT of 48  C for 2 min should be enough to achieve the
the result of loss of AA content. benefit of extra shelf life.
 E. Aguayo et al. / Postharvest Biology and Technology 110 (2015) 158–165 163

Table 6
Main phenolic compounds (mg kg 1) of apple slices that were cold water (4  C, 2 min) or hot water treated (HWT, 48  C or 55  C, 2 min), then either dipped in 0 or 6% CaAsc.
Slices were packaged in bags at ambient atmosphere, and stored up to 28 d at 4  C.

Storage time HWT CaAsc treatment Chlorogenic Quercetin derivatives Epicatechin Coumaric acid Procyanidins (B1, B2, C1) Phloridzin Total phenol
0d 4 C 0% 81.6a 10.2 51.5 80.1 194.3 176.6 610.7
6% 81.1 12.0 49.5 77.8 192.1 160.9 573.4

48 C 0% 85.9 15.4 52.7 69.4 185.1 174.0 589.1
6% 81.8 17.0 51.1 68.9 182.2 155.5 526.1
55  C 0% 89.3 16.3 47.4 75.2 181.7 175.5 585.3
6% 81.9 18.6 51.4 70.7 185.6 160.3 562.4

7d 4 C 0% 80.1 13.1 42.5 66.1 188.2 119.7 486.7

6% 78.0 14.0 39.2 66.3 163.6 122.5 466.6

48 C 0% 78.2 17.3 40.3 67.0 167.1 122.7 476.1
6% 80.9 21.0 40.0 70.2 170.7 119.0 478.7
55  C 0% 83.8 18.9 41.9 68.3 168.5 127.3 508.7
6% 82.9 20.3 42.3 70.4 179.4 134.5 518.1

14 d 4 C 0% 79.0 11.0 40.4 69.3 179.2 121.6 472.5

6% 78.0 12.2 38.4 68.5 147.4 116.0 458.4

48 C 0% 77.5 18.1 37.8 68.9 156.2 114.5 458.6
6% 80.2 18.3 40.5 65.6 163.3 120.9 482.8
55  C 0% 73.6 15.9 40.0 64.9 161.7 118.6 470.2
6% 82.5 21.0 40.4 71.3 161.0 126.6 482.9

21 d 4 C 0% 81.2 16.1 40.0 72.8 161.5 118.8 534.2

6% 80.1 15.3 37.3 65.2 151.6 108.2 441.7

48 C 0% 76.9 15.4 38.3 68.4 152.8 110.9 454.5
6% 80.5 18.0 40.3 61.9 157.3 108.5 481.6
55  C 0% 73.5 18.3 38.8 64.0 155.8 119.6 466.8
6% 79.7 18.9 39.0 64.3 153.2 120.6 472.6

28 d 4 C 0% 81.5 15.3 41.2 70.2 165.5 111.9 507.6

6% 79.2 16.1 38.5 62.4 158.6 100.3 476.3

48 C 0% 77.7 19.9 38.5 63.2 145.6 108.3 477.6
6% 80.2 22.1 38.2 53.5 155.1 105.6 469.4
55  C 0% 70.8 15.3 35.0 65.6 152.1 114.2 449.4
6% 80.1 16.5 41.2 60.9 151.5 119.4 516.2

Time (3.1) (1.2) (1.4) (2.0) (5.3) (5.4) (15.1)

HWT NS (0.9) NS (1.6) (4.1) (4.2) (11.7)
% CaAsc NS (0.8) NS (1.3) NS (3.4) NS
Time * HWT NS (2.1) NS (3.5) NS NS NS
Time * CaAsc NS NS NS (2.9) (5.8) (7.7) (21.3)
HWT * CaAsc NS NS (1.5) (2.2) NS NS (16.5)
Time * HWT * CaAsc NS NS NS NS NS NS (36.9)
a
Values are means (n = 3); NS, not significant. LSD values are in brackets at P < 0.05. T: Time. HWT: Water treatment. CaAsc: Calcium ascorbate.

