Journal of Food Composition and Analysis 24 (2011) 11–16

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Journal of Food Composition and Analysis

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

Original Article

Antioxidants and other chemical and physical characteristics of two

strawberry cultivars at different ripeness stages
Lı́via de L. de O. Pineli a,*, Celso L. Moretti b, Marcos S. dos Santos c, Alinne B. Campos c,
Andréia V. Brasileiro c, Andressa C. Córdova c, Marileusa D. Chiarello c
a
College of Health Sciences, Department of Nutrition, University of Brasilia, 70910-900, Brasilia, DF, Brazil
b
Embrapa Vegetables, Rod. BR 060, km 09, C.P. 218, 70359-970, Brası´lia, DF, Brazil
c
Catholic University of Brasilia, 71966-700, Brasilia, DF, Brazil

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

Article history: The present work evaluated the quality and antioxidant characteristics of ‘Osogrande’ and ‘Camino Real’
Received 6 March 2010 strawberries at different ripeness stages. Strawberries (Fragaria x ananassa Duch.) were harvested,
Received in revised form 13 May 2010 selected, graded according to ripeness (green, pink or 3/4 ripe and ripe) and evaluated for pH, total
Accepted 18 May 2010
soluble solids, total titratable acidity, sugar/acid ratio, vitamin C, total phenolic compounds, total ellagic
Available online 30 July 2010
acid, total and individual anthocyanins and antioxidant activity by 2,2-diphenyl-1-picrylhydrazyl
(DPPH) and ferric reducing antioxidant power (FRAP). The highest total soluble solid content was found
Keywords:
for pink (7.58 Brix) and ripe (7.98 Brix) ‘Osogrande’ strawberries. At pink stage, this cultivar showed
Fragaria x ananassa Duch.
higher amounts of total phenolics (2909.48 mg kg1 FW) and total ellagic acid (454.16 mg kg1 FW).
Strawberry
Osogrande Pink ‘Camino Real’ strawberries presented the highest content of vitamin C (528.55 mg kg1 FW).
Camino Real Antioxidant activity was higher for ‘Osogrande’ cultivar, at green stage, according to DPPH
Ripeness (11.91 mmol BHT g1 FW) and FRAP (36.75 mmol ferrous sulphate g1 FW) assays and at ripe stage,
Quality only for DPPH assay (12.83 mmol BHT g1 FW). Anthocyanins increased along ripening, with more
Phenolics elevated concentrations in ripe ‘Camino Real’ strawberries (292.9 mg kg1 FW). Cyanindin-3-glucoside
Vitamin C showed a higher concentration for the same treatment (17.23 mg kg1 FW), which might contribute to a
Antioxidant activity
more redish color. Although ripe berries have a better flavor and are more appreciated, higher
Food analysis
antioxidant contents and activities were observed at pink stage in which higher amounts of total
Food composition
phenolics, total ellagic acid and vitamin C were noticed for both cultivars.
ß 2010 Elsevier Inc. All rights reserved.

1. Introduction Genotype and environmental conditions can influence physical

and chemical characteristics of strawberries (Pinto et al., 2008;
Antioxidants from fruits and vegetables are considered an Hernanz et al., 2007; Ayala-Zavala et al., 2004; Cordenunsi et al.,
important protection factor against oxidative stress and its 2002). Harvest maturity and postharvest conditions are some of
deleterious consequences to human health (Battino et al., 2009). the factors that may lead to changes in sensory and nutritional
Strawberry (Fragaria x ananassa Duch.), a very popular berry with qualities of strawberries. Strawberries are frequently harvested at
high visual appeal and desirable flavor, is also considered a good 3/4 ripe (pink) or even 1/2 ripe (green) stages, in order to prevent
source of antioxidants, mainly given to its high vitamin C and postharvest losses due to softening and fungal decay. As fruits are
phenolic contents. Phenolic classes commonly found in strawberries non-climateric, full ripeness is not reached and they are consumed
are hydroxybenzoic acids (gallic and ellagic acids), hydroxycinnamic without the best sensory characteristics. Consumers may usually
acids (p-cumaric), hydrolysable tannins (ellagitannins), flavonols eat strawberries at any of the maturation stages. Therefore, the
(quercetin, kaempferol and myricetin), flavan-3-ols (catequins, effect of ripening on antioxidants and quality is a major issue.
epicatechins), and anthocyanins, being pelargonidin-3-glycoside, Every other year new genotypes, mainly from California and
the most important flavonoid pigment (Tulipani et al., 2008; Seeram Florida breeding programs, are introduced in Brazil. ‘Camino Real’
et al., 2006). is a recent strawberry cultivar developed in California (Shaw and
Larson, 2002). It is characterized by large, firm fruits, with dark-red
peel and pulp, and good flavor. It has been recently introduced in
the Brazilian market with significant production in the savannah
* Corresponding author. Tel.: +55 61 33072548/2510; fax: +55 61 32733676. areas. Limited information about ‘Camino Real’ strawberries is
E-mail address: liviapineli@unb.br (Lı́via de L. de O. Pineli). available.

