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Eur Food Res Technol (2009) 228:623–631
DOI 10.1007/s00217-008-0971-2
123
ORIGINAL PAPER
Comparative study of phenolic content and antioxidant activity
of strawberry puree, clear, and cloudy juices
Jan Oszmiajski · Aneta Wojdyio
Received: 8 October 2007 / Revised: 28 September 2008 / Accepted: 30 September 2008 / Published online: 28 October 2008
© Springer-Verlag 2008
Abstract The aim of this study was to determine the con-
centrations of phenolic compounds and their antioxidant
activity in diVerent kinds of juice: clear, cloudy, and puree
which were made from three diVerent strawberry cultivars
(Elkat, Kent, and Senga Sengana). The anthocyanins, p-
coumaric acid, ellagic acid, quercetin, keampferol deriva-
tives, (+)-catechin, proanthocyanidins content and degree
of proanthocyanidin polymerization, were determined both
in the fresh and after 6 months of storage at 4 and 30 °C.
Freshly produced juices contained higher amounts of phen-
olics, especially of anthocyanins and proanthocyanidins,
than those stored for 6 months at 4 and 30 °C. The process-
ing of the clear juice showed the higher loss of all phenolic
compounds. The antioxidant capacity was the smallest for
clear, and the highest for the puree juices. This was
assessed by measurements made with diVerent antioxidant
activity assays: ABTS and FRAP. The puree of strawberry
juice had signiWcantly higher levels of the phenolic com-
pounds and showed more antioxidant activity than the clear
and cloudy juices, before and after storage in all strawberry
cultivars.
Keywords Fragaria x ananassa Duch · Proanthocyanidin ·
Phloroglucinolysis · Antioxidant activity · Storage time
Introduction
Strawberries (Fragaria x ananassa Duch.) are an important
crop in certain temperate area such as, Central Europe.
They are widely consumed, both fresh and in processed
forms such as juices, which may further be stored. These
attractive fruits are favored for their excellent taste, and can
be considered a very rich source of bioactive phenolic com-
pounds including: hydroxycinnamic acids, ellagic acid,
ellagitannins, Xavan-3-ols, Xavonols, and anthocyanins [1].
Compared with other fruits, strawberries possess high anti-
oxidant activity [2]. Guo et al. [3] found that strawberries
had 1.3 times the antioxidant activity of oranges, twice that
of red grapes, Wve times that of apples and bananas, and
thirteen times that of honeydew. Antioxidant activity of
strawberry phenolics could participate in the prevention of
cancer, cardiovascular and other chronic diseases [4].
Recently, the antioxidant activity of strawberry extracts,
independent of in vitro antiproliferative activity, has been
shown with the use of the HepG2 human liver cancer cells
[5]. Reduction in antioxidant activity during processing and
storage [6] may reduce the beneWcial eVects of such food
products on health. Injury of raw material tissue and expo-
sure to oxygen, enzymes, light, and heat may reduce the
antioxidant compound content as well. Strawberry pheno-
lics such as pelargonidin, ellagic acid, p-coumaric acid,
quercetin, and keampferol derivatives are very instable and
undergo destruction during fruit transformation, especially
during the juice and nectar production process.
Previous studies have focused on the free phenolic con-
tent and antioxidant activity of strawberry products [7, 8].
However, antioxidants can exist in both free and bound
forms. Free anthocyanins, hydroxycinnamic acids, (+)-cate-
chin, and Xavonol glycoside do not bind to strawberry cell
walls while proanthocyanidins polymers bound selectively
J. Oszmiajski (&) · A. Wojdyio
Department of Fruit and Vegetable Processing,
Wroclaw Environmental and Life Science University,
25 Norwida Street, 50-375 Wrociaw, Poland
e-mail: oszm@wnoz.up.wroc.pl
A. Wojdyio
e-mail: anetajb@op.pl
624 Eur Food Res Technol (2009) 228:623–631
123
to polysaccharides and proteins before an example of this is
in apples [9].
Proanthocyanidins are also found in strawberry fruits as
procyanidin and propelargonidin derivatives, (+)-catechin
and (¡)-epicatechin contribute 93.8% of constituent units,
which is in accordance with the predominance of the procy-
anidins in strawberry [10].
Herbert et al. [11] suggested that proanthocyanidin con-
tent in strawberries can be used as an indicator of gray mold
resistance, and can be used to screen strawberry selections
and cultivars in order to improve their shelf-life and qual-
ity. The proanthocyanidins are receiving increasing atten-
tion owing to their much more potent antioxidant properties
than those of simple monomeric phenolics, which may cor-
respond to a health-protective action [12]. Little is known
about the structural features that aVect the bioavailability
and metabolism of proanthocyanidins within the body.
Sano et al. [13] showed that some oligomeric proanthocy-
anidins are absorbed, and bioavailable in the human body,
can be detected in human plasma as early as 2 h after inges-
tion of grape seed extract. Trimers have been shown to be
absorbed through the human intestinal cell line Caco-2
[14]. For this reason, identiWcation of heterogeneous pro-
anthocyanidins, especially the low oligomers, are empha-
sized. Incubating proanthocyanidins in vitro with human
colonic microXora can result in complete degradation to
hydroxylated derivatives of phenylacetic, phenylpropionic
and phenylvaleric acids [15] compounds that had previ-
ously been shown to arise from degradation of Xavonoid
monomers.
