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© 2004 Hindawi Publishing Corporation
Journal of Biomedicine and Biotechnology • 2004:5 (2004) 248–252 • PII. S1110724304406123 • http://jbb.hindawi.com
RESEARCH ARTICLE
The Change of Total Anthocyanins in Blueberries and
Their Antioxidant Effect After Drying and Freezing
Virachnee Lohachoompol, George Srzednicki,
∗
and John Craske
Food Science and Technology, School of Chemical Engineering and Industrial Chemistry,
The University of New South Wales, Sydney, NSW 2052, Australia
Received 26 April 2004; revised 11 June 2004; accepted 29 June 2004
This study examined the effects of freezing, storage, and cabinet drying on the anthocyanin content and antioxidant activity of blue-
berries (Vaccinium cory mbosum L). Fresh samples were stored for two weeks at 5
◦
C while frozen samples were kept for up to three
months at
−20
◦
C. There were two drying treatments, one including osmotic pretreatment followed by cabinet drying and the other
involving only cabinet drying. Total anthocyanins found in fresh blueberries were 7.2
± 0.5 mg/g dry matter, expressed as cyanidin
3-rutinoside equivalents. In comparison with fresh s amples, total anthocyanins in untreated and pretreated dried blueberries were
significantly reduced to 4.3
± 0.1 mg/g solid content, 41% loss, and 3.7 ± 0.2 mg/g solid content, 49% loss, respectively. Osmotic
treatment followed by a thermal treatment had a greater effect on anthocyanin loss than the thermal treatment alone. In contrast,
the frozen samples did not show any significant decrease in anthocyanin level during three months of storage. Measurement of the
antioxidant activity of anthocyanin extracts from blueberries showed there was no significant difference between fresh, dried, and
frozen blueberries.
INTRODUCTION
Anthocyanins, natur al pigments which are respon-
sible for the blue, purple, violet, and red colours of
fruit, are one of the major flavonoid classes [1]. The
major sources of anthocyanins in edible plants are
families Vitaceae (grape) and Rosaceae (cherry, plum,
raspberry, strawberry, blackberry, apple, peach, etc.).
Other plant families which contain anthocyanin pig-
ments are Solanaceae (tamarillo and eggplant), Sax-
ifragaceae (red and black currants), Cruciferae (red
cabbage), and Ericaceae (blueberry and cranberry)
[2]. Blueberries contain the following anthocyanins:
malvidin 3-galactoside, delphinidin 3-galactoside, del-
phinidin 3-arabinoside, petunidin 3-galactoside, petuni-
din 3-arabinoside, malvidin 3-arabino-side, cyanidin 3-
glucoside, cyanidin 3-galactoside, cyanidin 3-arabinoside,
delphinidin 3-glucoside, malvidin 3-glucoside, peoni-
din 3-glucoside, peonidin 3-galactoside, peonidin 3-
arabinoside, and peonidin 3-glucoside [3].
Flavonoids such as flavones, isoflavones, flavonones,
anthocyanins, and catechins have strong antioxidant ca-
pacity [4]. These compounds can be found in cereal
grains, tubers, tea, coffee, fruits, and vegetables. The
berries, such as blueberries, are significant sources of an-
thocyanins. Methanol extracts from Vaccinium angusti-
folium L and cultivar Fundy possess higher antioxidant
activity than sweet cherry, potato (purple), wheat germ,
and ginseng root [5].
The consumption of wild blueberries, a food source
with high in vitro antioxidant properties, is associated
with a diet-induced increase in ex vivo serum antioxi-
dant status [6]. Anthocyanins in grape juice reduced in
vitro oxidation of human low-density lipoprotein (LDL)
[7]. Potential bioavailability, in humans, of several an-
thocyanins from red wine was tested. Within 12 hours
after 300 mL of wine consumption, 1.5%–5.1% of the
ingested anthocyanins were found in urine. Two com-
pounds among the wine anthocyanins were unchanged
while the others, which were considered by the authors,
seemed to have undergone molecular modifications [8].
Frozen, liquid-extracted, and freeze-dried powders, made
from wild blueberries, were used in a study of bioac tive
properties, which included antioxidant activity, cardio-
protective capacity, and cancer chemoprevention activity.
Many fractions of the extr acts showed antioxidant activi-
ties, especially those rich in anthocyanins and proantho-
cyanidins [9]. Anthocyanins have been found to signifi-
cantly suppress the growth of cultured tumour cells and
have been shown to have greater inhibitor y effect than
other flavonoids [10, 11].