0889-1575/$ – see front matter ß 2010 Elsevier Inc. All rights reserved.
doi:10.1016/j.jfca.2010.05.004
12 L.L.O. Pineli et al. / Journal of Food Composition and Analysis 24 (2011) 11–16

The objective of this work was to investigate the effects of 2.4. Extraction and measurement of phenolic compounds and total
harvest maturity on some physical and chemical characteristics antioxidant capacity assays
and ‘in vitro’ antioxidant activity of ‘Osogrande’ and ‘Camino Real’
strawberries. 2.4.1. Sample extraction
Sliced frozen strawberries (75 g) were triturated in ice bath.
2. Materials and methods Samples of homogenized tissues were reserved at 18 8C for the
analysis of total anthocyanins (item 2.5) at the same day. For the
2.1. Fruit harvest preparation of the extracts, 10 g of the crushed tissues were
homogenized using a Utraturrax Turratec TE102E (Tecnal, Brazil) for
Strawberry fruits, cultivars Osogrande and Camino Real, grown 2 min at 15,000 rpm while cooled in ice, with 100 mL of 80% acetone,
in the Federal District (158410 S, 488100 W, 1200 m altitude) were as described by Shin et al. (2007), with some modifications. The
harvested on the same commercial field, located in Brazlândia. homogenate was filtered through Whatman #1 paper and acetone
Fruits were graded for size and external color, sorted to eliminate was evaporated using a rotary evaporator at 45 8C for 30 min.
damaged material and transported, under refrigeration, to the Deionized water was added to each sample in order to bring it to the
Postharvest Laboratory of Embrapa Vegetables (30 km). The same final volume of 10 mL. The extracts were kept frozen at 80 8C until
day, they were classified according to harvest maturity (1/2 ripe or analysis and were used for the measurement of phenolic compounds
green, 3/4 ripe or pink and full ripe) and packed into vented crystal and total antioxidant activity assays. All extractions were done in
clamshell (PET) boxes. Each box (experimental unit) presented duplicate, and the subsequent assays were run in triplicate.
300  10 g or 21  1 g strawberries. Frozen samples were then
quickly transported to the Food Science and Technology Laboratory, 2.4.2. Total phenolic compounds (TPC)
Catholic University of Brasilia. Fruits from each experimental unit Total phenolic compounds were quantified using a modified
(box) were cut into pieces (calix removed), packed in polyethylene Folin-Ciocalteau colorimetric method (Singleton et al., 1999). A
bags (portions of 75 g) with a vacuum sealer (Selovac, model 200B, 0.2 mL aliquot of the 40-fold water diluted strawberry extract was
Brazil), and stored at 80 8C. The experimental units were analyzed added to a 15 mL tube and 0.2 mL of 1:10 Folin-Ciocalteau
for soluble solids content, total titratable acidity, total vitamin C reagent:water solution was added to the mixture. The tube was
content, total phenolic compounds, total antioxidant activity assays, allowed to stand at room temperature for 1 min. Then, 2 mL of 7.5%
total and individual anthocyanins, and total ellagic acid contents. All (w/v) Na2CO3 were added to the mixture. After 2 h at room
analyses were carried out within 2 months. temperature, absorbance was measured at 765 nm versus a blank.
The results were expressed as mg gallic acid eq. kg1 FW. Calibra-
2.2. Total soluble solids (TSS) and total titratable acidity (TTA) tion was performed by analysing the standard gallic acid (Sigma–
Aldrich CO., USA) three times at five different concentrations, in the
Sliced frozen strawberries (75 g) were homogenized (Ultraturrax range of 10–100 mg (R2 = 0.9979).
Turratec TE102E, Tecnal, Brazil) for 2 min at 15,000 rpm while cooled
