Effects of temperature on the chemical composition and antioxidant activity of three strawberry cultivars

Food
Chemistry
Food Chemistry 91 (2005) 113–121
www.elsevier.com/locate/foodchem
E?ects of temperature on the chemical composition and
antioxidant activity of three strawberry cultivars
Beatriz Rosana Cordenunsi, Maria Ine´s Genovese, Joa˜o Roberto Oliveira do Nascimento,
Neuza Mariko Aymoto Hassimotto, Ricardo Jose´ dos Santos, Franco Maria Lajolo *
Laborato´rio de Qu?´mica, Bioqu?´mica e Biologia Molecular de Alimentos, Departamento de Alimentos e Nutric¸a˜o Experimental,
FCF, Universidade de Sa˜o Paulo, Av. Prof. Lineu Prestes 580, Bloco 14, 05508-900 Sa˜o Paulo, SP, Brazil
Received 8 March 2004; received in revised form 27 May 2004; accepted 27 May 2004
Abstract
Three strawberry cultivars (Dover, Campineiro, and Oso Grande), grown in the same commercial plantation, were harvested at
the ripe stage and stored at 6, 16 and 25 °C, for 6 days. During this period, chemical composition and antioxidant activity were
evaluated. Results showed an increase in total soluble sugars, anthocyanin and vitamin C contents, indicating that a new biosyn-
thesis had taken place during storage. Low temperature negatively a?ected anthocyanin and vitamin C accumulation, and positively
a?ected soluble sugars, while ?avonols, ellagic acid and total phenolic contents remained almost the same or even decreased at all
temperatures. Despite di?erences in anthocyanin content between varieties and its increase during storage (higher with increasing
temperature), there was no di?erence in the antioxidant activity between cultivars, which decreased after harvesting, independently
of the temperature of storage. Variations in the proportion dehydroascorbic acid/ascorbic acid (DHA/AA) showed that there were
di?erences between cultivars concerning adaptation of the fruit to low temperatures. The data obtained here indicate that cold stor-
age is an e?ective way to maintain strawberry quality, but a compromise between sensorial and nutritional values can be achieved at
16 °C, for all the cultivars.
Ó 2004 Elsevier Ltd. All rights reserved.
Keywords: Strawberries; Storage; Anthocyanins; Soluble sugars; Antioxidant activity; Vitamin C; Flavonols
1. Introduction
processing. On the other side, the preference for fresh
fruits is challenging because they have a very short
Strawberries are a good source of ascorbic acid (AA),
shelf-life, due to their sensitivity to fungal attack and ex-
anthocyanins and ?avonols and, among the fruits, they
cessive texture softening caused by the natural ripening
have one of the highest antioxidant activities evaluated
process.
by oxygen radical absorbance capacity (Cordenunsi,
To circumvent the losses associated with handling
Nascimento, Genovese, & Lajolo, 2002; Wang, Cao, &
and storage of strawberry, and other small fruits, some
Prior, 1996). Like other fruits, strawberries can be con-
postharvest conditions, such as low temperature or high
sumed ‘‘in natura’’, which turns out to be advantageous
CO2 concentration, as well as controlled atmosphere or
to consumers since there are no nutritional losses due to
a combination of both processes, are widely used to ex-
tend the shelf-life (Gil, Holcroft, & Kader, 1997; Pelayo,
Ebeler, & Kader, 2003). Since there are some injuries as-
*
sociated with postharvest handling (Manning, 1996),
Corresponding author. Tel.: +55-11-3818-3647; fax: + 55-11-3815-
fruit quality is evaluated in terms of its main sensorial
4410.
E-mail address: fmlajolo@usp.br (F.M. Lajolo).
attributes, in order to maintain consumer acceptance.
0308-8146/$ - see front matter Ó 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.foodchem.2004.05.054