Blueberries are commercialised in different ways,
mainly as fresh or frozen products. Freezing and dry-
ing are two possible methods to preserve blueberries but
the severit y of both processes might destroy anthocyanins
or their antioxidant effects. Blueberries are known for
their bioa ctive properties such as antioxidant activity,
cardiovascular protection, antidiabetic properties, vision
2004:5 (2004) Anthocyanins in Blueberr ies and Their Antioxidant Effect 249
improvement properties, and inhibition of carcinogenesis
and mutagenesis [12]. Thus, the aim of this study was to
determine and to compare total anthocyanins and their
antioxidant effects in frozen or dried blueberries and to
compare them with the values found in fresh berries.
MATERIALS AND METHODS
Samples
Fresh blueberries (Vaccinium corymbosum L) were
supplied by Blueberry Farms of Australia P/L, Corindi
Beach, New South Wales, Australia.
Treatments
Fresh blueberries were kept at 5
◦
Cforuptotwoweeks
before extraction (FR2). Several batches of blueberries
were frozen and kept at
−20
◦
C up to 3 months. The sam-
ples were taken and examined at 1-month (FZ1M) and 3-
month (FZ3M) storage. There were 2 replicates for each
sampling point.
Two batches of blueberries weighing 1 kg each were
dried. The first batch, PT, had been treated with 60% w/w
sugar and 1% w/w NaCl solution for 4 hours and slowly
dried in a cabinet dryer at 90
◦
Cfor90minutes,followed
by 70
◦
Cfor120minutes,andfinally50
◦
C for 120 min-
utes. The second batch, UN, was dried directly without
any pretreatment using the same temperature profile.
Dry matter was determined by drying 5–10 g blue-
berry sample in a vacuum oven at 70
◦
C, 85 kPa for 72
hours. The dried blueberries were weighed again and the
dried matter that remained was determined. Total antho-
cyanins and antioxidant effect from dried samples (UN
and PT) were compared with those of frozen and fresh
samples.
Anthocyanin extraction
Samples weighing 20 g of fresh, frozen, and propor-
tionally reduced amounts (based on moisture loss during
drying) of dried blueberr ies were blended in a food pro-
cessor for 1 minute with 150 mL of a mixture of methanol,
acetic acid, and distilled water (M:A:W) at a ratio of
25:1:24. Frozen blueberries were thawed in a refrigera-
tor (at about 5
◦
C) overnight prior to the extraction. Half
of the well-blended solution was centrifuged at 21 900 g
(12 000 rpm) for 20 minutes at 20
◦
C. The remaining
residue from centrifugation after the supernatant was re-
moved was mixed thoroughly with 75 mL M:A:W, cen-
trifuged, and the super natant was separated. Each sample
was extr acted 3 times. The clear liquid from the 3 extrac-
tions was evaporated under vacuum at 35
◦
C. The residue
from vacuum evaporation was redissolved with 5 mL of
3% (w/v) formic acid in water. This aqueous solution was
adsorbed on a C18 Sep-Pak cartridge. The cartridge was
washed with 5 mL of 3% (w/v) formic acid in water and
eluted with 3.5 mL of 3% (w/v) formic acid in methanol.
The anthocyanins eluted from the cartridge were evapo-
rated under vacuum at 35
◦
C until dryness [13].
1
0.8
0.6
0.4
0.2
0
260 310 360 410 460 510 560 610
Absorbance
Wavelength (nm)
538, 0.423
Figure 1. Scan spectrum of blueberry extracts in MeOH:HCl.
Determination of total anthocyanins
The residue was diluted to the volume of 25 mL by
mixing with the mixture of methanol and 0.1 M HCl at
a ratio of 85:15 (MeOH:HCl). The anthocyanin solution
was diluted to the appropriate concentration for mea-
surement of absorbance in the Cary 100 scanning UV-
Vis spectrophotometer using 1 cm path length quartz cells
at 538 nm. Total anthocyanins were expressed as cyani-
din 3-rutinoside equivalents [14]. The molar absorptivity
of cyanidin 3-rutinoside was equal to 31085 at 530 nm in
MeOH:HCl. This molar absorptivity has been determined
experimentally.
