Postharvest Biology and Technology 45 (2007) 110

Phenolic contents and other health and sensory related properties of

apple fruit (Malus domestica Borkh., cv. Aroma):
Effect of postharvest UV-B irradiation
Sidsel Fiskaa Hagen a,b, , Grethe Iren A. Borge a , Gunnar B. Bengtsson a , Wolfgang Bilger d ,
Arvid Berge c , Karin Haffner c, , Knut Asbjrn Solhaug b
a Matforsk AS, Norwegian Food Research Institute, Osloveien 1, NO-1430 Aas, Norway
b Norwegian University of Life Sciences, Department of Ecology and Natural Resource Management, P.O. Box 5003, NO-1432 Aas, Norway
c Norwegian University of Life Sciences, Department of Plant and Environmental Sciences, P.O. Box 5003, NO-1432 Aas, Norway
d University of Kiel, Institute of Botany, Olshausenstrasse 40, DE-24098 Kiel, Germany

Received 29 June 2006; accepted 2 February 2007

Abstract
The effects of postharvest irradiation with visible light and UV-B radiation on several health and sensory related properties, including antioxidant
capacity (ORAC assay), phenolic compounds, total phenols, ascorbic acid, skin colour, soluble solids and titratable acidity, were measured in
Aroma apples and the relationships between these properties were evaluated. The kinetics of flavonoid accumulation during irradiation were
measured with a non-destructive method based on chlorophyll fluorescence. The response to irradiation was compared between peel and flesh of
apples harvested from the inner (shade-grown) and outer (sun-exposed) canopy of the tree. The antioxidant capacity, the sum of phenols (HPLC)
and the content of anthocyanins, quercetin glycosides, chlorogenic acid and ascorbic acid increased upon postharvest irradiation. The accumulation
of flavonols started earlier and increased to a higher level than that of anthocyanins. A combination of visible light and UV-B radiation was the
most effective irradiation treatment and the response was greatest for the peel of the shade-grown apples. The apple flesh showed no response to
any of the irradiation treatments. Postharvest irradiation improved the apple skin colour, but did not influence the level of soluble solids or titratable
acidity in the apples. No visible damage or substantial weight loss was found in the apples after the irradiation treatments. The results suggest
that postharvest irradiation can be utilised to improve the health value and colour appearance of apples without changing important taste-related
parameters or causing damage to the fruit. Principal component analysis of the data showed that principal component 1 explained 72% of the
total variation and was closely related to the skin colour, sum of the phenols, total phenols, the content of ascorbic acid and also the level of
soluble solids and antioxidant capacity. Principal component 2 explained 12% of the total variation and was primarily related to titratable acidity.
The antioxidant capacity in the peel was better correlated with the sum of the phenols (r = 0.70, P < 0.001) than with the content of ascorbic acid
(r = 0.54, P < 0.001). The red to green colour values (a* ) of the apple skin were closely correlated with the sum of the phenols (r = 0.91, P < 0.001)
and ascorbic acid content (r = 0.83, P < 0.001) in the apple peel.
2007 Elsevier B.V. All rights reserved.

Keywords: Anthocyanins; Chlorophyll fluorescence; Flavonoids; l-Ascorbic acid; Vitamin C; Antioxidant capacity; ORAC; Polyphenols; Storage

1. Introduction

A diet rich in apple fruit is considered beneficial for human

Corresponding author at: Matforsk AS, Norwegian Food Research Institute,
Osloveien 1, NO-1430 Aas, Norway. Tel.: +47 64 97 04 12;
fax: +47 64 97 03 33.
E-mail address: sidsel.hagen@gmail.com (S.F. Hagen).
 This paper is dedicated to the memory of our dear colleague and co-author
Karin Haffner, recently deceased.
health (Boyer and Liu, 2004). Apple fruit contain several health
and sensory related constituents including dietary fibre, sug-
ars, vitamins and phenolic compounds. Vitamin C (ascorbic
and dehydroascorbic acids) is a powerful antioxidant that must
be ingested for survival (Padayatty et al., 2003). The antioxi-
dant capacity of apple is, however, mostly attributed to phenolic
compounds such as flavonoids and phenolic acids (Eberhardt