in ice bath, and centrifuged at 15 8C at 17,600  g for 15 min at 4 8C 2.4.3. Antioxidant activity
(Quimis Q222RM, Brazil). An aliquot was used to determine TSS with
a refractometer (Quimis Q767, Brazil). Result was expressed as 8Brix. 2.4.3.1. 2,2-Diphenyl-1-picrylhydrazyl (DPPH) radical-scavenging
TTA was determined by titration after diluting each 5 mL aliquot of activity. The antioxidant activity was determined by the DPPH
strawberry juice to 50 mL with distilled water. Titration was carried radical-scavenging method according to Rufino et al. (2007). Aliquots
out to pH 8.2 using a 0.1 mol L1 NaOH solution. Results were of 0.1 mL of the previously water diluted (100, 50 and 25 mg mL1)
expressed as meq. citric acid kg1 of fresh weight (FW). TSS, as % of strawberry extracts were mixed to 3.9 mL of 0.06 mM DPPH. The
total soluble solids or 8Brix, and TTA, converted to % of citric acid, solutions were incubated at 25 8C for 25 min. Absorbance was then
were combined to find sugar/acid ratio (SAR). recorded at 517 nm using methanol as a blank and methanolic
solutions of BHT at five concentrations (25–200 mg mL1) in the
2.3. Determination of total vitamin C place of strawberry extracts, as a control. Total antioxidant activity
was expressed as mmol BHT eq. g1 fruit (FW).
Total vitamin C content was quantified in accordance with the
dinitrophenylhydrazine (DNPH) method, modified by Nunes et al. 2.4.3.2. Ferric reducing antioxidant power (FRAP). The antioxidant
(1995). Sliced frozen strawberries (75 g) were triturated in ice activity by FRAP assay was determined according to the method
bath, and 1 g of frozen crushed tissue, for each replicate, was described by Rufino et al. (2006). In the darkness, a 90 mL aliquot of
homogenized using a homogenizer Utraturrax Turratec TE102E each aqueous extract dilution (40, 20 and 10 mg mL1) was mixed
(Tecnal, Brazil) for 2 min at 15,000 rpm while cooled in ice, with with 270 mL of distilled water and 2.7 mL of FRAP reagent. Tubes
19 mL of a mixture of 6% (w/v) metaphosphoric acid in 2 mol L1 were vortexed and incubated at 37 8C for 30 min. Absorbance was
acetic acid. The mixture was centrifuged at 17,600  g for 15 min determined at 595 nm using FRAP reagent as blank and 500–
at 4 8C. The supernatant was filtered through Whatman #1 filter 2000 mM ferrous sulphate solutions substituting strawberry
paper. An aliquot of 0.05 mL of 0.2% (w/v) 2,6-dichlorophenol- extracts as a control. Results were expressed as mmol ferrous
indophenol (DCPIP) was added to 1 mL of the supernatant and sulphate g1fruit (FW).
incubated at room temperature for 1 h. Thiourea solution (2%, w/v)
in 5% metaphosphoric (w/v) acid and 0.5 mL of 2% (w/v) DNPH in 2.5. Determination of total anthocyanins
4.5 mol L1 sulphuric acid were added and the solution was
incubated at 60 8C for 3 h. Tubes were placed in an ice bath and Total anthocyanins were determined according to Shin et al.
2.5 mL of ice cold 90% sulphuric acid were slowly added. Tubes (2007), with some modifications. Sliced frozen strawberries (75 g)
were vortexed and total vitamin C was measured by absorbance at were triturated (item 2.4.1) and 1 g, for each replicate, was added
540 nm. The concentration was calculated using a standard curve to 10 mL of methanol containing HCl (0.5%, v/v), homogenized for
of ascorbic acid, performed with 5 triplicate points between 1 and 3 min at 1500 rpm using a homogenizer Ultraturrax Turratec
40 mg (R2 = 0.9977). Concentrations of vitamin C were expressed as TE102E (Tecnal, Brazil), and held at 4 8C for 1 h in the darkness. The
mg ascorbic acid kg1 FW. slurry was centrifuged at 17,600  g for 15 min at 4 8C. The
 L.L.O. Pineli et al. / Journal of Food Composition and Analysis 24 (2011) 11–16 13