Quantitative data on proanthocyanidins found in the lit-
erature are often underestimated; the extraction from the
crude materials is not quantitatively accurate because of the
ability of proanthocyanidins to bind to cell wall polysac-
charides [16]. These complexes involve the formation of H-
bonds and hydrophobic interactions, the latter being
favored by the existence of hydrophobic cavities and cre-
vasses. AYnity is strongly inXuenced by the molecular
weight of polyphenols, and chemical composition of poly-
saccharides [9].
The phloroglucinolysis reaction can be applied directly
to crude strawberry materials to give pertinent information
on the structure and concentration of proanthocyanidins.
This method has been chosen here for the determination of
procyanidins in puree, clear and cloudy strawberry juices.
The aim of this study was to determine the types and
amounts of phenolic compounds in diVerent strawberry
juices and to correlate these data with antiradical activity.
Our objective was to monitor changes in phenolic content,
antioxidant activity during the processing and storage and
storage at diVerent temperatures of the juices obtained from
three diVerent strawberry cultivars (Elkat, Kent, and Senga
Sengana which are commonly used in the Polish juice
industry). The eVect of clariWcation and solid particles con-
tent in strawberry juices on phenolic content and antioxi-
dant activity after processing and storage was determined.
Materials and methods
Chemicals
Trolox (6-hydroxy-2,5,7,8-tetramethylchroman-2-carbox-
ylic acid), 2,2⬘azinobis-(3-ethylbenzthiazoline-6-sulphonic
acid (ABTS); potassium persulfate, (¡)-epicatechin, (+)-
catechin, acetic acid, benzyl mercaptan (toluene -thiol),
and methanol were purchased from Sigma-Aldrich (Stein-
heim, Germany). Chlorogenic acid, elagic acid, p-coumaric
acid, quercetin, kaempferol, cyaniding-3-glucoside, pelarg-
onidin-3-glucoside, pelargonidin-3-rutinoside were pur-
chased from Extrasynthese (Lyon Nord, France).
Plant material
Samples of three strawberry (Fragaria x ananassa) culti-
vars (Elkat, Kent and Senga Sengana) were harvested (in
Momcisko near Wrociaw, Poland) at processing maturity
and the fresh fruits were processed in lab scale at Wroclaw
University on June 2006. Prior to juices preparation the
samples were washed and carefully sorted for the same
degree of maturity, size and color.
Preparation of fresh strawberry to polyphenol analysis
After harvest, the whole strawberry fruits (about 1 kg for
each variety) were cut directly in liquid nitrogen, and
freeze-dried (24 h). The homogeneous powder was
obtained by crushing the dried tissues with the use of closed
laboratory mill to avoid hydration and analyzed.
Preparation of strawberry puree, cloudy and clear juices
The whole fresh strawberry fruits (2 kg each cultivars for
each replication) were ground in a Thermomix laboratory
mill (Wuppertal, Vorwerk, Germany) for 20 s at exactly the
same rotation speed for each samples. The 2 kg mush was
mixed with 2 kg distilled water, homogenized to obtain and
homogeneous pulp and divided in two parts. First part
(1,200 g) was heated in a microwave oven (Amica Wronki,
Poland) of 700 W for 5 min and the product temperature
after treatment was of 90 °C. The hot Wlling was poured into
0.2 L jars with meniscus and cooled to 20 °C. This sample
was called as puree juice. The second part was immediately
pressed to yield 75% (weight) juice in a Zodiak laboratory
hydraulic press with pressed cloth (SRSE, Warsaw, Poland).
The juice was heated in a microwave oven for 5 min and the
Eur Food Res Technol (2009) 228:623–631 625
123
product temperature after treatment was of 90 °C. The juice
was divided in two lots. For the Wrst lot (cloudy strawberry
juice) was poured into 0.2-L jars in the same way as puree
juice. The second lot (clear juice), was taken after the micro-
wave heating, cooled to 45 °C, eventually treated with pec-
tinase and stirred for 0.5 h at 40 °C (0.2 mL/kg Pectinex
Color; Novo Nordisk Ferment Ltd, Dittingen, Switzerland).
Following enzymatic treatment, the juice was clariWed,
using: Gelatin (0.25 g/L) + Baykisol 30 (6.0 mL/L) + SIHA-
Active-Bentonite G (1.0 g/L) (Begerow GmbH&Co, Ger-
many). This sample was then centrifuged at 12,100£g for
10 min and the clear juice was then pasteurized in the same
way as the puree and cloudy juice. All products used for clar-
iWcation were prepared by methods proposed by the pro-
ducer. The amounts of each clariWcation product were chosen
after preliminary testing. Three replicates of strawberry puree
and juices preparation were carried out. After processing and
storage at 4, or 30 °C in the dark, for 6 months, the straw-
berry products were subjected to analyses.
HPLC analysis of polyphenols
Before analysis the strawberry products were mixed with
methanol (1:1) and centrifuged at 20,878£g. The analysis
of anthocyanins, Xavan-3-ols, phenolic acids and Xavonol
glycosides was carried out with a L-7455 liquid chromatog-
raphy with a diode array detector and an L-7100 quaternary
pump equipped with a D-7000 HSM multisolvent delivery
system (Merck-Hitachi, Tokyo, Japan). Separation was per-
formed in a Phenomenex (Torrance, CA, USA). Synergi
Fusion RP-80A column (250 mm £4.6 mm, 4 m); the
oven temperature was set at 30 °C. The mobile phase was
composed of solvent A (45 mL formic acid and 955 mL
water) and solvent B (acetonitrile). The program began
with a linear gradient from 0 to 25% B in 36 min, followed
by washing and reconditioning of the column. The Xow rate
was 1 mL/min and the runs were monitored at wavelengths
of 280 nm (Xavan-3-ols), 320 nm (hydroxycinnamates),
360 nm (Xavonol glycosides and ellagic acid), and 510 nm
(anthocyanins). Photodiode array spectra were measured
over the wavelength range 200–600 nm in steps of 2 nm.
