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Abstract

The so called ‘Superfruits’ are linked to small red fruitberries growing in tropical climates which have highlevels of phenolic compounds, vitamins which havebeneficial health effects. This study aimed to assessthe evolution of chromatic parameters and associatethem to the antioxidant parameters in three stages ofripening of acerola fruits. As ripening increased, a de-crease in the levels of phytochemical compounds wasobserved - the unripe fruits showed the highest levelsof total phenols and antioxidant activity. The highestlevels of total monomeric anthocyanin content werefound in semi-ripe fruit. In terms of fruit juice, thesame trend was observed as for fruit, only differing inthe levels of anthocyanin, where the juice of ripefruits presented the highest levels.
222 November/December 2013
RAW MATERIAL
The so called ‘Superfruits’ are linked to small red fruit
berries growing in tropical climates which have high
levels of phenolic compounds, vitamins which have
beneficial health effects. This study aimed to assess
the evolution of chromatic parameters and associate
them to the antioxidant parameters in three stages of
ripening of acerola fruits. As ripening increased, a de-
crease in the levels of phytochemical compounds was
observed - the unripe fruits showed the highest levels
of total phenols and antioxidant activity. The highest
levels of total monomeric anthocyanin content were
found in semi-ripe fruit. In terms of fruit juice, the
same trend was observed as for fruit, only differing in
the levels of anthocyanin, where the juice of ripe
fruits presented the highest levels.
INTRODUCTION
The concept of ‘superfruit’ is linked to small red fruits
with high contents of bioactive compounds beneficial to
human health, such as polyphenols and vitamins.
Acerola, açai, pomegranate, guarana and pitaya are now
all popular internationally (Jamin, 2009). Although not
considered a specific group or category of fruit, they
continue to dominate the market thanks to their nutri-
tional benefits and potential health appeals (Nuijten,
2010). Although many are consumed fresh, attention
should be given to processing because if pectinolytic
preparations contain antocianase activity, this may
weakens the final product.
Small red fruits and their juices are known carriers of
high levels of anthocyanins and there have been recent
Malpighia glabra L.
EVOLUTION OF THE CHROMATIC AND ANTIOXIDANT PARAMETERS
IN THREE DIFFERENTE STAGES OF ACEROLA RIPENING
reports concerning the anthocyanin content of blackcur-
rants (Ribes bvnigrum L.) as a function of the commer-
cial importance of its juice used in the preparation of ap-
petizers (Weidel; Schantz; Richling, 2011).
The acerola (Malpighia glabra L.) is mainly produced in
Brazil but it is native to northern South America and
Central America. It has many names, including the cherry
of Antilles, of Barbados and of Brazil. Its oldest name is
the ‘cherry of West Indies’, reminiscent of the Era from
the grand voyages (Netto, 1986, Guedes et al., 2011). Its
composition in the form of juice has been reported by
Chaves et al., (2004), and it is considered acidic, with
more than 1000 mg of ascorbic acid pro 100 g. It is a fruit
containing malic acid, although it also has concentra-
tions of citric and succinic acids. A rating of 12 geno-
types revealed variations of between 835-1820 mg%
with respect to ascorbic acid, and from 0.69 to 1.65 % in
terms of total titratable acidity, demonstrating signifi-
cant difference among samples (Matsura et al., 2001).
According to Matta and Cabral (2010), acerola juice can
be characterised as having high acidity, i.e. a low pH, but
with low sugar content. They point out that the vitamin
C content may be 20 times that of oranges; however,
they did not provide definitions as to appearance, colour
or anthocyanin.
S. Avila, A.A.F. Zielinski, C. Goltz, A. Nogueira, G. Wosiacki
• ACEROLA • ANTHOCYANIN • PHENOLIC CONTENT • SUPERFRUITS •
Fig. 1a: Different stages of acerola ripening (Malpighia glabra L.).
© all figures Wosiacki Fig. 1b: Acerola (Malpighia glabra L.).