absorbance of the supernatant was recorded at 515 nm. Total reduced pressure through Whatman #1 filter paper, and the
anthocyanins content was calculated using the extinction coeffi- combined fractions were evaporated under vacuum at 40 8C to
cient (e) equal to 3.6  104 mol1 m1. Total anthocyanin content 20 mL in a rotatory evaporator and made up to 50 mL with water.
was expressed as mg pelargonidin-3-glucoside eq. kg1 FW. An aliquot of 25 mL of the extract was added to a 1 g polyamide
column (CC6, Macherey-Nagel, German) preconditioned with
2.6. Determination of total ellagic acid methanol (20 mL) and water (60 mL). The column was washed
with water (20 mL) and further eluted with methanol (40 mL), to
Extraction and hydrolysis of ellagitannins methods were obtain the neutral flavonols, and with methanol/ammonia
modified from Pinto et al. (2008). Frozen strawberries (75 g) were (99.5:0.5), to elute the acidic flavonols. The neutral fraction was
lyophilized (Jouan, model LP3, pressure of 7.7  102 mB and evaporated to dryness, redissolved in methanol (1 mL), filtered
temperature of 47 8C) for 4 days in the darkness. The moisture of through 0.22 mm PTFE filters (Millipore Ltd., Bedford, MA, USA),
fresh strawberries and the residual moisture of lyophilized and analyzed by HPLC. Identification and quantification of
strawberries were determined by gravimetry, after drying at anthocyanins were achieved using analytical reversed-phase HPLC
105 8C until constant weight. The results were used to convert data coupled with DAD detector (Shimadzu, SPD-M10A DAD, Germany),
from dry basis to fresh basis. Samples of lyophilized strawberry at 520 nm. The column used was a 5 mm ODS3 reversed phase
powder (0.5 g, for each replicate) were extracted three times in 80% silica (250 mm  4.6 mm i.d., 5 mm, Prodigy ODS3 reversed-phase
methanol (50 mL the first time, 25 mL the next two times) at silica (Phenomenex Ltd., USA). Elution solvents were: A, water:-
15,000 rpm for 1 min (Ultraturrax homogenizer Turratec TE 102E, tetrahydrofuran:trifluoroacetic acid 98:2:0.1 and B, acetonitrile.
Brazil), while cooled in ice bath, and vacuum filtered through Solvent gradient was in the initial proportion of 10% B, increasing
Whatman #1 filter paper. An aliquot of 2 mL of the combined to 17% B after 5 min, to 20% B after a further 8 min, to 23% B after
extracts was dried with gaseous nitrogen and 2 mL of 2N TFA were 5 min to 33% B after 5 min, to 50% B after other 5 min, and to 90% B
added. Hydrolysis was performed in a glycerin bath at 120 8C for after 10 min. Gradient was, then, decreased to 10% B after 10 min
60 min. Hydrolised extracts were rotaevaporated at 95 8C for and kept for other 10 min for equilibration. Calibration was
2 min, ressuspended at 1 mL of HPLC grade methanol, filtered in performed by injecting the standards three times at five different
0.22 mm PTFE filters, and analyzed by HPLC. Identification and concentrations, in the range of 0.1–10 mg. External standards were
quantification of total ellagic acid were achieved using analytical pelargonidin-3-glucoside (Plg-3g, R2 = 0.9983) and cyanidin-3-
reversed-phase HPLC in a Varian Pro Star system with autosampler glucoside (Cy-3g, R2 = 0.9982), both from Sigma Chemical Co. (St.
and ternary pump and UV–vis detector. Some extracts were also Louis, USA). Results were expressed as mg kg1 FW.
analyzed in a HPLC coupled with DAD detector (Shimadzu, SPD-
M10A DAD, Germany), in order to confirm the structures 2.8. Statistical analysis
previously identified by time retention with the UV –vis detector.
The column used was 250 mm  4.6 mm, i.d., 5 mm, Prodigy ODS3 Experiment for ripeness stages effects was carried out using a
reversed-phase silica (Phenomenex Ltd., USA) and elution solvents completely randomized design, with 6 treatments arranged in a
were A, water:tetrahydrofuran:trifluoroacetic acid (98:2:0.1) and 2  3 factorial scheme (two cultivars, three ripeness stages) and
B, acetonitrile. Solvent gradient was: 17% B for 2 min, increasing to three replicates. The experimental unit was a box with 300  10 g
25% B after 5 min, to 35% B after a further 8 min and to 50% B after of strawberries and all analysis were run in triplicate. Data were
5 min. Samples were injected in duplicate. Calibration was submitted to analysis of variance and means were compared by the
performed by injecting the external standard ellagic acid (Sigma least significant difference test (p  0.05). Principal component
Chemical Co., USA) three times at five different concentrations, in analysis (PCA) and cluster analysis (Ward’s Agglomeration method
the range of 0.1–10 mg (R2 = 0.9997). Results were expressed as applied, proximity by dissimilarity—Euclidean distance) were per-
mg kg1 FW. formed in XLSTAT software (Addinsoft, France) for 18 observations (3
replicates of each treatment) and 9 variables. PCA used Pearson
2.7. Determination of pelargonidin-3-glucoside and cyanidin-3- correlation matrix data, to examine the ability of the variables
glucoside by HPLC obtained to differentiate among treatments, and elucidate the effects
of ripening for both cultivars.
Extraction of individual anthocyanins, pelargonindin-3-gluco-
side and cyanidin-3-glucoside, was performed according to Pinto 3. Results and discussion
et al. (2008), with some modifications. Lyophilized sample
powders (0.5 g for each replicate) were extracted three times in 3.1. Quality variables
a solvent mixture (50 mL the first time, 25 mL the next two times)
comprising methanol/water/acetic acid (70:30:5) at 15,000 rpm Ripe strawberry flavor is partially conditioned by the sugars and
for 1 min (Ultraturrax homogenizer Turratec TE 102E, Brazil), acids ratio (SAR). The acidity of ripe strawberries grown in the
while cooled in ice bath. The homogenate was filtered under Brazilian state of São Paulo (southeast region) is close to 160,