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B.R. Cordenunsi et al. / Food Chemistry 91 (2005) 113–121
In this way, organoleptic parameters, such as texture,
Temperatures around 0 °C are considered the best for
freshness and colour are considered important, but the
strawberry storage because they cause few changes in
nutritional or functional issues are not considered. As
quality. However, the commercialization and post-mar-
a result, there are few studies focussing on the e?ects
ket storage usually occur at higher temperatures. These
of
strawberry
storage
conditions
on
nutritional
higher temperatures can a?ect, not only the strawberry
parameters.
shelf-life, but also its nutritional value, in terms of solu-
Controlled atmosphere has the undeniable bene?t of
ble sugars, vitamin C and antioxidant compounds. This
controlling postharvest decay of fruits, but a CO2-en-
study presents the temperature responses of three straw-
riched atmosphere with low O2 concentration can a?ect
berry cultivars in relation to nutritional parameters and
total ascorbic acid and anthocyanin contents adversely,
antioxidant capacity.
with a negative consequence in fruit colour and nutri-
tional value (Holcroft & Kader, 1999). It is already
known that anthocyanin synthesis continues after har-
2. Material and methods
vest and also low temperature storage (Kalt, Forney,
Martin, & Prior, 1999), but it is inhibited in fruits stored
2.1. Material
in high CO2 concentrations. Holcroft and Kader (1999)
found a negative e?ect of the atmosphere on the antho-
Strawberry fruits (Fragaria ananassa Duch.), of the
cyanin concentration and on the activities of the key en-
cultivars Dover, Campineiro (Brazilian cultivar) and
zymes
of
the
anthocyanin
synthesis
pathway:
Oso Grande, were harvested in the same commercial
phenylalanine ammonia lyase and UDP-glucose: ?avo-
plantation located in Atibaia (Sa˜o Paulo State), at the
noid glucosyltransferase. Also Kalt et al. (1999) con-
stage 5 (full size 3/4 red). Fruits of each variety were di-
cluded that low temperatures could a?ect anthocyanin
vided in three groups and stored at 6, 16 °C and room
synthesis during storage of small fruits, inclusive of
temperature ($25 °C), for 1–6 days. Samples of at least
strawberries.
40 fruits of each period were picked, made into pieces,
Like anthocyanins, the amount of AA is also depend-
immediately frozen in liquid nitrogen and stored at
ent on the strawberry cultivar and ripening degree, al-
À80 °C. At the time of analysis, samples were thorough-
though the average content (60 mg/100 g) is high
ly homogenized by powdering in liquid nitrogen. The
enough to consider strawberry as one of the richest
controls corresponded to fruits frozen at the day of har-
sources of AA among fruits (Cordenunsi et al., 2002).
vesting. Sampling of the stored strawberries was limited
To retain the initial AA content during storage, temper-
by the natural senescence of the fruits.
ature management is the most important factor to be
taken into account. In this respect the speed of lowering
the temperature after harvest and the temperature of
2.2. Carbohydrate determination
storage are crucial (Lee & Kader, 2000). Ascorbic acid
is the predominant form of vitamin C present in fruits,
Soluble sugars were extracted three times with 80%
and the primary oxidation product. L-Dehydroascorbic
ethanol at 80 °C. After centrifugation, the supernatants
acid (DHA) is also important because it also has biolog-
were combined and the ethanol was evaporated under
ical activity. The average content of DHA in fruits is less
vacuum. The soluble sugar content was analyzed by
than 10% of total vitamin C content and, to our knowl-
high pressure liquid chromatography with pulse amper-
edge, only one report indicated an increase of the DHA/
ometric detection (HPLC-PAD – Dionex, Sunnyvale,
AA ratio during storage (Lee & Kader, 2000). Since the
CA, USA), using a PA1 column (Dionex, Sunnyvale,
oxidized form is more prone to decomposition, leading
CA, USA) in an isocratic run of 18 mM NaOH during
to the loss of biological activity, the changes in AA
25 min. Total soluble sugars were obtained as the sum
forms are important in both, technological and nutri-
of glucose, fructose and sucrose values.
tional terms.
There is a controversy in the literature about the in-
?uence of vitamin C content on the antioxidant capacity
2.3. Ascorbic acid determination
of vegetables. On the other hand, there is a consensus
that the antioxidant capacity is directly correlated with
Total AA was extracted with metaphosphoric acid
phenolic compounds, especially ?avonoids (Connor,
(1% w/v) and analyzed by reversed-phase HPLC in a
Luby, Hancock, Berkheimer, & Hanson, 2002; Robards,
Hewlett–Packard 1100 system coupled to a diode array
Prenzler, Tucker, Swatsitang, & Glover, 1999; Wang
detector. The column used was a l-Bondapak (300
et al., 1996). Although the antioxidant compounds have
mm · 3.9 mm i.d., Waters, Milford, USA), and elution
an important role in human health, only a few reports
(?ow rate of 1.5 ml/min) was performed, under isocratic
have focussed on their changes in fresh fruit stored at
conditions, with 0.2 M sodium acetate/acetic acid bu?er
low temperature.
(pH 4.2), monitored at 262 nm. Total AA was estimated