4.2. Colour could indicate that 55  C for 2 min induces a superficial damage in
the apple flesh thus slightly reducing the lightless and colour. For
The increases in L* and  h values using 6% of CaAsc dips example, Glowacz et al. (2013) reported that HWT at 50  C for 120 s
indicates that it is an effective way to reduce browning in resulted in the greatest membrane damage (20%) compared to
accordance with prior research (Son et al., 2001; Rojas-Grau et al., spinach leaves treated at 40  C up to 120 s, 45  C up to 60 s and 50  C
2006; Tortoe et al., 2007). AA reduces browning incidence by for 30 s. In contrast to our results, Abreu et al. (2003) and
reducing o-quinones back to phenolic compounds prior to Koukounaras et al. (2008) found that in fresh-cut pear and peaches,
polymerization and subsequent formation of coloured pigments respectively, that mild heat pre-treatments were effective in
(McEvily et al., 1992). Additionally, a formulation containing avoiding the cut surface browning in fresh-cut fruits. No difference
calcium and ascorbic acid acts partly to prevent cell and membrane was observed in PPO activity for the control and for heated peach
breakdown (Toivonen and Brummell, 2008) and consequently slices (Koukounaras et al., 2008). Glowacz et al. (2013) also found
reduces the release of PPO or its substrates thus improving the that a chroma increased in spinach leaves with increasing
colour preservation of fresh-cut product (Pérez-Cabrera et al., temperature of the treatments (40  C vs 45  C vs 50  C). The hue
2011). Others researchers have found that dipping treatments with angle decreased with time of storage mainly because the
Ca ascorbate and heat shock (60  C for 1 min) caused PPO inhibition effectiveness of ascorbic acid in anti-browning is temporary. The
and were less affected by discolouration (Barbagallo et al., 2012). In enzymatic browning can re-generate after the ascorbic acid from
the present experiment, we did not measure the levels of PPO the CaAsc or from the proper apple tissue has been completely
enzyme but the use of heat treatment only resulted in a slight reduced to dehydroascorbic acid.
reduction in L* and  h values and increased chroma when 55  C
HWT was used. The combination of HWT and CaAsc maintained 4.3. Sensory changes
the colour of fresh-cut apple with significantly less browning than
with the HWT without CaAsc. Djioua et al. (2009) also found Appearance and overall acceptability was strongly improved by
greater percentage of L* loss in mango slices when whole mangoes dipping in CaAsc solutions since slices not treated with CaAsc had a
were subjected to HWT (46  C/75 min and 50  C/30 min). This very short shelf life (<7 d). However, the best treatments were the
164 E. Aguayo et al. / Postharvest Biology and Technology 110 (2015) 158–165