Antioxidant effects
The antioxidant ac tivity of the anthocyanin extr acts
was measured using a free radical method of Brand-
Williams et al [15]. The free radical used in this study
was 2, 2-diphenyl-1-picrylhydrazyl (DPPH). The UV-
1601 UV-Vis spectrophotometer was used to determine
the concentration of DPPH. 3.9 mL of 6
× 10
−5
mol/L
DPPH in methanol (spectrophotometric grade) were put
into the disposable cuvette with 0.1 mL of anthocyanin
extract. The decrease of absorbance was measured at 0
minute, 1 minute, and every 5 minutes at 515 nm for 2
hours or until the absorbance became steady. The remain-
ing DPPH concentration (C
DPPH
) was calculated using the
following equation [15]:
C
DPPH
=
Abs
515
+2.58 × 10
−3
×
12509
−1
. (1)
Statistical analysis
The data were analysed by analysis of v ariance
(ANOVA) method and Duncan, multiple-range test at 5%
level of significance using SPSS.
RESULTS AND DISCUSSION
Total anthocyanins
A spectrum of the blueberry extract is presented in
Figure 1. In the anthocyanin extracts, the peak in the vis-
ible region was recorded at 538 nm wh ile the peaks in the
UV range were at 280 and 320 nm. The presence of these
250 Virachnee Lohachoompol et al 2004:5 (2004)
Table 1. Anthocyanin content in evaluated samples.
Blueberry s amples
Total anthocyanins
mg/g dry matter
Fresh blueberri es (FR0) 7.2 ± 0.5
a∗∗
Fresh blueberr i es 2-week
5.7
±0.5
ab
storage at 5
◦
C(FR2)
Untreated dried (UN) 4.3
± 0.1
bc
Pretreated dried (PT) 3.7 ± 0.2
c
Stored frozen for 1 month (FZ1M) 8.1 ± 0.1
a
Stored frozen for 3 months (FZ3M) 7.9 ± 1.3
a
∗
Total anthocyanin as cyanidin 3-rutinoside equivalent.
∗∗
Mean ± standard deviation of duplicate samples. The means that have
the same superscript are not significantly different (P <.05).
peaks reflects the fact that blueberr ies contain a mix of
anthocyanins and other phenolic compounds.
One sample of fresh blueberries, (FR0), was extracted
immediately after having been received from the grower
while another sample, (FR2), was kept at 5
◦
Cfor2weeks
before extraction. Total anthocyanins in FR2 were slightly
lower than those in FR0; namely, 5.7 and 7.2 mg/g dry
matter, respectively (Table 1 ). The blueberries that had
been stored for two weeks at 5
◦
Cweresofter.Inastudyby
Sapers and Phillips [16], the leakage of anthocyanins was
proportional to the percentage of soft berries in the whole
sample. In comparison with other fruits such as plums,
the concentration of anthocyanins found in 2-week refrig-
erated blueberries was higher (5.7
± 0.5 mg/g dry matter)
than that found in fresh plums, which was in the range of
2.6–5.2 mg/g dry matter [ 14 ].
The total anthocyanin content in both dried samples
UN and PT was less than that in fresh berr ies (Tabl e 1).
The percentage of loss of anthocyanins in UN was 41%
while it increased to 49% in PT. Slightly higher reduc-
tion in anthocyanin content was observed in PT than in
UN. However, the difference was not statistically signifi-
cant. This difference was caused by the 4-hour pretreating
step that leached out some anthocyanins. Anthocyanin
leakage might happen due to dewaxing, w hich was caused
by stirring and soaking during the osmotic pretreatment.
This observation is comparable to the study by Sapers
and Phillips [16] who found that dewaxing weakened the
berry cuticle and allowed the skin to rupture. This permit-
ted some leakage from the exposed edges or undersurface
of the torn skin to the osmotic solution that caused an-
thocyanin loss before the drying step. Also, the thermal
processing destroyed some anthocyanins. A study about
the evolution of anthocyanins in raspberries during jam
making, in which heat was used, showed that 17%–40%
of anthocyanins were lost [17].
As for the appearance, the PT samples presented a
more shiny aspect than the UN samples. Furthermore, the
moisture content after drying of PT (33.6% wet basis) was
lower than that of UN (36.9% wet basis).
100
90
80
70
60
50
40
30
20
10
0
0 20 40 60 80 100 120
Remaining DPPH radical (%)
Time (min)
FR0
FR2
UN
PT
FZ1M
FZ3M
Figure 2. Kinetic behaviour of reducing DPPH radical of an-
thocyanins found in blueberry extract after the following treat-
ments: FR0 : fresh blueberries; FR2: fresh blueberries kept at 5
◦
C
for two weeks; UN : untreated blueberries dried in a cabinet
dryer; PT : osmotically pretreated blueberries dried in a cabinet
dryer; FZ1M : frozen blueberries kept at
−20
◦
C for 1 month;
FZ3M : frozen blueberries kept at
−20
◦
C for 3 months.