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B.R. Cordenunsi et al. / Food Chemistry 91 (2005) 113–121
115
after reduction of DHA with 10 mM 1,4-dithiothreitol
2.6. Antioxidant activity
(DTT). Dehydroascorbic acid was calculated as the dif-
ference between total-AA and reduced form of AA.
Powdered frozen samples ($1 g, in triplicate) were ex-
tracted twice in cold 70% aqueous methanol containing
5% acetic acid (10 ml portion each time) with a Brink-
2.4. Flavonol, anthocyanin and ellagic acid contents
mann Homogenizer (PolytronÒ – Kinematica GmbH,
Kriens-Luzern, Sweden). The homogenate was centri-
Extraction of ?avonoids was performed as previously
fuged at 10,000g for 10 min at 4 °C and made up to
reported (Cordenunsi et al., 2002). Frozen samples ($30
25 ml with 70% aqueous methanol containing 5% acetic
g, in duplicate) were extracted three times in cold meth-
acid. For the antioxidant assay the extracts were diluted
anol:water:acetic acid (70:30:5) with a Brinkmann
400 times with the extraction solution. The antioxidant
Homogenizer
(PolytronÒ
–
Kinematica
GmbH,
activity was determined according to the b-carotene
Kriens-Luzern, Sweden). The homogenate was ?ltered
bleaching method, following a modi?cation of the pro-
under reduced pressure through ?lter paper (Whatman
cedure described by Marco (1968). A 20 ll aliquot of
no 1) and the combined fractions were evaporated under
a b-carotene (Sigma Chemicals Co., St. Louis, USA) so-
vacuum at 40 °C to $20 ml in a RotavaporÒ RE 120
lution (100 mg/50 ml of chloroform) was added to a 200
(Bu¨chi, Flawil, Sweden) and made up to 25 ml with wa-
ml-Erlenmeyer ?ask containing 1.0 ml of chloroform,
ter. An aliquot of 10 ml of the extract was added to a 1
0.4 ml of linoleic acid and 0.4 ml of Tween 40 (Sigma
g-polyamide SC6 column (Macherey-Nagel Gmbh &
Chemicals Co., St. Louis, USA), and the chloroform
Co. Germany) preconditioned with methanol (20 ml)
was evaporated to dryness under nitrogen. Oxygenated
and water (60 ml). The column was washed with water
distilled water (100 ml) was added to the dried mixture,
(20 ml) and further eluted with 40 ml methanol, fol-
mixing thoroughly. Aliquots (100 ll) of the strawberry
lowed by 40 ml of methanol:ammonia (99.5:0.5). These
extracts or 70% MeOH (as the control) were added to
fractions were evaporated to dryness under reduced
2.9 ml of this b-carotene solution in cuvettes, and mixed
pressure at 40 °C, redissolved in methanol (1 ml) and ?l-
well. The samples were then subjected to thermal autox-
tered through 0.22 lm PTFE ?lters (Millipore Ltd.,
idation at 50 °C. The absorbance of the solution at 470
Bedford, USA). Identi?cation and quanti?cation was
nm was immediately measured after mixture and 2 h ox-
achieved by using analytical reversed-phase HPLC in a
idation with a Hewlett-Packard 8453 spectrophotome-
Hewlett–Packard 1100 system with autosampler and
ter. Antioxidant activity was calculated as the percent
quaternary pump coupled to a diode array detector.
inhibition relative to the control.
The column used was a Prodigy 5l ODS3 reversed
phase silica (250 mm · 4.6 mm i.d., Phenomenex Ltd.)
and elution solvents were: A, water:tetrahydrofuran:
2.7. Statistical analysis
tri?uoroacetic acid 98:2:0.1 and B, acetonitrile. Solvent
gradient was the same as used by Price, Prosser, Riche-
Statistical analysis was done by using the Statistica
tin, and Rhodes (1999), except for the separation of
software package version 5.0 (StatSoft, Inc., Tulsa,
acidic ?avonols, where the initial % B was 25%, in order
USA). Di?erences between means were ?rst analyzed
to allow separation of ellagic acid from quercetin glu-
by ANOVA test and then LSD (least signi?cant di?er-
curonide. Samples were injected in duplicate and ?avon-
ence) test (P < 0.05).
ols were quanti?ed using quercetin and kaempferol
(Sigma Chemicals Co., St. Louis, USA) as external
standards. For anthocyanins, standard solutions of pel-
argonidin chloride (Extrasynthe`se, Genay, France) and
3. Results and discussion
cyanidin-3-rutinoside (Apin Chemicals Ltd., Abingdon,
UK) were used. Ellagic acid was from Sigma Chemicals
3.1. Total soluble sugars (TSS)
Co. Calibration was performed by injecting the standard
three times at ?ve di?erent concentrations. Results were
At the ?rst day of storage the amount of total soluble
expressed as mg/100 g fresh weight (FW).
sugars was under 5 g/100 g (FW) for all cultivars (Fig. 1)
and, as can be seen, lower temperatures had a positive
e?ect on the TSS contents. Except for the Dover cultivar
2.5. Total phenolics
stored at 6 °C, the Campineiro and Oso Grande had
their TSS increased up 30%, when stored in a cold room.
Total phenols were determined according to the
In a previous experiment (Cordenunsi, Nascimento, &
method of Swain and Hillis (1959), using the Folin–
Lajolo, 2003), Campineiro fruits kept at 6 °C for a week
Ciocalteau reagent. Results were expressed as mg cate-
showed no changes in TSS values, while Dover had an
chin/100 g FW.
increase of 10% and Oso Grande had a decrease of