Table 1
Chemical characterization of ‘Osogrande’ and ‘Camino Real’ strawberries at different ripeness stages.

Treatment Total titratable acidity Total soluble solids pH Sugar/acid ratiob

(TTA, mg kg1 FW)a (TSS, 8Brix)

Green Osogrande 130.47  5.33a 6.0  0.0c 3.17  0.08c 7.7  0.8a
Pink Osogrande 116.47  10.73b 7.5  0.5ab 3.29  0.07ab 9.4  2.3a
Ripe Osogrande 130.17  13.39a 7.9  0.2a 3.27  0.05abc 9.6  0.9a
Green Camino Real 140.61  11.18a 5.0  0.06d 3.06  0.04d 5.6  0.5b
Pink Camino Real 135.98  7.05a 7.1  0.06b 3.19  0.05bc 7.5  1.1ab
Ripe Camino Real 111.64  2.72b 6.3  0.58c 3.37  0.08a 8.4  0.2a
a
Citric acid equivalent; bto calculate sugar/acid ratio (TSS/TTA), total titratable acidity (TTA) unit is first converted to g 100 g1 citric acid equivalent (%); values are expressed
as means  SD. Means in the same column with common letters are not significantly different (p < 0.05), according to LSD test.
14 L.L.O. Pineli et al. / Journal of Food Composition and Analysis 24 (2011) 11–16