November/December 2013
The use of some tropical fruit as a potential source of
phenolic compounds such as anthocyanins for use as
food colourant is highly relevant (Falcão et al., 2007;
Bordignon Jr. et al., 2009). The anthocyanin antioxidant
related to the bluish-red colour of blueberries is called
delphinidin, and other known anthocyanins include
cyanidin (red-orange), pelargonidin (orange), malvidin
(blue-red) and peodinina (red) (Mazza; Miniati, 1993). The
content of these compounds in fruits increases with ma-
turity, forming complexes with metals and other
bioflavonoids (Castañeda-Ovando et al., 2009). The
colour of fruit is an important indicator of the presence
of phenolic compounds from the class of anthocyanidin,
which are soluble glycosides of anthocyanin (Lima et al.,
2000; Castañeda-Ovando et al., 2009) and of biochemical
changes that occur during maturation, in particular
changes in pH and the oxidation-reduction state of the
cells.
Thus, the colour of the fruit is also sensitive to antioxi-
dant activity, the natural power of fruit (Wolfstädter,
2009). Functional foods or beverages that are naturally
coloured and rich in phenolic compounds follow a global
trend - that of healthy industrialised foods.
This study aimed to assess the evolution of chromatic pa-
rameters in three samples of acerola (Malpighia glabra
L.), well differentiated in terms of maturity, and to assess
the relationship with phenolic compounds, including an-
thocyanins and antioxidant activity.
MATERIALS AND METHODS
The fruits were harvested from a small-scale acerola
orchard located at the coordinates Latitude 25° 5.9 '37
"S, Longitude 50° 10'2 56" (Ponta Grossa - PR). Fruits
were harvested immature (> 75 % of the peel green),
semi-ripe (> 75 % with peel of an orange colour) and
ripe (100 % of the peel red in colour).
Extraction of anthocyanin: Parts of fruit (peel and pulp)
were separated and blanched and the solubles were ex-
tracted from them by vacuum filtration, with an acidified
methanol mixture (99:1, MeOH: HCl conc.) with
Whatman N.2 paper in a Büchner funnel. The extract was
concentrated under vacuum and at low temperature up
to 10 % of the initial volume, filtered in 0.45 µm mem-
brane (Millipore) and stored in amber bottles at 4.0 ± 1.0
°C (Lees, Francis, 1972).
Extraction of total phenol: Parts of fruit (peel and pulp)
were extracted with the mixture (80:20:1, 70 ° ethanol:
distilled water: 3 % formic acid), and subjected to meas-
urement of phenolic compounds and antioxidant activity
(Ferric Reducing Ability of Plasma – FRAP).
RAW MATERIAL
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224 November/December 2013
RAW MATERIAL
Processing of juice: The raw materials were washed in
running water and the badly damaged specimens were
rejected.
Those that remained were then crushed in a mixer and
the juice was extracted by centrifugal forces at 10,000
rpm for 15 minutes.
Chemical analyses: The phenolic compounds were
quantified by the Folin-Ciocalteu reagent using catechin
as standard (200 g/mL) (Singleton, Rossi, 1965). The
quantification of total monomeric anthocyanin was de-
termined by pH differential (Giusti; Wrolstad, 2001) and
the results expressed in mg of cyanidin 3-glycoside/100g
fruit. For antioxidant activity the FRAP methodology as
described by Benzie and Strain (1996) and by Pulido,
Bravo and Saura-Calisto (2000) was used. The results
were expressed as mMol of the total equivalent antioxi-
dant capacity (TEAC) per g of sample using Trolox stan-
dard (1000 mol).
Colorimetric analysis: The parameters of colour devel-
oped by Glories (1984) were used for the evaluation of
chromatic characteristics of the extracts as in the follow-
ing formula:
Global colour hue of sample: H
(A420 m/A520 m)
Intensity of colour of sample: IC
(A420 m+A520 m+A620 m)
Statistical analyses: The results of the experimental
procedures were analysed using analysis of variance
(ANOVA) with 95% reliability, and if there were differ-
ences these were distinguished by differential Tukey tests
at 5 % probability using STATISTICA 7.0 for Windows
(Statsoft, Inc.) statistical software.