0925-5214/$ see front matter 2007 Elsevier B.V. All rights reserved.
doi:10.1016/j.postharvbio.2007.02.002
2 S.F. Hagen et al. / Postharvest Biology and Technology 45 (2007) 110
et al., 2000; Lee et al., 2003). There is strong evidence for a
preventative effect of phenolic compounds on age-related dis-
eases, including cardiovascular disease and cancer (Boyer and
Liu, 2004; Kroon and Williamson, 2005).
The major classes of flavonoids present in apple fruit are
flavonols (quercetin glycosides), flavanols (catechin, epicat-
echin and procyanidins), dihydrochalcones (phloridzin) and
anthocyanins (cyanidin glycosides), whereas the major phe-
nolic acid is chlorogenic acid (Awad et al., 2000; Tsao et
al., 2003). The different classes of phenolic compounds are
unevenly distributed within the apple fruit (Awad et al., 2000)
and demonstrate non-uniform behaviour during fruit develop-
ment and in response to external factors (Awad et al., 2001).
The synthesis of these compounds is, in addition to genetic
background and developmental stage, influenced by environ-
mental factors such as nutrient availability, temperature and
in particular, light (Saure, 1990; Treutter, 2001). Many of the
enzymes involved in flavonoid synthesis are induced by light
(Lancaster, 1992; Treutter, 2001) and the total concentration of
flavonoids has been shown to be higher in apple skin that has
been exposed to sun radiation (Awad et al., 2001). Flavonoids
accumulated in the apple skin are efficient sunscreens that pro-
tect the cells against UV-B-induced damage (Solovchenko and
Schmitz-Eiberger, 2003) and are also involved in plant defence
in other stress situations, such as wounding or pathogen attack
(Treutter, 2001). Anthocyanins, co-pigmented by other phenolic
compounds, are responsible for the wide range of red colours
in apple skin (Lancaster, 1992). The redness in apple skin is for
many apple cultivars one of the most important factors in cus-
tomer acceptance (Saure, 1990). Phenols in apple are reported
to be stable during long-term storage in both normal air and
controlled atmospheres (van der Sluis et al., 2001; Awad and de
Jager, 2003), whereas ascorbic acid has been shown to decline
(Meberg et al., 2000).
Previously, postharvest irradiation has been used to study
red colour development and accumulation of selected phenols,
mainly anthocyanins, in apple skin (e.g. Arakawa et al., 1985;
Reay, 1999; Lancaster et al., 2000; Reay and Lancaster, 2001;
Ubi et al., 2006). The aim of the present study was to explore the
effect of postharvest irradiation not only on the major classes of
phenolic compounds, but also on other important health and sen-
sory related properties in the peel and flesh of red, sun-exposed
and green, shade-grown Aroma apples. The level of flavonoids,
chlorogenic acid, total phenols, ascorbic acid, antioxidant capac-
ity, soluble solids, titratable acidity and skin colour has been
measured and their interrelationships have been evaluated. Also,
a newly developed, non-destructive method based on chloro-
phyll fluorescence (Bilger et al., 2001; Hagen et al., 2006) has
been used to determine the kinetics of flavonoid accumulation
during the period of irradiation.
2. Materials and methods
2.1. Chemicals
Chlorogenic (5 -caffeoylquinic) acid, (+)-catechin, ()-
epicatechin, quercetin-3-rhamnoside, l-ascorbic acid, gallic
acid, Folin-Ciocalteus phenol reagent, -PE (-phycoerythrin)
and AAPH (2,2 -azobis (2-amidinopropane) dihydrochloride)
were purchased from Sigma Chemical Co. (St. Louis,
MO, USA). Quercetin-3-galactoside and quercetin-3-glucoside
were from Carl Roth GmbH & Co. (Karlsruhe, Ger-
many). Procyanidins B1 and B2 were from Extrasynthese
(Genay Cedex, France). Phloridzin dihydrate and Trolox (6-
hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid) were
from Fluka Chemie GmbH (Buchs, Switzerland). Cyanidin-3-
galactoside was from Polyphenols Laboratories AS (Sandnes,
Norway). All other chemicals were of analytical grade.
2.2. Plant material and irradiation
Apples (Malus domestica Borkh., cv. Aroma) were harvested
the 17 September 2002 at commercial maturity from 20-year-old
trees grafted on MM.106 rootstocks in the orchard of the Nor-
wegian University of Life Sciences (59 30 N, 10 47 E, 100 m
altitude). Apples with no signs of damage were harvested from
the outer and inner parts of the tree canopy, resulting in a selec-
tion of dark red (sun-exposed) and pale green (shade-grown)
apples. After harvest, the apples were stored for 8 weeks in total
darkness at 2 C and 98% relative air humidity (RH).
A total of 84 apples was used in the experiment. A zero
time sample of six sun-exposed and six shade-grown apples was
randomly picked and prepared for analysis. The remaining 72
apples were transferred to a climate room. The apples, 12 sun-
exposed and 12 shade-grown apples for each treatment, were
then irradiated with a combination of visible light and UV-B
radiation (Vis + UV-B) or visible light alone (Vis) or covered
with a black cloth during the entire experiment (dark). The sun-
exposed apples were irradiated on the side that had been most
exposed to the sun, whereas the shade-grown apples were irra-
diated on the palest side of the fruit. After the experiment, six
sun-exposed and six shade-grown apples from each treatment
were randomly chosen and prepared for analysis. All apples
were weighed before and after the experiment.
The apples were irradiated for 12 h per day during a period
of 10 days at 10 C and 9598% RH. Visible light was pro-
vided by a high pressure mercury lamp (Philips HPI 400 W)
installed 120 cm above the apples. A shade screen made of nar-
row strips of aluminium reduced the light level to 2530 mol
photons m2 s1 and UV radiation from the lamp was cut off
at 380 nm by an acrylic plate. UV-B radiation was provided by
two UV-B fluorescent tubes (Philips TL 20W/12RS) installed
50 cm above the apples. A 0.13 mm cellulose diacetate film cut
off UV-C radiation below 290 nm. The UV-B radiation at the
surface of the apples was measured to be 0.17 W m2 with a
Skye SKU430 sensor (Skye instruments Ltd., Powys, UK). The
irradiance from the UV-B fluorescent tubes was later measured
simultaneously with both the Skye UV-B sensor and an Optronic