Retention times and spectra were compared with those of
pure standards within that range.
The amounts of the diVerent phenolics in the samples
were determined by HPLC. Calibration curves were con-
structed with (+)-catechin, p-coumaric acid, ellagic acid,
isoquercitin and pelargonidin-3-O-glucoside as standards
(Extrasynthese, France).
Analysis of proanthocyanidins by phloroglucinolysis
Direct phloroglucinolysis of freeze-dried strawberry juices
was performed as described by Kennedy et al. [17].
Portions (0.5 mL) of juices were precisely measured into
2.2 mL Eppendorf vials and freeze-dried, then 0.8 mL of
the methanolic solution of phloroglucinol (75 g/L) and
ascorbic acid (15 g/L) were added. After addition of 0.4 mL
of methanolic HCl (0.3 M), the vials were closed and incu-
bated for 30 min at 50 °C with vortexing every 10 min. The
reaction was stopped by placing the vials in an ice bath,
with drawing of 0.5 mL of the reaction medium and dilut-
ing with 0.5 mL of sodium acetate buVer 0.2 M. Next the
vials were cooled in ice water and centrifuged immediately
at 20,000£g for 10 min at 4 °C. Samples were stored at
4 °C before reverse phase HPLC (RP-HPLC) analysis. All
incubations were done in triplicate. Phloroglucinolysis
products were separated in a Atlantis T3, 5-m, 100A col-
umn (250 mm £4.6 mm; Waters). The liquid chromatog-
raphy was a Waters (Milford, MA, USA) system equipped
with diode array and scanning Xuorescence detectors. Sol-
vent A (25 mL aqueous acetic acid and 975 mL water) and
solvent B (acetonitrile) were used in the following gradient:
initial, 5% B; 0–15 min, 10% B linear; 15–25 min, 60% B
linear; followed by washing and reconditioning of the col-
umn. A Xow rate of 1 mL/min and an oven temperature of
15 °C were observed with the injection of the Wltrate
(20 L) on the HPLC system.
Compounds, for which reference standards were avail-
able, were identiWed on chromatograms according to their
retention times and UV–visible spectra. The Xuorescence
detection was recorded at excitation wavelength 278 nm
and emission wavelength 360 nm. The calibration curves
which were based on peak area were established using
(+)-catechin, (¡)-epicatechin, (+)-catechin and (¡)-epicate-
chin–phloroglucinol adducts standards. The average degree
of polymerization was measured by calculating the molar
ratio of all the Xavan-3-ol units (phloroglucinol
adducts + terminal units) to (¡)-epicatechin and (+)-cate-
chin which correspond to terminal units. QuantiWcation
(mg/L of nectars) of the (+)-catechin, (¡)-epicatechin, (+)
catechin and (¡)-epicatechin–phloroglucinol adducts was
achieved by using the calibration curves of the correspond-
ing catechin and procyanidin standards (Extrasynthese
France) (Fig. 1).
Extraction of polyphenol compounds for antioxidant
activity analysis
About 10 g of each puree, clear and cloudy juices were
weighed into a test tube for antioxidant property analysis. A
total of 25 mL of 80% aqueous solution of methanol with
1% HCl was added, and the suspension was stirred slightly.
Tubes were sonicated twice for 15 min and left at 4 °C.
After 24 h the extract was centrifuged for 10 min (10 min,
1,500£g), and supernatants were collected at 4 °C until use
(within 24 h).
626 Eur Food Res Technol (2009) 228:623–631
123
ABTS·+ radical scavenging spectrophotometric assay
The free-radical scavenging activity was determined by
ABTS radical cation decolorization assay according to the
method of Re et al. [18]. The ABTS was dissolved in water
to a 7 mM concentration. The ABTS radical cation
(ABTS·+) was produced by reacting ABTS stock solution
with 2.45 mM potassium persulfate (Wnal concentration).
Next and the mixture was left to stand in the dark at room
temperature (20 §2 °C) for 12–16 h before the use. The
radical was stable in this form for more than 2 days when
stored in dark at room temperature. Before analysis the
ABTS·+ solution was diluted with bidistilled water to an
absorbance of 0.700 (§0.02) at 734 nm. Aliquots of 30 L
of sample extract were added supernatant to 3.0 mL of
diluted ABTS·+ solution [A734 nm = 0.700 (§0.02)] and
the absorbance was read exactly 6 min after initial mixing.
All determinations were performed in triplicate. Standard
curve was prepared using diVerent concentrations of
Trolox. The results of the assay were expressed as Trolox
equivalent antioxidant capacity (TEAC).
Ferric reducing/antioxidant power assay
The total antioxidant power of extracts was determined
using the ferric reducing ability of plasma (FRAP) assay by
Benzie et al. [19]. BrieXy, the FRAP reagent was prepared
by mixing acetate buVer (300 M, pH 3.6), a solution of
10 M TPTZ in 40 M HCl, and 20 M FeCl3 at 10:1:1 (v/
v/v). The FRAP reagent (300 L) and sample extracts
(10 L) were added to each well and mixed thoroughly.
The absorbance was taken at 593 nm after 10 min. Standard
curve was prepared using diVerent concentrations of
Trolox. All solutions were used on the day of preparation.
All determinations were performed in triplicates. The
results were corrected for dilution and expressed in M
Trolox/100 g dry weight (dw).