Anthocyanin contents of frozen samples were found
stable over 3 months of storage (Table 1). The fruits,
which were stored frozen for 1 month (FZ1M) and 3
months (FZ3M), showed no significant difference from
FR0.
Antioxidant effect
The results of the kinetic behaviour of blueberry ex-
tracts are shown in Figure 2. After adding the blueberry
extract to the DPPH solution, the absorbance was in-
creased due to the colour of the extracts. The slope of the
equations may be a useful parameter to define the antioxi-
dant capacity. The steeper the slope, the lower the amount
of antioxidant that is necessary to decrease by 50% the
initial DPPH concentration [18]. The steepest slope was
that of FZ3M (Ta ble 2 ). This means a lower amount of the
extract was necessary to decrease the initial DPPH con-
centration. FZ1M showed the lowest antioxidant activity
(though not the lowest anthocyanin content), while there
was no significant difference in antioxidant effect between
FZ3M and FR0.
Antioxidant activity can also be assessed by the oxygen
radical absorbance capacity (ORAC). The ORAC method
estimates the antioxidant capacity of a sample by taking
the oxidation reaction to completion whereas DPPH esti-
mates the stable free radical and thus is more appropriate
to characterise the antioxidant activity in a food sample.
In a study of the commercial frozen lowbush blueberries,
which contained lower levels (60%–80%) of “blue” than
the other samples, it was found that the antioxidant ac-
tivity (ORAC) was comparable to that of the fresh fruits
[19]. This result supported an earlier study on variation
in ORAC based on variety, maturity, and source, done by
Prior et al [20].
2004:5 (2004) Anthocyanins in Blueberr ies and Their Antioxidant Effect 251
Table 2. Average slope values of blueberry extracts.
Blueberry extracts
Slope
∗
(% DPPH/min)
FR0 −0.0110
ab
FR2 −0.01035
ab
UN −0.0103
ab
PT −0.0116
ab
FZ1M −0.0076
a
FZ3M −0.0145
b
∗
Slopes that have the same superscript are not significantly different (P<.05).
As for the dried products, UN and PT, samples showed
no significant difference in antioxidant activity from
the fresh berries even though the anthocyanin contents
shown in Tabl e 1 were lower than those in the fresh sam-
ples. According to similar studies [20, 21], the correlation
coefficient between ORAC and the total phenolics was
higher than the correlation coefficient between ORAC and
total anthocyanins. In a study of total phenolics in blue-
berries [22], chlorogenic acid, a major colourless phenolic
of blueberries, was found at the level of 60–100 mg/100 g
of fresh berries and significantly contributed to ORAC
[23]. The anthocyanins breakdown products from dr ying
process might act as antioxidants without being affected
by the thermal process.
CONCLUSIONS
The amount of total anthocyanins in the frozen sam-
ples, expressed as cyanidin 3-r utinoside equivalents, was
not significantly different from that in the fresh samples.
In contrast, the concentration of anthocyanins in dried
blueberries (UN and PT) was significantly reduced in
comparison with that in fresh blueberries while antioxi-
dant activity of the extracts did not differ from that of the
fresh fruit. Fruit drying resulted in reduction of the to-
tal anthocyanin level by 41%. When drying was preceded
with osmotic dehydration, 49% of anthocyanins were lost.
There was no significant difference in antioxidant activity
between the anthocyanin extracts of the frozen or dried
samples and the fresh fruit. Antioxidant activity in blue-
berries is an appealing characteristic to consumers. Any
processing method that maintains the level of compounds
known for their health benefits will be of interest to the
food industries.
ACKNOWLEDGMENT
The authors would like to thank Blueberry Farms of
Australia, Corindi Beach, New South Wales, Australia, for
providing blueberries used in this study.
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∗
Corresponding author.
E-mail: g.srzednicki@unsw.edu.au
Fax: +61 2 9385 5931; Tel: +61 2 9385 4355
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manuscript or a topic for inclusion in this issue.