model 756 spectroradiometer (Optronic Laboratories, Orlando,
FL, USA). Based on a calibration factor from this comparison,
the biologically effective UV-B irradiation was estimated to be
approximately 0.20 W m2 for the UV-B treatment. A weight-
ing function normalised to 1 at 300 nm (Green et al., 1974) was
used for the calculations.
 S.F. Hagen et al. / Postharvest Biology and Technology 45 (2007) 110
2.3. Chlorophyll fluorescence measurements
Chlorophyll fluorescence measurements were performed five
times during the 10-day experiment. The procedure was an
adaptation of a method by Bilger et al. (2001) and was per-
formed with a UV-A-PAM Chlorophyll Fluorometer (Gademann
Instruments, Wurzburg, Germany) and a PAM-2000 Fluo-
rometer (Walz, Effeltrich, Germany). Chlorophyll fluorescence
was measured and relative absorbance values were calcu-
lated as described by Hagen et al. (2006). The relative blue
light absorbance is positively correlated with the content of
anthocyanins in the apple skin, whereas the relative UV-A
absorbance is positively correlated with the content of UV-
absorbing flavonoids (total flavonoids) in the apple skin (Hagen
et al., 2006).
2.4. Colour measurements
The colour of the apple skin was measured non-destructively
on all apples prior to chemical analysis. The measurements were
performed with a LabScan XE HunterLab (Hunter Associates
Laboratories, Inc., Reston, Virginia, USA) on the same area as
for the chlorophyll fluorescence measurements. Colour appear-
ance was given as L* , a* and b* values. L* measures lightness
and varies from 100 for perfect white to zero for black, approx-
imately as the eye would evaluate it, a* measures redness when
positive and greenness when negative and b* measures yellow-
ness when positive and blueness when negative.
2.5. Chemical analyses
2.5.1. Sample preparation
Plugs of skin and flesh were cut from the apples with a cork
borer (17 mm diameter). One plug, for phenol extracts, was cut
from the same area as subjected to the chlorophyll fluorescence
measurements and two plugs, one for ascorbic acid and one
for soluble solids and titratable acidity analyses, were cut from
the adjacent area. The plugs (approximately 2025 mm long)
were immediately frozen in liquid nitrogen, put in plastic tubes
(10 mL) and flushed with nitrogen before storage at 80 C.
2.5.2. Extraction of phenols
Peel (0.2 g; approximately 1 mm thick) or flesh (0.6 g;
approximately 10 mm of the outer part of the apples) was cut
from the frozen plug, cut into six pieces and rapidly trans-
ferred to a tube with 1 mL 0.01 M HCl in methanol. The sample
was exposed to sonication for 30 min in an ultrasonic bath
(CTU150, Coax Teknik, Lynge, Denmark) followed by 30 min
shaking (1400 rpm) in a CM-9 Mixer (SARSTEDT, Numbrecht,
Germany). The extract was transferred to another tube before
re-extraction of the peel or flesh with the same procedure as
described above. The entire procedure was carried out at 04 C
while shaded from incident light.
2.5.3. HPLC of phenols
Quantification of phenols from extracts was carried out on an
Agilent Technologies (Waldbronn, Germany) 1100 Series HPLC
3
system equipped with a photodiode array detector (DAD). The
procedure has been described by Hagen et al. (2006). Quantifi-
cation was based on external standard calibration curves. The
concentration of the phenolic compounds was expressed as mg
per 100 g fresh weight of sample.
2.5.4. Total phenols
Total phenols were determined according to the Folin
Ciocalteu method (Waterhouse, 2002). The phenol extracts were
appropriately diluted and mixed with FolinCiocalteu reagent.
The samples were incubated for 1 min before sodium carbonate
(7.5%, w/v) was added. The mixture was then incubated for 2 h
at room temperature before absorbance was measured at 765 nm
in a UV-Vis spectrophotometer (Model 5384, Agilent Technolo-
gies). All samples were analysed in triplicates. Total phenols
were expressed as milligram gallic acid equivalents (GAE) per
100 g fresh weight of sample.
2.5.5. Antioxidant capacity
Antioxidant capacity was determined by the ORAC (oxy-
gen radical absorbance capacity) assay according to the method
of Cao and Prior (1999) with some modifications (Aaby et
al., 2004). The phenol extracts were appropriately diluted with
75 mM phosphate buffer, pH 7.4. The ORAC assay was car-
ried out in duplicates with four dilutions on a Wallac 1420
Victor2 96-well plate reader (EG & Wallac, Turku, Finland) with
a fluorescence filter (excitation 540 nm, emission 565 nm). -PE
(16.7 nM final conc.) was the fluorescence and target molecule.
Free radical generation was initiated by AAPH (4 mM final
conc.). The reaction was conducted at 37 C with Trolox (1 M
final conc.) as control standard and phosphate buffer as blank
sample. After addition of AAPH, the fluorescence was recorded
every 3 min until the fluorescence was less than 5% of the ini-
tial reading. Final results were calculated using the differences
of areas under the -PE decay curves between the blank and
the sample. Antioxidant capacity was expressed as mol Trolox
equivalents (TE) per gram fresh weight of sample.
2.5.6. Ascorbic acid
l-Ascorbic acid was determined according to the method
described by Sanchez-Mata et al. (2000) with minor modi-
fications. Frozen peel (0.2 g) or flesh (0.61.0 g), separated
from the apple plug, was transferred to a tube with 3 mL
4.5% meta-phosphoric acid and homogenised using a Polytron
PT 1200 (Kinematica AG, Luzern, Switzerland). The samples
were centrifuged for 10 min at 13,800 g and 4 C before
filtered through a folded cellulose filter (No. 595 , Schle-
icher & Schull GmbH, Dassel, Germany) and further through a
0.45 m Millex HA filter (Millipore, Molsheim, France). HPLC
analysis was performed on an Agilent Technologies system
comprising a HP1100 liquid chromatograph, a thermostated
autosampler (4 C) and a DAD. The separation was conducted
on an Aqua C18/C2 column (250 mm 4.6 mm, 5 m) from
Phenomenex (Aschaffenburg, Germany). The mobile phase was
0.05 M KH2 PO4 adjusted to pH 2.6 with 0.5 M H2 SO4 for iso-
cratic elution at 25 C and the flow rate was 1.0 mL/min. The
injection volume was 5 L and the run time was set to 5 min.
4 S.F. Hagen et al. / Postharvest Biology and Technology 45 (2007) 110

