Innovative Food Science and Emerging Technologies 8 (2007) 347 – 352
www.elsevier.com/locate/ifset
Evaluation of colour and stability of anthocyanins from
tropical fruits in an isotonic soft drink system
Veridiana Vera de Rosso, Adriana Z. Mercadante ⁎
Department of Food Science, Faculty of Food Engineering, State University of Campinas (UNICAMP), PO Box 6121, CEP 13083-862, Campinas, SP, Brazil
Abstract
Due to the growing market of food products associated to good health, the colour changes and stability of anthocyanin extracts from acerola,
containing high level of ascorbic acid, and from açai, rich in flavonoids, were evaluated in an isotonic soft drink like system and in buffer solution.
The degradation of anthocyanins from both sources followed first-order kinetics in all the systems, under air, either in the presence or absence of
light. Addition of sugars and salts had a negative effect on the anthocyanin stability, being the rate constant (kobs) values in isotonic soft drink
system 6.0 × 10− 2 h− 1 for acerola and 7.3 × 10− 4 h− 1 for açai, both in the dark. In the presence of light, the anthocyanin degradation was 1.2 times
faster for acerola and 1.6 times faster for açai in soft drink isotonic systems, as compared to their respective buffer solutions. The highest stability
observed in all açai systems was correlated to its high total flavonoid content and absence of ascorbic acid. The gradual degradation of red colour
during storage of all systems was verified by the decrease of a⁎ values, accompanied with decreased colour intensity (decrease in C⁎ values) and
tonality changes from red to yellow colour, as the h values increased during the experiment time.
© 2007 Elsevier Ltd. All rights reserved.
Keywords: Acerola; Açai; Anthocyanin; Stability; Isotonic Beverage model system
Industrial relevance: Functional foods and addition of bioactive compounds to processed foods and drinks are a worldwide growing market. Thus, the aim of this
study was to evaluate the stability of added anthocyanins from acerola and açai to an isotonic soft drink system and to study the effect of fluorescent light, mimicking
the supermarket conditions, in such systems. Since colour is of fundamental importance for the acceptance of a food by consumers, the colour fading that occurs in
these systems was also verified.
1. Introduction
Anthocyanins pigments are responsible for the red-purple to
blue colours of many flowers, fruits, vegetables, and grains, the
fruits and vegetables anthocyanin-rich extracts being used as
food colourants. In the United States, 4 of the 26 colourants that
are exempt from certification and approved for food use are
anthocyanin-derived: grape skin extract, grape colour extract,
fruit juice, and vegetable juice (Wrolstad, 2004). Although
grape marc constituted a very abundant source of anthocyanins
(Mazza & Miniati, 1993), other fruits such as elderberries, black
currants, chokeberries, raspberries, blackberries and also red
cabbage have been used as sources of anthocyanins colourants
for food products and beverages (Wrolstad, 2004).
⁎ Corresponding author. Fax: +55 19 3521 2153.
E-mail address: [email protected] (A.Z. Mercadante).
1466-8564/$ - see front matter © 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.ifset.2007.03.008
Besides the colourant property, anthocyanins have been
found to exhibit potential therapeutic effect as anti-inflammatory, radiation-protective, chemoprotective, vasoprotective
(Kong, Chia, Goh, Chia, & Brouillard, 2003), inhibition of
LDL oxidation and also prophylactic action, such as decrease
risks of cardiovascular diseases (Seeram & Nair, 2002).
Acerola (Malpighia emarginata DC.) and açai (Euterpe
oleracea Mart.) belonging, respectively, to the Malpighiaceae
and Arecaceae families, are tropical fruits recognized as
functional foods. Acerola shows high contents of ascorbic
acid (AA) (Vendramini & Trugo, 2000; Assis, Lima, & FariaOliveira, 2001) and of carotenoids (De Rosso & Mercadante,
2005), besides the presence of the anthocyanins cyanidin-3-αO-rhamnoside and pelargonidin-3-α-O-rhamnoside (Hanamura, Hagiwara, & Kawagishi, 2005); açai is considered an
energetic fruit, rich in the anthocyanins cyanidin-3-glucoside
and cyanidin-3-rutinoside (Gallori, Bilia, Bergonzi, Barbosa, &
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V.V. de Rosso, A.Z. Mercadante / Innovative Food Science and Emerging Technologies 8 (2007) 347–352
Vincieri, 2004), dietary fibers and iron (Sangronis, Teixeira,
Otero, Guerra, & Hidalgo, 2006).