Manuscript Due November 1, 2006
Acceptance Notification February 1, 2007
Final Manuscript Due April 1, 2007
Publication Date 3rd Quarter, 2007
GUEST EDITORS:
Hossein A. Ghanbari, Panacea Pharmaceuticals, Inc.,
Gaithersburg, MD 20877, USA; hag@panaceapharma.com
Michael S. Lebowitz, Panacea Pharmaceuticals, Inc.,
Gaithersburg, MD 20877, USA; msl@panaceapharma.com
Hindawi Publishing Corporation
http://www.hindawi.com
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8
th
International Meeting on
Molecular Epidemiology and
Evolutionary Genetics in
Infectious Diseases
(MEEGID - VII)
(Cosponsored by CDC, USA,
IRD and CNRS, France, University of Mahidol and
French Embassy, Bangkok, Thailand)
BANGKOK, THAILAND
AFTER THE SUCCESSFUL LAUNCH OF INFECTION, GENETICS AND EVOLUTION (ELSEVIER),
NOW COVERED BY MEDLINE AND INSTITUTE FOR SCIENTIFIC INFORMATION
Objectives of The
Meeting:
First, to integrate epidemiology, molecular biology, genomics, proteomics,
bioinformatics and population biology in areas of diagnosis, strain typing, species
identification, pathogenesis, antigenic variation, drug and vaccine resistance, host
(animal and human) susceptibility, and vector specificity.
Second, to foster interactions between epidemiologists, clinicians, field and laboratory
scientists working on hosts, parasites, yeast and fungi, bacteria, viruses, and vectors
of medical, veterinary and agronomical interest.
Third, to provide health care providers, public health professionals, policy makers,
epidemiologists and laboratory scientists, and program managers an opportunity to
discuss the use of the genetic tools and methodologies that are needed to meet the
challenges of diagnosis and management of emerging, re-emerging, and endemic
infectious diseases.
Special emphasis will be given to diseases of special interest to the subregion (bird
flu, SARS, malaria, dengue)
Place:
Bangkok, Thailand
When:
30
th
November-3
rd
December 2006
.
For further information please contact:
Michel Tibayrenc
IRD Representative
Office
French Embassy
29, Thanon Sathorn Tai
Bangkok 10120, Thailand
Phone +662 6272190
Fax +6626272194
Michel.Tibayrenc@ird.fr
Abstract Submission Deadline: September 30th, 2006
Registration Fee: 200.00 euros or equivalent in other currencies; Students pay only meals and coffee breaks;
For more information, see
http://www.th.ird.fr
M
odern drug
Discovery & Development Summit
11 Full Conferences!
Systems Biology
in Drug Discovery
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in Drug Discovery
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and Drug Discovery
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in Drug Discovery
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Medicinal Chemistry
Epigenetic Cancer
Therapy
Stem Cell Research
and Regenerative
Medicine
Drug Safety and
Pharmacovigilance
Biotech and Pharma
Licensing and
Deal-Making
December 4-6, 2006
Pennsylvania Convention Center
Explore
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Gain access to the key players in
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Over 250 talks from experts within
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Call 626-256-6405 or Visit www.gtcbio.com for more information
SPECIAL OFFER: Register 2, the 3rd Goes Free!
Abdelali Haoudi, Ph.D.
Eastern Virginia Medical School
Department of Microbiology and
Molecular Cell Biology
Lewis Hall 3011
Norfolk, VA 23501, USA
E-mail: haoudia@evms.edu
Dear Colleague,
The Journal of Biomedicine and Biotechnology (JBB) is an international journal publishing
original work in all areas of biomedicine. We also welcome papers from geneticists and
molecular biologists covering the area of animal, bacterial, and plant genetics. In addition,
contributions on medical informatics, medical technology, and commercial biology are
considered as well.
JBB is primarily devoted to original research papers, but will also publish review articles, forum
discussion articles, editorials, book reviews, and meeting reports. It is indexed in a number of
databases, among which are ISI Science Citation Index Expanded, Biotechnology Citation Index,
the Directory of Open Access Journals (DOAJ), PubMed, Elsevier BIOBASE, and Scopus.
Moreover, all of JBB’s articles are included in PubMed Central.
Since JBB is an open access journal, the full text of all articles will be freely available on the
journal’s website immediately after publication. In addition, authors will retain the copyright of
their papers by signing a Creative Commons Attribution License, which gives the readers the
right to download, print, and redistribute any article as long as it is properly cited. The Open
Access Publishing model has proven to be very successful with JBB and increased the downloads
of its articles.
The most recent Impact Factor for the journal is 1.812 according to the 2005 Journal Citation
Reports® released by Thomson Scientific in 2006.
As the Editor-in-Chief of the Journal of Biomedicine and Biotechnology, I would like to invite
you to submit your papers for publication in the journal and maximize your readership and the
impact of your research articles.
You can submit your contributions via the publisher’s electronic Manuscript Tracking System
located at http://www.hindawi.com/mts/.
Please do not hesitate to contact me at jbb@hindawi.com if you need further information.
Sincerely yours,
Dr. Abdelali Haoudi
Editor-in-Chief of JBB