l-Ascorbic acid was measured at 245 nm and quantified with

the use of an external standard curve.

2.5.7. Soluble solids and titratable acidity

The measurements were performed on mixed samples of skin
and flesh. The apple plug was cut in eight pieces and crushed
with a glass rod during thawing in a tube before centrifugation for
30 min at 13,800 g and 4 C. The supernatant (juice) was used
for the analyses. Soluble solids (in apple mainly sucrose with
smaller amounts of glucose, fructose and organic acids) in the
juice were measured at 20 C with a digital RE40 Refractome-
ter (Mettler-Toledo AG, Greifensee, Switzerland) and expressed
as Brix, which is equivalent to weight percent of sucrose.
Titratable acidity was measured as the amount of 0.1 M NaOH
to reach pH 8.1 in a sample diluted 10 with distilled water
using an automated METTLER DL25 Titrator (Mettler-Toledo
AG) and calculated as gram malic acid equivalents per 100 g
juice.
2.6. Statistical analysis
One-way analysis of variance (ANOVA), general linear
model (GLM) for repeated measurements, least significant dif-
ference (LSD) post hoc test and standard Pearsons correlation
were performed with significance level = 0.05 on original
data (N = 48) using the statistical package SPSS version 13.0
(SPPS Inc., Chicago, IL, USA). To study the main tendencies
of variation among the health and sensory related properties,
principal component analysis (PCA) was performed using The
Unscrambler, version 9.2 (Camo Process AS, Oslo, Norway).
PCA projects the information carried by the original variables
onto a smaller number of underlying (latent) variables called
principal components (PC). Plotting the principal components
can reveal interrelationships between the variables (loading plot)
and detect sample patterns, groupings, similarities or differ-
ences (score plot). All variables were scaled to unit variance
and centred before PCA. Two outliers were removed. The
calibration model was validated by full scale cross valida-
tion.
Fig. 1. HPLC chromatograms of the phenolic compounds in an acidic methanol
extract of peel from sun-exposed Aroma apples. The peaks identified are pro-
cyanidin B1 (1), catechin (2), chlorogenic acid (3), cyanidin-3-galactoside (4),
procyanidin B2 (5), epicatechin (6), quercetin-3-galactoside (7), quercetin-3-
glucoside (8), quercetin-3-rhamnoside (9) and phloridzin (10).
epicatechin was higher in the peel than in the flesh of the apples
(P < 0.001). Chlorogenic acid was more concentrated in the flesh
than in the peel of the apples (P < 0.001). All flavonoids, but not
chlorogenic acid, were more abundant in the peel of sun-exposed
apples than in the peel of shade-grown apples (P < 0.001).
The flesh concentrations were similar for both growth
conditions.
The level of total phenols, ascorbic acid and antioxidant
capacity was higher in the peel than in the flesh of the apples
(P < 0.001) and was higher in the peel for the sun-exposed
apples than for the shade-grown apples (P < 0.01). The solu-
ble solids and titratable acidity values in mixed samples of peel
and flesh did not show any significant differences between the
shade-grown and sun-exposed apples (Table 1).
3.2. Effects of postharvest irradiation
3.2.1. General appearance and skin colour
There was no visual damage on any of the apples after the
irradiation treatments. The weight loss of the apples during
the experiment was less than 1% for all irradiation treatments.

3. Results

3.1. Distribution of chemical properties in apples

The phenolic compounds quantified in Aroma apples were

epicatechin, procyanidin B1 and B2, cyanidin-3-galactoside,
quercetin-3-galactoside, quercetin-3-glucoside, quercetin-3-
rhamnoside, phloridzin and chlorogenic acid. Catechin was
detected only in trace amounts and was not quantified in this
study. Procyanidin B2 was more abundant than procyanidin
B1. Quercetin-3-galactoside was the predominant quercetin gly-
coside over quercetin-3-glucoside and quercetin-3-rhamnoside
(Fig. 1).
Anthocyanins and quercetin glycosides were found exclu-
sively in the apple peel, whereas epicatechin, procyanidins, Fig. 2. Skin colour in shade-grown and sun-exposed apples (cv. Aroma) before
phloridzin and chlorogenic acid were found in both peel and and after postharvest irradiation. L* represents lightness (100 = white, 0 = black),
flesh of the apples (Table 1). The concentration of phloridzin and a* is the red (+) to green () axis and b* is the yellow (+) to blue () axis.
 S.F. Hagen et al. / Postharvest Biology and Technology 45 (2007) 110 5

Table 1
Health and sensory related properties in the peel and flesh of shade-grown and sun-exposed apples (cv. Aroma) before postharvest irradiationa
Parameters Shade-grown apples Sun-exposed apples