The addition of anthocyanic extracts from acerola and açai as
colourant and functional ingredient to isotonic soft drinks is an
interesting alternative due to the growing market of food
products associated to health and well-being. According to the
ACNielsen Global Services (2004), the worldwide sells of
isotonic and energetic soft drinks in 2004 increased by 10%
compared to those from 2003. Moreover, more than 77 million
litres of isotonic beverages were sold on the Brazilian market in
1996, along with a tendency for market increase of 4.8% per year
(López, 2002). However, many limitations exist for the
commercial application of anthocyanin extracts in food products
and beverages due to its low stability, which depends on its
chemical structure and concentration, pH, temperature, presence
of oxygen and light, as well as on the medium composition, such
as presence of ascorbic acid, co-pigments and sugars.
The effect of added sugar on the anthocyanin stability
depends on its structure, concentration and type of sugar.
Wrolstad, Skrede, Lea and Enersen (1990) reported that when
the sucrose concentration increased by 20%, the stability of the
anthocyanins from strawberry also increased. On the other
hand, at low concentration of sucrose (86 g/L) the degradation
of anthocyanins from red cabbage, blackcurrant and elderberry
extracts was higher in soft drinks compared to buffer systems
both at pH 3, whilst the opposite was observed for grape extract
(Dyrby, Westergaard, & Stapelfeldt, 2001). In another study, the
anthocyanins stability of grape marc, elderberry and blackcurrant extracts was lower in all sucrose (100 g/L) added
systems as compared to the control at pH values of 3, 4 and 5,
whereas the browning index did not change with addition of
sugar (Malien-Aubert, Dangles, & Amiot, 2001). Addition of
20 g/L of sucrose to a drink model system (pH 3) containing red
cabbage and grape extracts did not influence the thermal and
photo stability of the anthocyanins (Duhard, Garnier, &
Megard, 1997). Although most anthocyanic extracts showed
lower stability in sugar added systems, no statistical analysis
was carried out to verify the significance of this difference.
The aim of this study was to evaluate the influence of added
sugars and salts, simulating an isotonic soft drink, on the
stability of anthocyanins from acerola and açai, as well as to
study the effect of fluorescent light in such systems. Since
colour is of fundamental importance for the acceptance of a
food by consumers, the colour fading that occurs in these
systems was also verified.
2. Materials and methods
2.1. Materials
High performance liquid chromatography (HPLC) grade
solvents (EM Science, Darmstadt, Germany) were used. The
other reagents were all of analytical grade, being citric acid,
sodium phosphate dibasic, sodium benzoate, sucrose, fructose,
glucose, sodium citrate, sodium chloride, potassium chloride
and potassium phosphate monobasic from Labsynth (Diadema,
Brazil). The water was purified in the Milli-Q system
(Millipore, Bedford, MA) and the samples and solvents were
filtered through Millipore membranes (0.22 and 0.45 μm) prior
to HPLC analysis.
2.2. Anthocyanins extracts
Fresh acerola fruits (4 kg) from the region of Campinas, São
Paulo State, Brazil, were pulped in a knife pulper to remove
skins and seeds. The açai was obtained in the form of
commercial frozen pulp (1 kg) in Campinas. The fruit pulps
were exhaustively extracted with 1% HCl in methanol, solvent
to solid ratio of 3:1, with agitation provided by a homogenizer
(Metabo, Nürtingen, Germany), at room temperature. The
solution obtained was then filtered and vacuum concentrated
(T b 38 °C) until complete methanol evaporation. This concentrated crude extract of anthocyanins (CCE) was stored at
− 18 °C under nitrogen.
2.3. Preparation of model systems
Solutions using the acerola and açai CCE were prepared in
phosphate–citrate buffer and in an isotonic soft drink system,
both at pH 2.5. The composition of the isotonic soft drink was,
Fig. 1. Degradation kinetic curves of the anthocyanins from açai and acerola in citrate–phosphate buffer and isotonic soft drink model systems, both at pH 2.5, under
air and in the presence/absence of light.