ranging from 89.1 to 353.1 meq. citric acid kg1 FW, according to Tillmans method after extraction in oxalic acid. Pinto et al. (2008)
the results found by Cordenunsi et al. (2002). Total titratable and Cordenunsi et al. (2002) used 6% and 1% (w/v) metaphosphoric
acidity found (TTA) in the present study (Table 1) was in acid solution, respectively, as solvent of extraction, and dithio-
accordance with the literature (Shin et al., 2007; Cordenunsi threitol (10 mM) was applied in both works to avoid oxidation and
et al., 2002, 2003). ‘Osogrande’ strawberries were less acid at pink reduce dehydroascorbic to ascorbic acid. Some differences in the
stage. However, TTA in ‘Camino Real’ fruits was lower for fully proportion of samples and solvents, as well as in the time of
ripened berries. Values found for pH tended to be lower for extraction of the different studies were also noticeable.
‘Camino Real’, except in full ripe stage.
There were no significant differences for total soluble solids 3.3. Total phenolic compounds, ellagic acid and antioxidant activity
content at pink stage for both cultivars. However, at full ripeness, TSS
content for ‘Osogrande’ was higher than ‘Camino Real’ (Table 1). ‘Camino Real’ strawberries presented significantly lower
Sugar/acid ratio (SAR) was calculated and ‘Osogrande’ showed content of total phenolic compounds when compared to ‘Oso-
values ranging from 7.7 and 9.6, from green to ripe stage, whereas grande’ for all ripeness stages (Table 2). Additionally, there were no
values for ‘Camino Real’ ranged from 5.6 to 8.4. SAR in green ‘Camino differences for this variable during ripening of ‘Camino Real’ fruits.
Real’ strawberries were significantly lower than in the other On the other hand, a peak in the concentration of phenolics was
treatments, except for pink ‘Camino Real’ fruit. Cordenunsi et al. observed for ‘Osogrande’ at the pink stage, and the concentration
(2002) observed a higher SAR for ‘Osogrande’ fruits (9.2), whereas was higher than the one previously reported by Cordenunsi et al.
Olsson et al. (2004) found a variation in SAR from 8.9 to 9.9 and from (2002). However, Cordenunsi et al. (2005) reported an even higher
6.7 to 8.0 for Honeoye and Senga Sengana cultivars, respectively. content for ‘Osogrande’ in another set of experiments. Differences
in methodology (HPLC screening and spectrophotometry) as well
3.2. Vitamin C as environmental conditions could explain the observed variation.
The results found in this work are comparable to those observed by
The highest value for total Vitamin C content was determined Scalzo et al. (2005), for six different cultivars of ripe strawberries
for ‘Camino Real’ strawberries at pink stage (Table 2). The content (Don, Idea, Camarosa, Onda, Patty and Sveva), grown in Italy.
of this vitamin increased 2- and 1.6-fold from green to pink stage in Nevertheless, Skupien and Oszmianski (2004) evaluated other six
‘Osogrande’ and ‘Camino Real’ cultivars, respectively. However, it cultivars in Poland (Kent, Elsanta, Selva, Elkat, Dukat and Senga
decreased between 14% and 49% from pink to ripe stage. Kafkas Sengana) and the results were higher than the observed in the
et al. (2006) evaluated quality variables in strawberries at different previous studies reported, being 1.82–2.55-fold higher than total
ripeness stages and found out that ascorbic acid content always phenolics determined for ripe ‘Camino Real’ strawberries in the
increased during ripening. The authors also found that such present study. The comparison among studies also reveals the high
increasing was strongly genotype dependent. The amount of total variability among genotypes.
vitamin C found by Cordenunsi et al. (2002) in strawberries ranged Ellagic acid is considered the most important phenolic
from 401 mg kg1 FW (cv. Dover) to 853 mg kg1 FW (cv. Campi- compound in strawberries (Häkkinen et al., 1999). There is a
neiro), showing a wide variation among cultivars. The result particular interest in ellagic acid due to evidences of its potential
reported by these workers for ‘Osogrande’ was higher than the chemoprotective, anti-inflammatory and antibacterial effects
ones found in the present study. Pinto et al. (2008) found a content (Vattem and Shetty, 2005). Total ellagic acid (TEA) changed with
of vitamin C 2.1-fold higher in ‘Osogrande’ full-ripe strawberries ripeness stages, but the behavior was not the same for both
harvested in the winter of 2005 in São Paulo State (Brazil), genotypes. In ‘Osogrande’ strawberries this phenolic increased
compared to the same cultivar in the present work. Values found from green to pink stage and decreased to the lowest value at full
by Skupien and Oszmianski (2004) for strawberries grown in ripeness. For ‘Camino Real’ strawberries no differences were found
Poland were about 16–87% higher than values found in ripe for this phenolic acid between green and pink stages, but full-ripe
‘Camino Real’ in this work. Genotype and environmental condi- fruit also exhibited a decrease in TEA. When cultivars were
tions as well as differences related to methods of extraction and compared, the highest content was observed for ‘Osogrande’
analysis used in the different published papers must be considered berries at the pink stage and the lowest, for ripe ‘Camino Real’ fruits
in order to understand and justify the wide range in vitamin C (Table 2). Olsson et al. (2004) did not find differences in ellagic acid
content of strawberries reported above. concentrations during ripening of ‘Honeoye’ and ‘Senga Sengana’
Concerned to the analytical methods, it was found that Kafkas strawberries. Pinto et al. (2008) reported a significant variation
et al. (2006) extracted ascorbic acid from the samples with among full ripe strawberry cultivars for TEA. The values found for
aqueous metaphosphoric acid (3%) and analyzed it by HPLC–DAD ripe ‘Camino Real’ are slightly lower than the lowest content
whereas Skupien and Oszmianski (2004) used the volumetric reported by Pinto et al. (2008), for ripe ‘Toyonoka’ strawberries.