Peel Flesh Peel Flesh

Epicatechin (mg/100 g fw) 23.5 0.8 a 14.1 2.0 b 41.5 4.0 c 14.2 1.4 b
Procyanidins (mg/100 g fw)b 13.8 2.0 a 14.3 2.1 a 23.6 2.5 b 19.0 2.1 ab
Phloridzin (mg/100 g fw) 1.3 0.2 a 0.5 0.1 b 3.5 0.3 c 0.5 0.1 b
Quercetins (mg/100 g fw)c 0.6 0.1 a ND a 62.3 11.4 b ND a
Anthocyanin (mg/100 g fw)d ND a ND a 49.5 10.7 b ND a
Sum of flavonoids (mg/100 g fw) 39.3 1.8 a 28.8 4.1 a 180 17 b 33.7 3.4 a
Chlorogenic acid (mg/100 g fw) 4.1 0.7 a 15.4 1.8 b 6.5 1.2 a 15.1 1.6 b
Sum of phenols (mg/100 g fw) 43.3 2.1 a 44.2 5.8 a 187 18 b 48.8 4.9 a
Total phenols (mg GAE/100 g fw)e 434 57 a 190 28 b 838 47 c 215 16 b
Ascorbic acid (mg/100 g fw) 1.1 0.5 a ND a 38.6 6.6 b 0.7 0.3 a
Antioxidant capacity (mol TE/g fw)f 4.5 0.6 a 1.9 0.3 b 9.1 1.2 c 2.8 0.2 b
Soluble solids ( Brix)g 9.8 0.4 a 10.9 0.6 a
Titratable acidity (g MAE/100 g fw)g 0.56 0.02 a 0.53 0.01 a
a Mean S.E.M. of five to six samples. Within each row, means with the same letters are not significantly different (P > 0.05). ND: not detectable.
b Sum of procyanidin B1 and procyanidin B2.
c Sum of quercetin-3-galactoside, quercetin-3-glucoside and quercetin-3-rhamnoside.
d Cyanidin-3-galactoside.
e Measured by the FolinCiocalteu assay. GAE: gallic acid equivalents.
f Measured by the ORAC assay. TE: Trolox equivalents.
g Measured in samples containing peel and flesh. MAE: malic acid equivalents.

The shade-grown apples treated with Vis + UV-B had devel-

oped a skin colour that was much redder, darker and less yellow
(P < 0.001) than the other groups of shade-grown apples (Fig. 2).
The skin of shade-grown apples exposed to Vis alone became
more yellow (P < 0.05) and less green (P < 0.01) during irra-
diation. The skin colour of the sun-exposed apples exposed to
Vis + UV-B was not different from that of the start-samples, but
was slightly darker and less yellow than the skin colour of the
sun-exposed apples exposed to Vis alone or kept in the dark
(P < 0.05).

3.2.2. Chlorophyll fluorescence

The relative absorbance of both blue light and UV-A
increased (P < 0.001) in the skin of both sun-exposed and shade-
grown apples during irradiation with Vis + UV-B (Fig. 3A and
B). The increases were highest for the shade-grown apples.
The relative UV-A absorbance in the skin of shade-grown
apples increased also when exposed to Vis alone (P < 0.001),
but to a lesser extent than upon the Vis + UV-B treatment.
The increase in relative UV-A absorbance started earlier and
advanced to a higher level than the increase in relative blue light
absorbance.

3.2.3. Phenols
Since no effect of postharvest irradiation was detected in
the apple flesh, only peel values are presented in Table 2.
Postharvest irradiation with Vis + UV-B enhanced the sum of
flavonoids (P < 0.001), sum of phenols (P < 0.001) and total
phenols (P < 0.05) in the peel of shade-grown apples. The con- Fig. 3. Relative absorbance of blue light (A) and UV-A radiation (B) in the
tent of quercetin glycosides in the peel of shade-grown apples skin of Aroma apples as calculated from repeated chlorophyll fluorescence
measurements during the period of postharvest irradiation. Relative blue light
increased upon treatment with Vis alone (P < 0.05), but was six- and UV-A absorbance in apple skin is positively correlated with the peel content
fold higher upon the Vis + UV-B treatment (P < 0.001). The latter of anthocyanins and UV-absorbing flavonoids, respectively (Hagen et al., 2006).
treatment resulted in a quercetin content nearly 70% of that in All data points are mean values S.E.M. of 12 samples.
6 S.F. Hagen et al. / Postharvest Biology and Technology 45 (2007) 110

Table 2
Effect of postharvest irradiation on health and sensory related properties in sun-exposed and shade-grown apples (cv. Aroma)a
Parameters Shade-grown apples Sun-exposed apples