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116
B.R. Cordenunsi et al. / Food Chemistry 91 (2005) 113–121
Dover
Campineiro

A 20% decrease in the sucrose content was observed
Oso Grande
6






































for all cultivars, except for 16 °C-Dover, but the amount
RT
RT






































RT






































5
at the last day of storage was still higher than that de-












































































scribed earlier (Cordenunsi et al., 2003). Since sucrose
4
-1






































is the primary source of glucose and fructose, the in-






































3







































100g
creased amount of these monossacharides could account






































g.
2
for the decrease in sucrose level. However, the TSS in-












































































1
crease indicates that sucrose synthesis had taken place












































































during cool-storage.
0









































16 oC






































There are three possible carbon sources for soluble
16 oC
16 oC






































5






































sugar synthesis after harvest: starch, organic acids and






































4
cell-wall disassembly. Since strawberry fruit has insu?-
-1












































































cient starch ($0.1%) to support this synthesis, organic
3







































100g






































acids and cell-wall are the more likely sources. The neg-
g.
2






































ative correlation between texture and TSS-increase, ob-






































1
served for Dover and Campineiro during cool-storage,






































0
could indicate that cell-wall disassembly plays an impor-






































6 oC
6 oC
6 oC






































tant role in sugar accumulation. The increase in xylose






































5






































content during cold storage of Campineiro and Dover






































4
cultivars (Fig. 2) also reinforces this idea. On the other
-1












































































3
hand, Oso Grande, which is a more stable cultivar in






































100g






































g.
terms of texture (Cordenunsi et al., 2003), showed the
2












































































highest increase of TSS (up to 30%). Since there were
1






































no marked changes in xylose content and the citric acid
0
content decreased (Cordenunsi et al., 2003), this organic
1
3
6
1
3
6
1
3
6
Days of storage
= sucrose
Dover
Campineiro
Oso Grande



0,20
= fructose






































RT
RT






































RT
= glucose












































































0,15






































+
+
= total soluble sugars






































-1












































































0,10

Fig. 1. Sucrose, fructose and glucose contents (g/100 g FW) of






































100g






































cultivars Dover, Oso Grande and Campineiro strawberries during
g.












































































storage at di?erent temperatures (6, 16 and 25 °C). Data presented are
0,05






































means of triplicate assays ± SE.












































































0,00









































16 oC






































16 oC
16 oC
20% over storage time. Comparable data reported in the












































































0,15






































literature showed a decrease between of 8% and 16% in






































-1






































TSS of strawberries kept at 5 °C in air (Gil et al., 1997;






































0,10







































100g
Pelayo et al., 2003). The apparent discrepancies between






































g.






































the previous and the present data are in agreement with






































0,05






































the results reported by Watson (Watson, Wright,












































































McBurney, Taylor, & Linforth, 2002), who found that
0,00
6 oC
6 oC
6 oC
sugar, citric acid and volatile compounds varied consid-


















































































































erably between the fruits and harvests.
0,15












































































The main soluble sugars found in all cultivars were
-1












































































0,10
fructose, glucose and sucrose (Fig. 1). At the ?rst day






































100g






































g.
of storage, the fructose to glucose ratio was almost the












































































0,05
same (1.1) for all cultivars, the sucrose content being al-












































































most half that of fructose. During storage at 16 °C, the






































0,00
fructose/glucose ratio increased for all cultivars, reach-
1
3
6
1
3
6
1
3
6
ing values around 1.4. The higher fructose content at
Days of storage
this temperature could be explained by the glucose con-
Fig. 2. Xylose content (g/100 g FW) of cultivars Dover, Oso Grande
sumption (by the respiration) which was inhibited at the
and Campineiro strawberries during storage at di?erent temperatures
lower storage temperature (6 °C).
(6, 16 and 25°C). Data presented are means of triplicate assays ± SE.

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B.R. Cordenunsi et al. / Food Chemistry 91 (2005) 113–121
117
acid could be an alternative carbon source for soluble
Table 1
sugar synthesis in cold-stored Oso Grande strawberries.
Ratio between pelargonidin and cyanidin glycosides in strawberry
cultivars and its change after storage at di?erent temperatures
3.2. Anthocyanins
Pelargodini