Table 2
Vitamin C, total phenolic compound (TPC), total ellagic acid (TEA) and in vitro antioxidant activity of ‘Osogrande’ and ‘Camino Real’ strawberries at different ripeness stages.

Treatment Vitamin C Total phenolic content Total ellagic acid TAAc–DPPHd TAAc–FRAPe
(mg kg1 FW)a (mg kg1 FW)b (mg kg1 FW) (mmol g1 FW) (mmol g1 FW)

Green Osogrande 231.61  23.25d 2169.44  163.98b 216.05  17.29c 11.91  1.42a 36.75  6.49a
Pink Osogrande 468.84  17.54b 2909.48  155.96a 454.16  31.33a 12.21  0.75a 34.30  3.00a,b
Ripe Osogrande 314.52  24.30c 2234.62  69.61b 194.03  21.27c,d 12.83  1.08a 27.37  5.77b,c
Green Camino Real 322.84  38.44c 1786.41  96.06c 308.55  26.02b 9.75 0.83b 26.50  2.00c
Pink Camino Real 528.55  10.28a 1853.25  117.79c 307.33  33.12b 12.01  0.98a 28.49  2.48b,c
Ripe Camino Real 465.78  38.45b 1743.47  226.05c 166.88  20.98d 10.10  0.43b 24.13  2.97c

Means in the same column with common letters are not significantly different (p < 0.05), according to LSD test.
a
Ascorbic acid equivalent.
b
Gallic acid equivalent.
c
Total antioxidant activity.
d
BHT equivalent.
e
Ferrous sulphate equivalent; values are expressed as means  SD.
 L.L.O. Pineli et al. / Journal of Food Composition and Analysis 24 (2011) 11–16 15

Table 3 increasing along ripening, and the highest value of both pigments
Total and individuals anthocyanins (pelargonidin-3-glucoside, P-3g and cyanindin-
was found in full ripe ‘Camino Real’ fruit. Besides P-3g and Cy-3g,
3-glucoside, Cy-3g) of ‘Osogrande’ and ‘Camino Real’ strawberries at different
ripeness stages. their aglycones were also investigated, but it was found only traces
of cyanidin in some samples, and no pelargonidin was detected.
Treatment Total P-3gb Cy-3gb
Once we consider total anthocyanins as the sum of individual
anthocyanins (mg kg1 FW) (mg kg1 FW)
(mg kg1 FW)a
anthocyanins evaluated by HPLC, it can be noticed higher values of
total anthocyanins by spectrophotometry, specially at pink stage,
Green Osogrande 23.5  3.5c 8.51  0.38d 2.09  0.45c
for both cultivars. Only ripe ‘Camino Real’ showed higher values of
Pink Osogrande 215.5  5.6b 70.32  1.59c 3.49  0.56bc
Ripe Osogrande 226.4  6.6b 189.36  10.28b 3.48  0.20bc total anthocyanins by HPLC. Is was observed that the ratio P-3g/
Green Camino Real 17.7  0.4c 9.94  2.17d 2.12  0.00c total anthocyanins (P-3g + Cy-3g) changed from 80.3% for green
Pink Camino Real 228.0  28.6b 121.45  11.17c 5.03  0.84b ‘Osogrande’ strawberries to 98.2% at full ripeness. For ‘Camino Real’
Ripe Camino Real 292.9  32.3a 323.11  70.62a 17.23  3.69a
the same ratio increased from 82.4% to 94.9%, which means a
a
Pelargonidin-3-glycoside equivalent by spectrophotometry, bdetermined by HPLC. higher proportion of Cy-3g in ripe ‘Camino Real’. This can
Values are expressed as means  SD. Means in the same column with common letters contribute for redish in ‘Camino Real’ cultivar, given that cyanidins
are not significantly different (p < 0.05), according to LSD test.
are redish whereas pelargonidin are a red-orange pigment.