Statistical analyses for individual phenolics in strawberry
products
Statistical analysis was conducted using Statistica version
6.0 (StatSoft Poland). SigniWcant diVerences (P·0.05)
between average responses were evaluated by using one-
way ANOVA with Duncan test. Principal component anal-
ysis (PCA) was performed using XLSTAT (Addinisoft,
France) was performed on mean values of 27 samples and 5
variables.
Results and discussion
The puree, clear and cloudy juice of strawberry were
obtained from three cultivars, Elkat, Kent, and Senga Seng-
ana. The results of the qualitative and quantitative phenolic
composition of the strawberry juices, as determined by the
HPLC method, are presented in Tables 1, 2, and 3. The
concentrations of anthocyanins, p-coumaric acid, ellagic
acid, quercetin, keampferol derivatives, (+)-catechin, and
proanthocyanidins as well as degree of polymerization of
proanthocyanidins were determined in raw material, fresh
products and after 6 months of storage at 4 and 30 °C.
The average phenolic contents of the three strawberry
cultivars, Elkat, Kent and Senga Sengana used as raw mate-
rial are shown in Table 1. The total phenolic content (con-
sidering the sum of all of the individual phenolics) ranged
from 243.3 mg/kg fresh fruits (Senga Sengana) to
290.4 mg/kg fresh fruits (Elkat, typically Polish cultivar).
Proanthocyanidins were the major polyphenols in all the
samples under study. Regarding proanthocyanidins, the
highest concentrations were found in Kent (235.5 mg/kg
fresh fruits) and the smallest in Senga Sengana (159.2 mg/
kg fresh fruits) cultivars. Ellagic acid is a hydrolytic prod-
uct of ellagitannins. The content of total ellagic acid
(ellagic acid and ellagic acid glucoside) ranged from 1.2–
1.3 mg/kg fresh fruits (Senga Sengana and Kent) to 3.5 mg/
kg fresh fruits (Elkat). The concentration of ellagic acid and
ellagic acid glucoside was the present at same as previously
reported in strawberry fruits [1, 7]. The concentration of
Xavonols such as kaempferol and quercetin ranged from 5.1
to 9.0 mg/kg. Lugasi and Hovari [20] reported that querce-
tin was present at a concentration of 10.0–53.0 mg/kg. Var-
iation in content of diVerent phenolic in strawberries may
be due to cultivar between maturity, size, present of achenes,
extraction solvent and procedure. Pelargonidin-3-O-glucoside
was predominant in all cultivars and ranged from 29.9 mg/kg
Fig. 1 Comparison of chromatograms (HPLC-FD) strawberry puree
and clear juice (dotted line) after phloroglucinolysis for fresh Elkat cul-
tivar. 1 Phloroglucinol–(+)-catechin; 2 phloroglucinol–(¡)-epicate-
chin; 3 (+)catechin; 4 (¡)epicatechin; n nonidentiWed
Eur Food Res Technol (2009) 228:623–631 627
123
for Kent strawberries to 68.2 mg/kg for Elkat. SigniWcant
diVerences in the concentration of p-coumaric acid among
diVerent cultivars (1.3 mg/kg fresh fruits for Kent and
8.1 mg/kg fresh fruits for Elkat) were found. The concen-
tration of p-coumaric acid found here in Senga Sengana
was twice that reported by Häkkinen and Törrönen [21].
Table 1 Concentration (mg/kg
of fresh weight) of the phenolic
compounds in raw material used
for strawberry products: clear,
cloudy and purees juices
Phenolic compound Senga Sengana Kent Elkat
Cyanidin-3-O-glucoside 2.9 §0.3f 2.0 §0.4e 3.4 §0.7f
Pelargonidin-3-O-glucoside 53.9 §3.2b 29.9 §1.2b 68.2 §3.8b
Pelargonidin-3-O-rutinoside 3.9 §0.5e 1.9 §0.7e 0.0 §0.0 g
Pelargonidin-3-O-malonyl-glucoside 6.9 §1.3c 4.7 §0.3d 12.4 §0.3c
p-Coumaric acid 6.9 §0.9c 1.3 §0.4f 8.1 §1.9d
Ellagic acid 1.2 §0.3 g 1.3 §0.6f 3.5 §0.8f