V.V. de Rosso, A.Z. Mercadante / Innovative Food Science and Emerging Technologies 8 (2007) 347–352
Table 1
Rate constant values (kobs) and half-life times (t1/2) for anthocyanin degradation
in citrate–phosphate buffer and isotonic soft drink systems added with acerola
and açai, at pH 2.5 under air and presence/absence of light
kobs (h− 1) a
Systems
Acerola — buffer
Acerola — isotonic soft drink
Açai — buffer
Açai — isotonic soft drink
a
Light
Dark
Light
Dark
Light
Dark
Light
Dark
−2
5.0 × 10
3.9 × 10− 2
6.6 × 10− 2
6.0 × 10− 2
7.6 × 10− 4
1.5 × 10− 4
1.3 × 10− 3
7.3 × 10− 4
t1/2 (h) a
13.7
17.5
10.6
11.4
909.1
6456.2
548.2
943.4
Average of two experiments.
per litre: 55 g of sucrose, 5.5 g of fructose, 5.5 g of glucose,
0.15 g of sodium benzoate, 3.0 g of citric acid, 0.14 g of sodium
citrate, 0.5 g of sodium chloride, 0.5 g of potassium chloride
and 0.4 g of potassium phosphate monobasic. For each fruit, the
same ratio of CCE mass to volume was used in both systems
and the initial absorbance (A0) of the anthocyanin solutions, ca.
0.8, was measured at the maximum absorption wavelength in
the visible region (λmax). The solutions were allowed to rest in
the absence of light for 3 h, to attain the equilibrium amongst the
different forms of anthocyanin, absorbance being monitored
every hour. The solutions were then distributed in screw-capped
Pyrex tubes (cut-off 310 nm) with a nominal volume of 10 mL.
These tubes were placed in a support between two fluorescent
white lamps (GE T8 32 W, General Electric, Rio de Janeiro,
Brazil), corresponding to 850 lx at the sample position, in a
room free of other light sources at a temperature of 20 ± 1 °C.
Other tubes of each solution were maintained in the same room
but in the absence of light, to serve as control.
beginning, middle and end of the experiment period. Immediately after aliquots were drawn, all water was removed under
nitrogen with addition of absolute ethanol. The dried anthocyanin extracts were re-dissolved in acidified methanol immediately before HPLC analysis. An HPLC equipment (Waters,
Milford, MA, USA) consisting of a quaternary solvent pumping
system, Rheodyne injector with a 20 μL loop, external oven
with temperature control, on-line degasser, diode array detector
(PDA) and Millennium data collection and processing system
was used. The anthocyanins were separated on a C18 Shim-pack
CLC–ODS column (5 μm, 250 × 4.6 mm) from Shimadzu
(Canby, OR, USA) using as mobile phase a linear gradient of
5% aqueous formic acid/methanol, going from 85:15 (v/v) to
20:80 in 25 min, the latter proportion being maintained for
further 15 min, at a flow rate of 1 mL/min and column
temperature maintained at 25 °C. The chromatograms were
processed at 280, 320 and 520 nm, and the spectra acquired
between 200 and 600 nm. The anthocyanin identification was
based on the literature (Gallori et al., 2004; Hanamura et al.,
2005).
2.5. Kinetic calculations and statistical analysis
The anthocyanin concentration (% residual anthocyanins)
was plotted against time (h) and a linear regression analysis was
used to determine the adequacy of the anthocyanin degradation
kinetic model. The degradation rate constant (kobs) was
determined from the first derivative of the curves plotted (Eq.
(4)) and the half-life time (t1/2) was calculated from Eq. (5).
½anthocyanin ¼ ½anthocyanin0 exp ðkobs t Þ
t1=2 ¼
2.4. Monitoring of the model systems
The stability of the anthocyanins was monitored using a
Beckman DU-70 spectrophotometer (Fullerton, CA, USA),
measuring the loss of the solution absorbance at λmax up to an
approximately 75% loss as compared to the initial absorbance.
In addition, the changes in colour of the anthocyanin systems
were determined from the CIELAB parameters using the Color
Quest XE colorimeter (Hunter Lab., Reston, VA, USA) equipped
with the light source D65 and observation angle of 10°. Using the
parameters L⁎ (lightness), a⁎ (red) and b⁎ (yellow), the values for
C⁎ (chroma), h (hue angle) and ▵E⁎ (total colour difference)
were calculated using Eqs. (1), (2) and (3).
h
i1=2
2
2
C ⁎ ¼ ða⁎Þ þðb⁎Þ
ð1Þ
h ¼ arctan ðb⁎=a⁎Þ
ð2Þ
h
i1=2
2
2
2
DE ⁎ ¼ ð DL⁎Þ þðDa⁎Þ þð▵b⁎Þ
:
ð3Þ
The relative losses of each anthocyanin in the different
systems were evaluated by HPLC, sampling aliquots at the
349
ln2
:
kobs
ð4Þ
ð5Þ
The software Microcal Origin 5.0 was used for all analyses,
both ANOVA and kinetic.