When TAA was measured using the DPPH method, no significant 3.5. Multivariate analysis
differences within ‘Osogrande’ strawberries at different ripeness
stages were found. On the other hand, ‘Camino Real’ strawberries had Principal component analysis (PCA) searches a linear combina-
the highest TAA at pink stage, as well as vitamin C content (Table 2). tion of the studied variables in order to maximize the total variance
Our results of DPPH are similar to those found by Pinto et al. (2008). explained. If the variables are highly correlated, they will be
When TAA was assayed using the FRAP method, ‘Osogrande’ combined into a component that will explain the highest quantity
fruits showed significantly higher amounts of antioxidants than of variance in the sample (observations). The second component
‘Camino Real’ berries at green stage. The lowest values of FRAP were will explain the second highest quantity of variance and will not be
found for ‘Camino Real’ at both green and ripe stages. Halvorsen et al. correlated to the first component (Favero et al., 2009).
(2002) investigated TAA in wild type and commercially grown Based on the theoretical arguments of the PCA described by
strawberries using FRAP assay. Fragaria vesca showed the highest Hair et al. (2005), the significant factor loading values higher than
values for TAA, varying from 66.7 to 70.1 mmol ferrous sulpha- or equal to 0.7 were used to identify the most important variables
te g1 FW, whereas in commercial varieties TAA ranged from 18.5 to and observations in each dimension, or principal components (PC).
23.4 mmol ferrous sulphate g1 FW. The results observed in the Factor loading values are the correlation of each variable with the
present study were higher than those reported for commercial PC. They are represented as vectors (positions) in the space
varieties but lower than values found for wild strawberries. resulted by the axes of the biplot graphic (Fig. 1a). In the graphic,
variables (Fig. 1a) and observations (Fig. 1b) that are close to each
3.4. Anthocyanins other, and in the same geometric plan of the biplot, are
interrelated, and distant from variables and observations to which
Total and individual anthocyanins contents are shown in Table they are not related, or even negatively related. The greater is the
3. It was observed that values of total anthocyanins (ANT) vector (distance from the origin of the axis), the greater is the
increased approximately 9–13-fold from green to pink stage for correlation of the variable with the PC represented in that
‘Osogrande’ and ‘Camino Real’, respectively. The latter cultivar dimension (axis).
showed the highest contents at pink and fully ripe stages. ANT Differences among the studied treatments in relation to
found at ripe stage (Table 3) for both cultivars can be considered ripeness and cultivar were enphasized by PCA, which contributed
low if compared to the results of total anthocyanins in strawberries to more detailed data and information in many measured variables
grown in São Paulo state (Brazil), reported by Cordenunsi et al. as well as into a smaller set of components. Two main PCs
(2002, 2003). Once again, differences can be associated with explained approximately 69.4% of total data variability, called PC1
environmental conditions as well as other growing practices. P-3g (39.7%) and PC2 (29.7%) (Fig. 1). Total and individual anthocyanins
and Cy-3g contents, determined by HPLC, followed ANT trend of positively associated to PC1, and ripe ‘Camino Real’ strawberries
[()TD$FIG]

Fig. 1. Principal Component Analysis and Clusters (1, 2, 3 and 4) in ‘Osogrande’ and ‘Camino Real’ at different ripeness stages. Observations (a) are Oso = ‘Osogrande’
strawberries; Cam = ‘Camino Real’ strawberries. Variables (b) are: ANT = anthocyanins; Cy-3g = cyanidin-3-glucoside; DPPH = DPPH assay; FRAP = FRAP assay; P-
3g = pelargonidin-3-gluoside; SAR = sugar/acid ratio; TEA = total ellagic acid; TPC = total phenolic compounds; VTC = vitamin C.
16 L.L.O. Pineli et al. / Journal of Food Composition and Analysis 24 (2011) 11–16

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