Flavonolsa3.5 §0.8e 9.0 §1.8c 5.1 §0.5e
(+)-Catechin 4.9 §1.0d 5.0 §0.4d 5.8 §1.1e
Proanthocyanidins 159.2 §4.7a 235.5 §5.8a 183.9 §3.8a
Total 243.3 290.6 290.4
aFlavonols (sum of kaempferol
and quercetin)
Table 2 Mean contents of phenolic compounds (in mg/L §SD) in clear, cloudy and puree strawberry juices during 6 months of storage in diVer-
ent conditions (4 and 30 °C)
C Clear juice, T cloudy juice, P puree; M months; L 4°C; H 30 °C
aValues are mean §standard deviation, n= 3; in each column, mean values with diVerent letters are signiWcantly diVerent at P<0.05
Cultivars Time and
conditions
of storage
p-Coumaric
acid
Ellagic acid Quercetin Kaempferol (+)-Catechin Proanthocyanidins Degree of
polymerization of
proanthocyanidins
Senga
Sengana
C-0M 29.4 §0.2da3.3 §0.4i 1.9 §0.3jk 1.8 §0.5b 20.3 §0.4efg 157.3 §2.3s 2.60r
T-0M 34.9 §0.4ab 2.4 §0.0ijk 1.5 §0.0jk 6.3 §0.2a 23.4 §1.6d 188.4 §7.4n 3.67m
P-0M 35.9 §1.1a 15.8 §1.3ef 3.1 §0.7ij 6.7 §0.5a 25.6 §2.4c 452.7 §3.4c 4.12k
C-6M-L 30.1 §1.1d 2.2 §0.3ijk 0.7 §0.1k 1.3 §0.1b 18.7 §1.8gh 93.9 §4.3x 4.97i
T-6M-L 34.1 §1.0b 2.6 §0.2ijk 1.4 §0.2jk 1.3 §0.3b 21.6 §1.2e 172.8 §6.5o 7.43b
P-6M-L 32.4 §2.3c 28.9 §1.9b 1.4 §0.3jk 2.3 §0.6b 20.5 §2.3ef 412.6 §1.8f 9.22a
C-6M-H 19.4 §3.2h 1.0 §0.1k 0.5 §0.0k 1.3 §0.7b 7.9 §1.2op 69.1 §1.1z 2.34s
T-6M-H 22.0 §0.1g 1.3 §0.0jk 0.7 §0.0k 0.9 §0.1b 17.3 §2.3hi 136.1 §1.9w 5.61g
P-6M-H 19.5 §0.0h 22.5 §1.4c 1.0 §0.0k 2.7 §0.8b 18.8 §4.5fgh 276.2 §0.5r 6.85d
Elkat C-0M 24.2 §0.2ef 6.9 §0.5g 3.0 §0.2ij 0.8 §0.0b 14.0 §2.3jk 264.2 §2.9k 2.60r
T-0M 25.7 §0.9e 2.3 §1.0ijk 8.2 §1.0de 0.8 §0.0b 13.4 §1.2kl 316.5 §3.4i 3.67m
P-0M 28.9 §1.9d 16.3 §2.2e 12.3 §1.7a 1.5 §0.1b 15.8 §0.8ij 592.5 §2.9a 4.12k
C-6M-L 22.7 §1.2fg 6.9 §0.7g 1.2 §0.3k 1.5 §0.4b 13.4 §1.4kl 167.9 §4.5p 2.73pr
T-6M-L 23.0 §1.3fg 2.2 §0.3ijk 7.1 §0.6ef 1.3 §0.2b 14.8 §1.6jk 224.3 §7.8l 3.58m
P-6M-L 25.2 §2.3e 32.5 §2.1a 11.6 §2.3ab 1.8 §0.6b 15.8 §2.3ij 498.6 §0.9b 5.15h
C-6M-H 16.3 §1.9j 3.9 §0.5hi 0.9 §1.1k 1.7 §0.5b 4.1 §0.5r 136.5 §2.3w 2.07f
T-6M-H 18.0 §2.9hi 2.0 §0.3ijk 2.2 §0.2jk 1.8 §0.3b 6.6 §0.7p 167.1 §1.2p 3.03o
P-6M-H 7.7 §0.0ij 23.3 §0.2c 0.8 §0.0k 2.2 §0.5b 7.7 §0.9p 404.3 §1.9g 4.33j
KENT C-0M 2.9 §1.8l 5.9 §1.1g 1.1 §0.3k 1.0 §0.6b 28.4 §1.5b 172.4 §9.2o 3.24n
T-0M 3.0 §1.1kl 3.4 §1.8i 6.4 §0.2fg 1.5 §0.2b 26.0 §1.2c 197.7 §6.7n 3.94l
P-0M 3.8 §0.4k 14.5 §1.4f 10.1 §0.1bc 2.2 §0.0b 39.4 §2.3a 449.7 §10.1d 7.12c
C-6M-L 2.3 §1.0kl 5.6 §0.5gh 1.1 §0.0k 1.0 §0.2b 9.5 §2.8no 160.7 §1.7r 2.82p
T-6M-L 3.1 §1.9kl 2.9 §0.1ij 5.6 §1.1fgh 1.4 §0.9b 11.8 §1.8lm 182.7 §7.9n 3.70m
P-6M-L 3.2 §1.8kl 28.7 §5.6b 9.4 §1.6cd 2.0 §0.6b 20.0 §2.8efg 433.4 §1.8e 6.52e
C-6M-H 1.8 §1.0l 3.1 §2.1ij 0.5 §0.1k 1.1 §0.2b 6.7 §2.9op 80.9 §1.5g 1.95t
T-6M-H 2.4 §1.0kl 5.2 §2.3gh 4.0 §0.2hi 1.2 §0.0b 10.2 §1.9mn 147.7 §0.3t 3.33n
P-6M-H 2.2 §0.7kl 20.2 §0.1d 5.2 §0.6gh 2.0 §0.7b 8.0 §0.1op 367.0 §1.1 h 5.93f
628 Eur Food Res Technol (2009) 228:623–631
123
Out of the three kinds of strawberry juices, the pheno-
lic compounds that showed the biggest diVerence were
proanthocyanidins (Tables 2, 3). The amount of these
compounds diVered signiWcantly between clear and puree
juices (i.e. cloudy juice still containing a suspension of
cell-wall fragments and cumulative), with a two- to three-
fold variation of their concentration. The fresh clear juice
from Senga Sengana, Elkat, and Kent cultivars contained
157.3, 264.2, and 172.4 mg/L of proanthocyanidins,
respectively. While the puree juice from Senga Sengana,
Elkat, and Kent cultivars contained 452.7, 592.5, and
449.7 mg/L, respectively. The degree of polymerization