Dark Vis Vis + UV-B Dark Vis Vis + UV-B

Epicatechin (mg/100 g fw) 18.3 2.5 22.9 2.3 24.9 1.6* 35.7 3.9 36.1 4.1 48.1 3.6*
Procyanidins (mg/100 g fw)b 11.9 2.2 12.3 1.9 11.9 1.1 22.4 2.6 20.4 2.7 26.7 1.6
Phloridzin (mg/100 g fw) 1.4 0.1 1.3 0.3 1.5 0.1 3.3 0.4 3.3 0.3 3.1 0.4
Quercetins (mg/100 g fw)c 2.5 1.2 8.3 0.4* 48.7 3.2*** 72.5 13.4 56.7 11.4 72.7 12.0
Anthocyanin (mg/100 g fw)d ND ND 8.9 1.5*** 30.1 5.5 32.5 3.9 47.1 6.8*
Sum of flavonoids (mg/100 g fw) 34.1 5.1 44.8 3.8 95.9 5.8*** 164 14 149 11 198 17
Chlorogenic acid (mg/100 g fw) 4.4 0.4 5.5 0.7 18.0 1.1*** 7.4 1.3 7.2 1.8 17.9 2.5***
Sum of phenols (mg/100 g fw) 38.5 5.4 50.3 4.4 114 7*** 171 15 156 11 216 18
Total phenols (mg GAE/100 g fw)e 355 45 399 37 507 26* 865 58 889 61 872 58
Ascorbic acid (mg/100 g fw) 1.7 0.9 7.8 2.9* 10.2 1.7** 41.7 4.9 30.1 3.2 44.4 5.0
Antioxidant capacity (mol TE/g fw)f 3.9 1.0 4.2 0.8 8.4 1.7* 9.5 0.5 7.0 1.6 13.7 2.0*
Soluble solids ( Brix) 8.7 0.9 9.2 0.3 9.4 0.4 11.9 0.2 10.8 0.2 11.2 0.5
Titratable acidity (g MAE/100 g fw)g 0.53 0.01 0.54 0.01 0.54 0.01 0.54 0.01 0.53 0.00 0.56 0.02
a Peel values, except for soluble solids and titratable acidity (mixed samples of peel and flesh). Data represents mean S.E.M. of five to six samples. Effect of

irradiation is tested against control (dark) for sun-exposed and shade-grown apples separately. Level of significance: *P 0.05; **P 0.01; ***P 0.001. ND: not
detectable.
b Sum of procyanidin B1 and procyanidin B2.
c Sum of quercetin-3-galactoside, quercetin-3-glucoside and quercetin-3-rhamnoside.
d Cyanidin-3-galactoside.
e Measured by the FolinCiocalteu assay. GAE: gallic acid equivalents.
f Measured by the ORAC assay. TE: trolox equivalents.
g MAE: malic acid equivalents.