combination of HWT (48 or 55  C) with CaAsc dips, which In samples with no added CaAsc, no significant difference in AA
extended the shelf life from 14 d (slices not heated) up to 21 d content or antioxidant activity changes was observed between
(heated slices). The ultimate shelf life limitation was related to heated and unheated apples slices. In support, others have found
sensory quality more than microbiological growth. that HWT had no effect on AA content in fresh-cut lettuce (Murata
Texture and taste from apple slices was also improved with et al., 2004), spinach (Gómez et al., 2008; Glowacz et al., 2013),
CaAsc dips. HWT had no significant effect on these changes. Dea mango slices (Dea et al., 2010), rocket leaves (Koukounaras et al.,
et al. (2010) also found there was no effect of heat treatment on the 2009) or fresh-cut onions (Siddiq et al., 2013).
firmness of mango slices when whole mango were immersed in In this experiment, the AA content and antioxidant capacity
hot water (46  C for 90 or 75 min). Softness and deformation of decreased with time of storage particularly in treatments with
stored oranges neither were influenced by HWT treatments (52  C highs level of ascorbic acid (HWT plus CaAsc dip). In previous work,
for 180 s and at 56  C for 20 s) (Strano et al., 2014). Aguayo et al. (2010) found that apple slices with lower antioxidant
The calcium present in CaAsc salt may be the main effect activity and ascorbic acid levels seemed to be more proportion-
influence on the retention of texture. However, many researchers ately stable than those with initial very high antioxidant activity.
have reported good results in maintaining or improving texture Kalt et al. (1999) showed that in vegetable cells most of the AA is
when a combination of HWT and calcium dipping is used (Aguayo located in the vacuole (a very low pH environment), together with
et al., 2008; Silveira et al., 2011) generally explained in terms of phenolic flavonoids. As Cocci et al. (2006) reported, it is probable
pectin esterase (PE) activation, cleaving the methoxyl groups from that exogenous AA in the dipped slices diffused into the apple
methylated galacturonic acid residues in pectin (Belitz and Grosch, tissue and not being located within vacuoles was therefore more
1986) which contain newly available carboxyl groups. The effect of exposed to oxygen and endogenous oxidases and thus more
HWT on firmness maintenance during storage has been reported rapidly oxidized.
for fresh-cut apple, pear, and melon (Kim et al., 1993; Abreu et al., Exogenous antioxidant treatments should be able to interfere
2003; Silveira et al., 2011). In our experiment, HWT had no a with senescence-related oxidation reactions. Aguayo et al. (2010)
negative effect on retention of texture (as measured subjectively) found that there is an efficient level of antioxidant content which
although HWT can inhibit the synthesis of cell wall hydrolytic decreases or even neutralizes the active oxygen species (AOS),
enzymes in whole apple fruit (Lurie et al., 1998). resulting in prevention of induced senescence and thus, extension
The improvement on taste using CaAsc was linked to the of shelf life. The antioxidant levels, as measured by FRAP, were
browning reduction activity of ascorbate temporarily avoiding the related to shelf life and it appears that a minimal antioxidant of
oxidation of phenolic compounds and keeping the endogenous about 2 g kg 1 may be needed level to maintain shelf life in
organic acid in apple slices at levels, which provide a good taste. In ‘Braeburn’ apple slices. In the present experiment, the longest
apple juice, Komthong et al. (2007) found that using AA increased residual minimum level of antioxidant was obtained through the
the two aldehydes, hexanal and trans-2-hexenal about 4 and 5-fold combination of hot water and 6% CaAsc. These apple slices, stored
obtaining a positive green odour of apple juice. in air at 4  C, achieved the longest shelf life being scored above the
All sensory parameters decreased with time of storage as limit of marketability at day 21.
consequence of the natural oxidative processes which are
particularly high in fresh-cut products. This quality loss with 4.6. Phenolic compounds
increased storage time was reduced by the combination of HWTs
and CaAsc dips. Exogenous CaAsc dips had small but significant effects on the
reduction of p-coumaric and procyanidins and phloridzin. The
4.4. Microbial counts decrease could be a consequence of formation of a complex of
ascorbic acid from CaAsc with natural products of coumaric acid
Many authors have shown application of calcium to fresh-cut (Kesinger and Stevens, 2009). The use of HWT increased the
fruit also reduces microbial growth, due to calcium increasing the quercetin and phloridzin levels but decreased coumaric and
rigidity of the cell wall and resistance to microbial enzymes. CaAsc procyanidins (B1, B2, C1) levels. HWT has been proposed to reduce
treatment, in combination with HWT, very efficiently reduced the browning in fresh-cut tissues because of the demonstrated effect
microbial growth. However, the use of high temperature HWT on delaying the wound-induced production of PAL (Saltveit, 2000).
could damage apple tissue and affect shelf life. Heat damage has In contrast, some researchers have reported that HWT enhanced
been previously reported in lettuce using mild heat treatment some enzyme activities associated with biosynthesis of phenolic
(50  C, 2 min), spinach leaves using HWT upper than 45  C for 60 s compounds in mandarins (Ghasemnezhad et al., 2008), muskmel-
or in whole apples using rinsing at 65  C for 20 s (Moreira et al., on fruit (Yuan et al., 2013) or peach (Spadoni et al., 2014). In our
2006; Maxin et al., 2012; Glowacz et al., 2013). experiment, the use of HWT did not provide a same response in all
the phenolic compounds. This could be attributed to differences in
4.5. Antioxidant activity (DDPH, FRAP) and ascorbic acid content the metabolism of different phenolic compounds by the apple
cells. Heat could have enhanced the phenolic extraction in apple
The use of CaAsc dips increased the AA content and slices. Gerard and Roberts (2004) found that increasing tempera-
consequently the antioxidant activity as measured by DPPH and ture during pressing from 40  C to 70  C allows increasing flavonoid
FRAP. This response was increased when dips were combined with content (50%) in apple juice. However, some phenolics such as
HWT. This increase is most likely due to the thermal effects of gas coumaric and procyanidin show lower stability (or higher
expansion in the hot water such that solutions are pulled into the susceptibility) to thermal degradation, autoxidation or breakdown
tissue due to contraction of the gas space with the cooler calcium (Ioannou et al., 2012), whilst others (such as quercetin) are more
ascorbate dip treatment. Alternatively, high temperature could stable upon heating (Odriozola-Serrano et al., 2008).
enhance the diffusion rate of ascorbic acid and increase the In general, heat treatments are effective at stopping or at least
solubility of ascorbic in water and apple tissue. This fact has been delaying “active” physiological processes (Woolf et al., 2012).
demonstrated with calcium salt as CaCl2 where hot water However, in our experiment, the beneficial effects of HWTs was
treatment (60  C, 1 min) had a significantly higher internal and most likely due to the physical thermal effects of moving from high
external free calcium concentration suggesting a possible en- to low temperature such that solutions are pulled into the tissue
hanced diffusion at higher temperatures (Aguayo et al., 2008). due to gas space contraction with the cooler calcium ascorbate dip
 E. Aguayo et al. / Postharvest Biology and Technology 110 (2015) 158–165 165

treatment. HWTs could enhance diffusion rate of ascorbic acid and Ioannou, I., Hafsa, I., Hamdi, S., Charbonnel, C., Ghoul, M., 2012. Review of the effects
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