(DP) was higher for puree (DP 4.1–7.1) than for clear
juice (DP 2.6–3.2). Strawberry polymeric Xavan-3-ols are
composed of (+)-catechin and (¡)-epicatechin which are
constitutive units of procyanidins. The chain extension
units and chain terminating units in procyanidins were
(+)-catechin and (¡)-epicatechin (Fig. 2). Polymeric pro-
anthocyanidins are the major class of polyphenolic com-
pounds in strawberry puree juice. The concentration of
proanthocyanidins was present at much higher levels as
previously reported for strawberry [22]. This diVerence is
due to the fact that our results take into account the poly-
mer and oligomer proanthocyanidins analysed by
phloroglucinolysis method. Previous studies had reported
only oligomer proanthocyanidin content analysed directly
by HPLC method [23, 24]. Puree juice also contained the
highest amount of free ellagic acid. The average concen-
tration ranged from 14.5 mg/L in Kent puree juice up to
16.3 mg/L in Elkat puree juice. After 6 months of storage
Table 3 Mean contents of anthocyanins (in mg/L §SD) in clear, cloudy and puree strawberry juices during 6 months of storage in diVerent con-
ditions (4 °C and 30 °C)
n= 3; in each column, mean values with diVerent letters are signiWcantly diVerent at P<0.05
C Clear juice, T cloudy juice, P puree; M months; L 4°C; H 30 °C
aValues are mean §standard deviation
Cultivars Time and
conditions
of storage
Cyanidin-
3-glucoside
Pelargonidin-
3-glucoside
Pelargonidin-
3-rutinoside
Pelargonidin-
3-malonylglucoside
Total
anthocyanins
Senga
Sengana
C-0M 9.5 §0.3bca218.7 §4.3d 9.7 §0.0a 14.9 §0.2g 252.8c
T-0M 9.7 §0.0bc 228.0 §1.9c 10.3 §1.4a 24.9 §1.7d 272.9c
P-0M 11.8 §1.8a 248.6 §1.8a 10.6 §2.9a 27.7 §1.7c 298.7a
C-6M-L 0.5 §0.1r 9.5 §1.9x 1.2 §0.1g 0.3 §0.1l 133.3k
T-6M-L 0.5 §0.0r 22.2 §4.3s 1.5 §1.0fg 0.5 §0.2kl 165.2h
P-6M-L 1.1 §0.1ij 29.7 §1.1r 1.4 §0.0g 0.7 §0.0kl 184.5g
C-6M-H 4.2 §0.1fg 118.7 §1.9k 5.5 §1.8c 5.0 §0.1j 11.5t
T-6M-H 6.7 §0.6d 141.5 §1.6h 6.8 §0.3b 10.1 §1.1h 24.6s
P-6M-H 8.9 §0.5bc 154.5 §2.9g 7.0 §0.4b 14.2 §0.3g 32.9r
Elkat C-0M 8.8 §1.1bc 207.6 §3.2f 0.0 §0.0h 22.4 §0.4e 238.7f
T-0M 8.8 §1.8bc 216.3 §2.3e 0.0 §0.0h 33.5 §0.6b 258.6d
P-0M 0.4 §0.4ab 237.2 §5.6b 0.0 §0.0h 37.4 §1.1a 285.0b
C-6M-L 4.1 §0.4fg 117.8 §1.7k 0.0 §0.0h 10.5 §1.0h 132.4k
T-6M-L 5.8 §0.3def 129.1 §1.9j 0.0 §0.0h 13.5 §1.8g 148.4j
P-6M-L 8.3 §0.2c 135.7 §1.4i 0.0 §0.0h 17.4 §1.5f 161.4i
C-6M-H 1.0 §0.1ij 6.0 §0.3y 0.0 §0.0h 2.5 §0.4k 9.4y
T-6M-H 0.7 §0.1ij 11.5 §0.1w 0.0 §0.0h 2.3 §0.4kl 14.6w
P-6M-H 1.1 §0.1ij 14.1 §1.6t 0.0 §0.0h 1.1 §0.1kl 16.3t
Kent C-0M 4.9 §0.4efg 102.1 §1.5c 3.8 §2.7de 7.4 §0.3i 118.2m
T-0M 4.6 §0.2efg 99.2 §3.8m 3.9 §0.2de 14.2 §2.0g 121.9l
P-0M 6.0 §1.1de 119.1 §8.1k 4.3 §0.7cd 17.7 §2.7f 147.1j
C-6M-L 3.9 §0.5gh 56.4 §3.6p 2.9 §0.3de 4.1 §1.1j 67.2p
T-6M-L 2.4 §0.7hi 69.9 §2.9o 2.7 §0.7ef 6.7 §1.7i 81.7o
P-6M-L 4.8 §0.4efg 83.2 §5.2n 2.9 §0.2de 11.4 §0.3h 102.3n
C-6M-H 0.0 §0.0j 1.1 §0.5z 0.1 §0.0h 0.2 §0.0l 1.5z
T-6M-H 0.1 §0.0j 6.1 §0.6y 0.1 §0.0h 1.5 §0.3kl 7.8y
P-6M-H 0.2 §0.1j 7.2 §0.2y 0.3 §0.0h 1.1 §0.1kl 8.8y
Eur Food Res Technol (2009) 228:623–631 629
123
at 4 °C, ellagic acid in Kent and Elkat puree juices
increased signiWcantly to 2.87 and 32.5 mg/L, respec-
tively.
Proanthocyanidins and ellagitannins are present at
higher concentrations in the puree juice (Tables 1, 2) than
in fresh fruits. Ellagic acid occurs in particularly high con-
centration in strawberry achenes [23] and it is liberated as a
hydrolytic product of ellagitannins in puree juices during
processing and storage.