3. Results and discussion
3.1. Influence of sugars/salts and light on the stability of
anthocyanin extract
In all the systems evaluated, the degradation of anthocyanins
from acerola and açai followed first-order kinetics (Fig. 1),
which model was applied to give the anthocyanin degradation
rate constants (Table 1). This behaviour had already been
reported in most anthocyanin systems, such as for strawberry
(Wrolstad et al., 1990) and blood orange extract in both
ethanolic and carbonated drinks (Katsaboxakis, Papanicolaou,
& Melanitou, 1998).
The addition of sugars and salts had a negative effect on the
anthocyanin stability, both from acerola and açai. In the dark,
the kobs values were 1.5 and 6.8 times faster, respectively, in
acerola ( p = 0.0497) and açai ( p b 0.0001) isotonic soft drink
system compared to their respective control solution. In the
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V.V. de Rosso, A.Z. Mercadante / Innovative Food Science and Emerging Technologies 8 (2007) 347–352
presence of light, the anthocyanin degradation was 1.2 times
faster for acerola ( p = 0.4977) and 1.6 times faster for açai
( p b 0.001) in soft drink isotonic systems, as compared to the
respective control.
As expected, light had a deleterious effect on anthocyanin
stability in all systems; however, this effect was not statistically
significant for the acerola systems, either in isotonic soft drink
( p = 0.6286) or in buffer solution ( p = 0.1150). This fact is
certainly due to the high ascorbic acid (AA) levels naturally
present in the acerola anthocyanic extracts (De Rosso &
Mercadante, 2007), since direct condensation reaction between
AA and anthocyanins (Wrolstad et al., 1990) can occur in all
acerola systems.
On the other hand, light had a significantly negative
influence in both açai added systems, isotonic soft drink
( p b 0.001) and buffer ( p b 0.001). In fact, since in the buffer
system the degradation rate of açai anthocyanins extract was 7.1
times faster under light than in the dark, and only 1.7 times
faster when in isotonic soft drink system, sugars/salts did have a
great influence on the stability of açai anthocyanins. This result
is most probably associated to the formation of sugar
degradation compounds due to the long time, 1200 h, taken in
this set of experiment. It is known that in acidic solutions the
most probable compounds derived from sugars are furfural and
5-hydroxymethylfurfural (HMF) (Shinoda, Komura, Homma,
& Murata, 2005), which in turn were proved to be responsible
for the highest degradation when added to the blackberry
anthocyanin buffer solution at pH 3.45 at 24 °C (DebickiPospisil, Lovric, Trinajstic, & Sabljic, 1983).
both sources are quite similar (Hanamura et al., 2005; Gallori
et al., 2004). In addition, since all systems started with ca. 0.8
absorbance unities, measured at λmax in the visible range,
differences in concentrations of the coloured forms can be
excluded as a factor of influence.
3.3. Changes in the relative composition of the anthocyanins
The decrease in the peak areas of each anthocyanin from both
sources were similar in the isotonic soft drink and buffer solutions,
and no intermediate degradation compounds absorbing at 520,
320 and 280 nm were detected in all systems. In previous studies
using HPLC–PDA, anthocyanin degradation products were also
not detected in red radish (Rodriguez-Saona, Giusti, & Wrolstad,
1999) and pomegranate (Martí, Pérez-Vicente, & García-Viguera,
2001) juice type model systems. Moreover, no monomeric or
dimeric adduct compounds consisting of malvidin 3-O-glucoside
units and furfural were detected by both HPLC–PDA and HPLC–
MS (Es-Safi et al., 2000).
As expected for first-order reaction (Fig. 1), the highest the
anthocyanin concentration the lowest the degradation loss. In
3.2. Influence of source of anthocyanin extract on stability
The anthocyanin stability from acerola was 82 times lower
than that from açai, in the isotonic soft drink system in dark
conditions. The great stability difference from both anthocyanin
sources is most probably due to the high contents of AA in the
anthocyanic extract from acerola (1921 mg/100 g) since no AA
was detected in açai (De Rosso & Mercadante, 2007). Another
factor that could also have contributed to the instability of
acerola anthocyanins is the formation of furfural and HMF from
AA, as previously observed in blood orange juice (Krifi,
Chouteau, Boudrant, & Metche, 2000).