the sun-exposed apples. The content of anthocyanins in the peel
of shade-grown apples had increased from not detectable to
2025% of the content in sun-exposed apples after treatment
with Vis + UV-B (P < 0.001). The peel content of anthocyanins
in sun-exposed apples and epicatechin in both shade-grown
and sun-exposed apples did not increase during irradiation,
but was higher after Vis + UV-B irradiation than after darkness
(P < 0.05). The content of chlorogenic acid more than doubled
in the peel of both sun-exposed and shade-grown apples after
treatment with Vis + UV-B (P < 0.001).
3.2.4. Antioxidant capacity and ascorbic acid
The antioxidant capacity increased significantly in the peel
of both sun-exposed and shade-grown apples after irradiation
with Vis + UV-B (P < 0.05) (Table 2). The content of ascorbic
acid in the peel of shade-grown apples increased significantly,
that is from 3% to 1825% of the content in sun-exposed apples,
after treatments with either Vis alone (P < 0.05) or Vis + UV-B
(P < 0.01).
3.2.5. Soluble solids and titratable acidity
No significant changes in the content of soluble solids or
titratable acidity were detected after postharvest irradiation
(Table 2).
3.3. Relationships between the measured properties of
apple peel
The data processed with PCA showed that the two first prin-
cipal components explained 84% of the total variation. The
correlation loadings demonstrate how the original variables were
related to each other (Fig. 4A). PC 1 explained 72% of the
total variation and appeared to be closely related to the skin
colour, the content of phenols, ascorbic acid and soluble solids
and the antioxidant capacity. PC 2 explained 12% of the total
variation and was related mainly to the titratable acidity, and
to a lesser extent also to the antioxidant capacity and amount
of soluble solids. When the single phenolic compounds were
included (not shown), the flavonoids were closely related to
PC 1, whereas chlorogenic acid was more related to PC 2. The
score plot demonstrates the distribution of the samples due to
PC 1 and 2 (Fig. 4B). The plot reveals three sample groups
separated by PC 1. The sun-exposed apples are described by pos-
itive values, the shade-grown apples by negative values and the
shade-grown apples treated with Vis + UV-B by values closer to
zero.
The sum of phenols identified and quantified by HPLC was
linearly correlated (r = 0.88, P < 0.001) with the total phenols
determined by the FolinCiocalteu assay. The antioxidant capac-
ity in the peel was better correlated with the sum of phenols
(r = 0.70, P < 0.001) than with the concentration of ascorbic
acid (r = 0.54, P < 0.001). The phenols were correlated with the
antioxidant capacity as follows: quercetin glycosides (r = 0.65),
epicatechin (r = 0.56), chlorogenic acid (r = 0.56), anthocyanin
(r = 0.55), phloridzin (r = 0.51), all with P < 0.001, and with pro-
cyanidins (r = 0.46, P < 0.01). The red to green colour values
(a* ) of the apple skin were positively correlated with the sum
of phenols (r = 0.91), total phenols (r = 0.85), content of ascor-
bic acid (r = 0.83), anthocyanins (r = 0.80) and soluble solids
(r = 0.66) and the antioxidant capacity (r = 0.63) in the apple peel
(P < 0.001 for all). The concentration of chlorogenic acid was
poorly correlated with the concentration of flavonoids (r = 0.44,
P < 0.01). Titratable acidity was not significantly correlated with
any of the other parameters measured.
 S.F. Hagen et al. / Postharvest Biology and Technology 45 (2007) 110
Fig. 4. Principal component analysis (PCA) correlation loadings (A) and score
plot (B) for health and sensory related properties in the peel of shade-grown and
sun-exposed apples (cv. Aroma) before and after postharvest irradiation. PC 1
explains 72% and PC 2 explains 12% of the total variation.
4. Discussion
This study shows that it is possible to increase the level of
not only phenolic compounds, but also other health and sensory
related properties of Aroma apples by postharvest irradia-
tion treatment. The intensity of red skin colour, the antioxidant
capacity, the total phenols and the content of anthocyanins,
quercetin glycosides, chlorogenic acid and ascorbic acid were
all enhanced. The combination of visible light and UV-B radia-
tion was found to be the most effective irradiation treatment and
only the apple peel, not the flesh, was affected. The treatment
did, however, not change important taste related properties.
As expected from the colour changes, only the apples exposed
to visible light and UV-B radiation combined had developed
a higher content of anthocyanins. This combination has been
previously found to be effective or even essential in the induc-
tion of anthocyanins in several red and non-red apple cultivars
(Arakawa et al., 1985; Reay, 1999; Lancaster et al., 2000;
Reay and Lancaster, 2001; Ubi et al., 2006), although Arakawa
et al. (1985) and Marais et al. (2001) demonstrated antho-
cyanin accumulation also upon irradiation with visible light
7
alone in Jonathan and Cripps Pink apples, respectively. The
light-regulated synthesis of anthocyanins in apple involves phy-
tochromes, specific UV-B photoreceptors and photosynthesis
(Saure, 1990) and is still not fully understood. The increase
of the colourless or slightly yellow flavonols upon postharvest
irradiation in Aroma apples was much higher when UV-B
was included than when irradiated with visible light alone,
whereas earlier work on five other cultivars has demonstrated
an accumulation of quercetin glycosides only after irradiation
with UV-B and visible light combined (Reay, 1999; Lancaster
et al., 2000; Reay and Lancaster, 2001). Postharvest irradiation
with Vis + UV-B had a stronger influence on the peel concen-
tration of chlorogenic acid than preharvest exposure to solar
radiation. In contrast, the content of epicatechin, procyanidins
and phloridzin in the present study was minimally influenced by
the irradiation treatments, even though the peel concentration of
these compounds was almost two-fold higher in the sun-exposed
apples than in the shade-grown apples. The results are in agree-
ment with Lancaster et al. (2000), who found increased levels of
chlorogenic acid, but not procyanidins, in the apple peel of five
cultivars after irradiation with UV-B and visible light. Takos et
al. (2006) reported a coordinated transcription of the flavonoid
biosynthetic pathway genes except for the genes coding for the
synthesis of proanthocyanidins (condensed tannins) in Cripps
Red apples. The concentration of chlorogenic acid, which orig-
inates from the same biosynthetic pathway as the flavonoids
(Treutter, 2001), in the present study was not correlated with the
concentration of any of the flavonoids.
The non-destructive chlorophyll fluorescence measurements,
performed directly on the skin of intact apples, mostly con-
firmed the results obtained with chemical analyses. However,
these measurements showed that the content of anthocyanins
and UV-absorbing flavonoids, mainly represented by quercetin
glycosides (Hagen et al., 2006), had increased also in the skin
of the sun-exposed apples exposed to Vis + UV-B by 1215%.
Chlorophyll fluorescence measurements were performed repeat-
edly on the same individual apples during the experiment and
are thus more powerful for detecting changes than destruc-
tive methods, where data from different apples are compared.
Furthermore, the fluorescence data were collected from twice
as many apples as for the chemical analysis. The chlorophyll
fluorescence measurements revealed that the accumulation of
UV-absorbing flavonoids started earlier and increased to a higher
level than the accumulation of anthocyanins. This, in addition to
the increase of quercetin glycosides but not anthocyanins upon