The processing of puree, and clear and cloudy juices,
had a less eVect on (+)-catechin, anthocyanin, p-coumaric
acid, quercetin, and keampferol derivative content (Table 2,
3). The diVerences due to heterogeneity of raw material
were limited by preparing the strawberry products from the
same homogenate strawberry pulps. Qualitative diVerences
were found in anthocyanin composition of the three culti-
vars. Four anthocyanins were identiWed in Senga Sengana
and Kent strawberry cultivars: cyanidin-3-O-glucoside,
pelargonidin-3-O-glucoside, pelargonidin-3-O-rutinoside,
and pelargonidin-3-O-malonylglucoside. Elkat cultivar
(Polish origin) had only three anthocyanins which did not
include the pelargonidin-3-O-rutinoside. The content of
anthocyanidins in strawberries and, then, in juices
depended on a series of factors, such as the stage of matu-
rity, cultivar, storage conditions. Pelargonidin-3-O-gluco-
side was predominant in all strawberry products and ranged
from 248.6 mg/L for Senga Sengana purée juice to
99.2 mg/L for Kent cloudy juice. The process had less
inXuence on the anthocyanidins concentrations than the
storage did (Tables 1, 3). All samples exhibited a dramatic
anthocyanidins loss when stored at the highest temperature
(30 °C) and much slower at 4 °C (Table 3). All strawberry
products stored for 6 months at 30 and at 4 °C had about
one-tenth and half of the initial concentrations, respec-
tively. The results were consistent with general Wndings
that monomeric pigment concentrations decrease during
storage [24, 25] and the stability of anthocyanins was mark-
edly inXuenced by temperature [26, 27].
Some protective eVects on anthocyanidins degradation
were observed in puree and cloudy juice in comparison to
clear juice. The presence of pectins in cloudy has protected
strawberry juices against anthocyanin degradations. Other
factors could be connected with co-pigmentation by high
concentrations of copigments, such as proanthocyanidins,
Fig. 2 Principal component analysis of strawberry juice (c clear;
t
cloudy; p puree) prepared from three cultivars (S Senga Sengana; E El-
kat; K Kent) before storage time (0) and after stored 6 months at 4 (4)
and 30 °C (30). The measured variables (phenolics: AN anthocyanins;
PR proanthocyanins; EAellagic acid; antioxidant capacity: ABTS and
FRAP) are shown on the same plot
Sc0
St0 Sp0
Sc4
St4 Sp4
Sc30 St30
Sp30
Ec0
Et0 Ep0
Ec4 Et4
Ep4
Ec30 Et3 0
Ep30
Kc0 Kt0
Kp0
Kc4 Kt4
Kp4
Kc30 Kt30
Kp30
AN
PR
EA
ABTS
FRAP
-2
-1
0
1
2
3
4
5
-3 -2 -1 0 1 2 3 4 5
PC 1 (73.5 %)
PC 2 (17.9 %)
Table 4 Antioxidant activities of strawberry juices as determined by
the ABTS and FRAP assays (M Trolox/100 mL) before and after
6 months of storage in diVerent conditions (4 and 30 °C)
n= 3; in each column, mean values with diVerent letters are signiW-
cantly diVerent at P<0.05
C Clear juice, T cloudy juice, P puree juice; M months; L 4°C; H 30 °C
aValues are mean §standard deviation
Variety Time and
conditions
of storage
ABTS FRAP
Senga
Sengana
C-0M 80.83 §1.34k 335.39 §1.43r
T-0M 73.06 §3.56m 380.11 §2.13n
P-0M 200.65 §2.00c 1021.26 §1.24b
C-6M-L 73.58 §1.43m 302.77 §3.56s
T-6M-L 69.43 §1.45n 364.87 §2.87o
P-6M-L 187.86 §1.11e 893.55 §2.87e
C-6M-H 53.89 §2.67r 261.72 §2.56x
T-6M-H 55.96 §1.65s 306.63 §3.01s
P-6M-H 107.36 §2.03g 630.06 §1.18g
Elkat C-0M 88.60 §2.87j 470.82 §2.45i
T-0M 101.04 §1.23h 514.67 §1.34
P-0M 248.54 §2.90a 1155.86 §4.67a
C-6M-L 78.76 §1.11l 295.92 §2.01t
T-6M-L 96.89 §3.02i 410.30 §2.56l
P-6M-L 195.83 §1.34d 712.84 §3.05f
C-6M-H 59.84 §1.09p 415.56 §1.17k
T-6M-H 69.69 §2.78n 510.69 §1.45h
P-6M-H 149.54 §2.45f 928.75 §1.46d
Kent C-0M 69.43 §1.45n 390.13 §1.92m
T-0M 95.85 §1.23i 511.16 §1.83h
P-0M 205.20 §2.98b 957.38 §1.89b
C-6M-L 68.91 §1.11n 356.81 §0.21o
T-6M-L 79.79 §1.67l 465.74 §3.00j
P-6M-L 193.91 §1.12d 924.06 §2.56d
C-6M-H 51.81 §0.23t 271.80 §1.35w
T-6M-H 63.21 §1.01o 369.08 §1.78o
P-6M-H 145.36 §1.59f 713.31 §1.09f
630 Eur Food Res Technol (2009) 228:623–631
123
which can be involved – stacking (co-pigmentation) and
formation of brownish covalent adducts [28, 29].