Another factor that should be taken in consideration is the
highest total flavonoid level in the açai anthocyanic extract
(537 mg catechin equivalent (CE)/100 g) compared to that found
in the acerola extract (53 mg CE/100 g) (De Rosso &
Mercadante, 2007). This type of compounds may have a
protective effect on the anthocyanins from açai, since there is a
competitive action of flavanols and anthocyanins in the
condensation process with furfural and HMF by the formation
of both flavanol–furfuryl and anthocyanin–furfuryl–flavanol
adducts (Es-Safi, Cheynier, & Moutounet, 2000).
Extracts containing acylated anthocyanins were more stable
than those extracts with non-acylated anthocyanins, even in
added sugar systems (Malien-Aubert et al., 2001). However, for
acerola and açai extracts, the influence of anthocyanin
structures can be neglected since the major anthocyanins in
Fig. 2. HPLC–PDA chromatograms obtained during the degradation of (A)
acerola (peak 1 — cyanidin-3-rhamnoside, peak 2 — pelargonidin-3-rhamnoside,
peak 3 — not identified and peak 4 — not identified), and (B) açai (peak 1 —
cyanidin-3-glucoside and peak 2 — cyanidin-3-rutinoside) anthocyanins in
isotonic soft drink system at pH 2.5, in the presence of light and air. Processed at
520 nm. See text for chromatographic conditions.
V.V. de Rosso, A.Z. Mercadante / Innovative Food Science and Emerging Technologies 8 (2007) 347–352
351
Fig. 3. Changes in the colour parameters values (A) a⁎, (B) hue angle (h), (C) chroma (C⁎) and (D) DE⁎ for the buffer (■) and isotonic soft drink (□) systems of
anthocyanin extracts from acerola and açai, at pH 2.5, in the presence of light and air.
the isotonic soft drink systems, the acerola anthocyanins
cyanidin-3-rhamnoside (peak 1), pelargonidin-3-rhamnoside
(peak 2) and peak 3, which represented, respectively, 77%,
14% and 7% of the total area at zero time, showed area losses of
76%, 92% and 98% after 20 h, whilst peak 4, presented as 3% of
the relative area at zero time, disappeared within 20 h (Fig. 2A).
Also in the isotonic systems, the açai anthocyanins cyanidin-3glucoside and cyanidin-3-rutinoside, which corresponded to
respectively 13% and 87% of the total area, decreased by 90%
and 76% within 1000 h (Fig. 2B) under light and air.
3.4. Changes in colour
The initial colour parameter values for the acerola buffer and
isotonic systems were, respectively, 68.30 and 60.90 for L⁎,
49.52 and 46.27 for a⁎, 28.40 and 33.22 for b⁎, 57.08 and
56.96 for C⁎, and 29.83 and 35.70 for h. In açai buffer and
isotonic systems these values were, respectively, 59.22 and
60.27 for L⁎, 41.69 and 43.76 for a⁎, 11.99 and 9.94 for b⁎,
43.37 and 44.87 for C⁎, and 16.04 and 12.79 for h. The initial
values show that the colour of all acerola systems was more
intense than that observed in the açai systems, and that the
tonality of the acerola systems tends to orange whereas that of
the açai systems tends to red. The influence of the isotonic soft
drink medium composition, compared to buffer, on the initial
global colour was higher for acerola (DE⁎ = 9.40) than for açai
(DE⁎ = 3.09).
The gradual degradation of red colour, visually observed in all
systems, was verified by the similar decrease of a⁎ values in the
isotonic soft drink and control systems (Fig. 3A). The red colour
intensity also decreased during storage of all systems, as can be
seen by the C⁎ values behaviour (Fig. 3B). These changes were
accompanied by the tonality changes from red to yellow colour, as
the h values increased during the experiment time (Fig. 3C). In
addition, since the values of DE⁎ N 10 indicate that anthocyanin
degradation can be easily perceived by human eyes (Gonnet,
2001), either isotonic soft drink and control systems showed
visual colour changes approximately after 5 and 300 h of storage
under light of acerola and açai systems, respectively (Fig. 3D).
4. Conclusion
Considering that addition of sugars and salts had a negative
effect on the anthocyanin stability from both sources, and that
the açai added systems were significantly more stable than those
of acerola, these results show that the viability of anthocyanin
natural extract addition to soft drinks strongly depends on the
composition of the anthocyanin source.
Acknowledgements
The authors thank the Brazilian Funding Agency FAPESP
(Fundação de Amparo à Pesquisa do Estado de São Paulo) for
the financial support.
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