irradiation with Vis alone, demonstrates an uncoordinated syn-
thesis of quercetin glycosides and anthocyanins in the apple
skin. Since the gene transcription for the synthesis of these com-
pounds appears to be regulated in a coordinated manner, an
additional post-transcriptional or post-translational regulation
of their synthesis has been suggested (Takos et al., 2006). Com-
petition for enzyme substrates in the synthesis of anthocyanins
and flavonols has been demonstrated in petunia flower petals
(Davies et al., 2003) and there is strong evidence for the exis-
tence of multi-enzyme complexes that facilitate channelling of
intermediates between active sites in plant cells (Winkel-Shirley,
2001).
8 S.F. Hagen et al. / Postharvest Biology and Technology 45 (2007) 110
That the highest increase of anthocyanins and quercetin gly-
cosides upon postharvest irradiation was found in the peel of the
shade-grown apples is in agreement with irradiation studies dis-
tinguishing between sun-exposed and shaded sides of Gala,
Royal Gala, Braeburn, Pacific Rose and Aurora apples
(Lancaster et al., 2000; Reay and Lancaster, 2001). Lancaster
et al. (2000) suggested that in apples with a high content of
anthocyanins and quercetin glycosides, either because of previ-
ous light exposure or genetic background, the UV-B response
may be saturated and a further increase of these compounds
may then not be possible. Accumulation of anthocyanins and
quercetin glycosides induced by postharvest irradiation was in
the above-mentioned experiments always highest at 20 C and
in most cases absent at 10 C (Lancaster et al., 2000; Reay and
Lancaster, 2001). Besides variation between apple cultivars, the
lack of response to irradiation treatments at 10 C in these stud-
ies could be due to a shorter experimental period than in the
present study. Even though low temperature is known to induce
anthocyanin synthesis in tree-attached apples, the lag-time for
anthocyanin production seems to increase under low tempera-
ture in detached apples (Saure, 1990). Low temperature during
irradiation may, however, be beneficial in preventing loss of fruit
quality. The apples had no symptoms of damage after the exper-
iment and the weight loss was very small. Previous literature
does not report the quality of the fruit after irradiation treat-
ments. Irradiation in the present experiment was carried out
at intervals of 12 h per day which may increase the apples
ability to recover from oxidative stress. In a possible appli-
cation, similar intervals allow the irradiation to be executed
at hours when workers are not present (e.g. in a commercial
store).
Soluble solids and titratable acidity are the most common
parameters measured in standard chemical analyses related to
fruit quality, and a change in the ratio between these parameters
can have a great impact on the taste of the apple. Despite the
fact that the flavonoid synthesis requires consumption of sug-
ars (Lister et al., 1997), postharvest irradiation did not have any
influence on the total sugar content in the apples. The apples have
some photosynthetic capacity themselves (Blanke and Lenz,
1989) and apple skin photosynthesis due to exposure to visi-
ble light may have provided the necessary assimilates for the
flavonoid synthesis.
The glycosylation of the flavonoid nucleus (aglycon) is con-
sidered to be the last step in the flavonoid biosynthesis and is
depending upon the specificity or level of specific glycosyltrans-
ferases present (Lister et al., 1997). The abundance ranking of
quercetin glycosides (galactoside > glucoside > rhamnoside) in
the apple skin was neither influenced by postharvest irradiation
nor by preharvest exposure to solar radiation, but has been previ-
ously found to differ between apple cultivars (Lister et al., 1997;
Lancaster et al., 2000).
The content of ascorbic acid in fruits and vegetables is largely
influenced by the temperature and access to light (Lee and Kader,
2000; Ma and Cheng, 2004) and has been found to decrease
during long-term storage of apples (Meberg et al., 2000). The
increase of ascorbic acid in the peel of shade-grown apples
upon postharvest irradiation suggests that light treatments may
be utilised to increase the content of ascorbic acid in sub-
optimal apples or to re-increase the level of ascorbic acid in
apples after long-term storage. Similar published results have
not been found, but Lu et al. (1991), who studied the effect
of UV-C irradiation on shelf-life and ripening of apples and
peaches, found that Golden Delicious apples exposed to UV-C
before storage had a higher content of ascorbic acid than non-
treated apples after storage for 30 days at room temperature.
Ma and Cheng (2004) found an increased level of ascorbic acid
in the shaded side of apples in the field after they had been
turned 180 and exposed to full sun. It is shown in the present
study that even low levels of visible light (2530 mol photons
m2 s1 ) given postharvest can increase the ascorbic acid con-
tent. Ascorbic acid has an important function in the xanthophyll
cycle that protects the photosynthetic apparatus against reactive
oxygen species produced at high light levels (Ma and Cheng,
2004). The contents of phenolic compounds and ascorbic acid
in the present study were very well correlated, probably due
to their light-regulated synthesis. However, unlike the synthe-
sis of flavonoids for which UV-B seems to be most important,
visible light may be most important for an increase in ascorbic
acid.
Postharvest irradiation increased the antioxidant capacity,
measured by the ORAC assay, in the peel of both sun-exposed
and shade-grown apples. The content of phenols in the peel of
Aroma apples was much higher than the content of ascorbic
acid and was also better correlated with the antioxidant capac-
ity. This supports the results by Eberhardt et al. (2000) and Lee
et al. (2003), who found that phytochemicals such as phenolic
compounds are the major source of antioxidants in apple rather
than other constituents such as vitamin C.
Compared to more widely distributed apple cultivars,
Aroma has a medium to relatively high content of phenolic
compounds (van der Sluis et al., 2001; Tsao et al., 2003). The
distribution of phenolic compounds within and between the peel
and the flesh of Aroma was found to be similar to that of other
cultivars. Since both flavonoids and ascorbic acid are more con-
centrated in the peel than in the flesh of apples, peeling an apple
before consumption will obviously decrease the health value of
the apple dramatically. Based on our data, a peel of 1 mm thick-
ness would contain only about 10% of the edible mass but nearly
30% of the antioxidant capacity and the phenolic compounds,
including 100% of the quercetin glycosides and anthocyanins,
and 85% of the ascorbic acid content in a medium sized and
sun-exposed Aroma apple (Table 3). Similar results have been
found by McGhie et al. (2005). The loss of health value due
to peeling will increase with peel thickness. However, in spec-
trophotometric transmission measurements of apple peel discs
with various thicknesses (data not presented), the highest con-
centration of flavonoids was found in the outer 0.25 mm of the
peel. That the red to green colour values (a* ) of the apple skin
were so well correlated with the peel content of phenolic com-
pounds and ascorbic acid in Aroma apples implies that the
colour of the apple skin may provide useful information about
the health value of the apples.
In conclusion, postharvest irradiation at certain wavelength
ranges can be utilised to improve the health value of Aroma
 S.F. Hagen et al. / Postharvest Biology and Technology 45 (2007) 110 9

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