The concentrations of phenolics such as p-coumaric
acid, quercetin, keampferol derivatives, and (+)-catechin
were lower than those of anthocyanins and proanthocyani-
dins in both fresh fruits and juices from all three strawberry
cultivars (Tables 1, 2). The puree juice was richer in these
phenolics than the clear and cloudy ones. Juices prepared
from Elkat and Senga Sengana contained about ten times
more p-coumaric acid than those prepared from Kent straw-
berry, especially for puree juices.
The concentration of proanthocyanidins and other pheno-
lic compound in the cloudy and puree juice was higher than
clear juice independently of the variety. The transfer rates
varied between the type of processing and also between the
cultivars. The increased transfer of proanthocyanidins from
clear to cloudy juice then to puree was accompanied by
higher degree of polymerization, and this is in agreement
with data for apple obtained by Le Bourvellec et al. [30].
All juices were examined for their antioxidant activity.
The antioxidant activities of clear and cloudy juices and
puree juice, assessed either by a radical scavenging assays
(ABTS) or a reducing power assay (FRAP) were signiW-
cantly diVerent (Table 4). The process strongly aVected the
antioxidant activity and this was closely dependent on the
content of phenolic compounds, as was conWrmed by Fer-
nandez-Pachon et al. [31] in their study on wine-process.
SigniWcant diVerences in the antioxidant activity among
clear, cloudy, and puree juices were also found after
6 months of storage at 4 and 30 °C. Clear, cloudy and puree
produced from all cultivars stored at 4 °C had signiWcantly
higher antioxidant capacity than products stored at 30 °C.
Our results are consistent with the results obtained by
Wicklund et al. [32] for strawberry jams stored for
3 months at 4 and 20 °C. Moreover, diVerences were ascer-
tained between varieties Senga Sengana juices had the low-
est ABTS and FRAP-values. In Wicklund et al. [32]
strawberry jam preserves prepared in laboratory with the
Senga Sengana variety had smaller FRAP.
The need for the use of diVerent methods/assay to mea-
sure the antioxidant activity is due to the diVerent chemical
mechanisms beyond each assays and to the fact that antiox-
idants could acts through diVerent modes of action [33].
The use of a single method provides only an estimate of the
capacity that is dependent upon time of reaction, mode of
action, and the complexity of the reaction kinetics. Second,
the potential for interaction/polymerization of phenolic
compounds may cause the antioxidant capacity of both fruit
samples and individual compounds to be underestimated.
Using at least two diVerent antioxidant methods to compare
fruits samples provides the opportunity to identify varia-
tions in response that may otherwise be missed.
Principal component analysis was preformed for all 27
samples using the mean values of six variables: three con-
centrations (total anthocyanins and proanthocyanidins, and
ellagic acid) and the two antioxidant capacities (ABTS,
FRAP). One the Wrst PC, which explained 73.5% of the
variance loaded the proanthocyanidins content, the ellagic
acid content and the antioxidant activity. Along the Wrs PC
samples of “puree juice” were separated from samples of
clear and cloudy juices regardless of the variety and storage
conditions. Moreover, within each single variety, cloudy
juices were separated from puree juices along PC1 for a
lower polyphenols content and antioxidant activity. The
puree juices indeed contained higher polyphenol concentra-
tions and had higher antioxidant capacities. On the second
PC which explained 17.9% of total variation, loaded the
anthocyanins content. Juices stored at diVerent tempera-
tures for diVerent durations, were separated the juices
stored at 30 °C. PCA permitted to evidence a very limited
impact of the cultivars on data structure, with some separa-
tion of Senga Sengana, from Elkat along PC1Kent. There
were signiWcant correlations (Table 5) between proanthocy-
anidins concentrations and ellagic acid concentrations and
the two antiradical assays, with Pearsons coeYcients of
0.870 and 0.944 for ABTS and FRAP, respectively, as well
as between ellagic acid and antioxidant capacity 0.729 and
0.771, respectively. However, the concentrations of pro-
anthocyanidins and ellagic acids were also strongly corre-
lated in this data set, in contrast to anthocyanins (Table 5).
The relationship between the various phenol classes could
be related to their response to the process (better transfer to
the puree juice for proanthocyanidins and ellagic acid due
either to the adsorption on cell wall remnants or extraction
Table 5 Correlation coeYcients of anthocyanins, procyanidins, ellagic acid and total antioxidant activity measured by ABTS and FRAP
Values in bold are signiWcantly diVerent from 0 with a signiWcance level alpha = 0.05
Variables Anthocyanins Proanthocyanidins Ellagic acid ABTS FRAP
Anthocyanins 0.428 0.055 0.172 0.306
Proanthocyanidins 0.428 0.805 0.870 0.944
Ellagic acid 0.055 0.805 0.729 0.771
ABTS 0.172 0.870 0.729 0.910
FRAP 0.306 0.944 0.771 0.910
Eur Food Res Technol (2009) 228:623–631 631
123
from fragmented seeds over time; higher sensitivity to stor-
age for anthocyanins).
Our results showed that the puree of strawberry juice had
signiWcantly higher levels of the phenolic compounds and
showed more antioxidant activity than the clear and cloudy
juices, before and after storage in all strawberry cultivars.
Therefore puree strawberry juice may be interesting from a
nutritional and, thus, commercial and pharmaceutical, per-
spective.
Acknowledgments Author’s very grateful to Dr. Maria T. Laux and
Dr. Kaitlyn Mitchell from Department of Molecular Medicine (Warren
Hall Cornell University, Ithaca, NY, US) for languages correction and
insightful comments on this manuscript. This work was supported by
the European Commission FOOD-CT-2004-513960 FLAVO.
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