RESULTS AND DISCUSSION
Evolution of chromatic parameters at the different
stages of maturation
Figure 2 shows the spectrophotometric characteristics of
the different fruit extracts (anthocyanin and phenol) and
of the acerola juice in different stages of maturation. The
absorbance readings of A420nm, A520nm and A620nm
allow a quantitative determination of yellow, red and
blue colouring, respectively (GLORIES, 1984). According
to Lima et al., (2012) colour is an attribute of quality in
fruit that can vary with the harvest season due to the
stage of maturation and exposure to the sun. The instru-
mental determination is interesting for the study of vari-
ation and comparison of the pigments present in fruit.
Figure 2 (A) shows the characteristics in relation to the
anthocyanin extracts, and it can be seen that the highest
levels of yellow and red colours are concentrated in the
skin, whereas in the pulp the highest levels are for the
colouring of blue. Figure 2 (B) shows the characteristics
of the phenolic extracts, and regarding the extract of an-
thocyanin, it was found that higher contents of yellow
and red were concentrated in the skin, while the blue
colouring varied for the different samples. The results
from juices are shown in Figure 2 (C) and it can be seen
that all the higher levels of colouration were found in the
juice produced from ripe fruit, but there was no signifi-
cant difference between that produced from semi-ripe
fruit. It can be concluded that for the production of
acerola juice, as all the extraction of pigmentation occurs
with semi-ripe berries they can be considered as mature
from an industrial point of view. Although they are a
good source of anthocyanin, the skin of the fruits is not
even mentioned in textbooks (Matta, Cabral, 2012).
Figure 1. Spectrophotometric characteristics of the acerola samples and juice (A) anthocyanin extract; (B) phenols extract; (C) juice.
Note: (A) I: semi-ripe skin; II: ripe skin; III: semi-ripe pulp; IV: ripe pulp. (B) I: ripe skin; II: semi-ripe skin; III: immature skin; IV: immature pulp;
V: semi-ripe pulp; VI: ripe pulp; (C) I: ripe; II: semi-ripe; III: immature.
(A) (B) (C)
November/December 2013 225
RAW MATERIAL
After completing the spectrophotometric characteristics
(Figure 1), Table 1 shows the characteristics of hues (H)
and colour intensity (CI). Regarding the anthocyanin ex-
tract, it was observed a significant difference between
the hue of the skin of ripe and semi-ripe fruits but there
was no such difference for the pulp. Values were not de-
termined for the immature fruit because at this stage of
maturation it was not possible to extract anthocyanins.
In the colour index there was no significant difference in
the extracts of the skin, whilst the semi-ripe pulp showed
the highest levels.
The phenolic extracts showed significant differences be-
tween them for the different stages of maturation for
both skin and for the pulp, with the highest hue in the
skin for the immature fruit, and in both the immature
and semi-ripe for the pulp. The juices produced were not
different significantly in hue between
ripe and semi-ripe and they were sig-
nificantly colour index in the differ-
ent stages.
According to Vendramini and Trugo
(2000) the colour of the fruit is not
only a sign of transformation of pig-
ments in the skin and pulp, but is also
correlated with complex biochemical
changes during the ripening of fruits.
Effect of different maturation
stages in levels of anthocyanins,
total phenolics and antioxidant
activity.
Table 2 presents the results of the lev-
els of anthocyanin, phenolic com-
pounds and antioxidant activity of
acerola fruit in three different stages
of maturation. The highest levels of
total monomeric anthocyanin (TMA)
in the skin were for semi-ripe fruit
with 23.03±3.91 mg/100g, while the
anthocyanin levels in the pulp were
not different significantly in the dif-
ferent stages of maturation. The se-
mi-ripe fruit also showed the highest
levels for total anthocyanin in fruits
with 24.45±27.30 mg/100g.
The levels of anthocyanin both in
semi-ripe and ripe fruit were within
the results presented by Lima et al.
(2000), where in six different selec-
tions of acerola (all considered
Anthocyanin extracts Phenol extracts Juices
Samples HCI HCI HCI
Ripe
CES 2.01±0.01
a
22.13±0.25
a
2.39±0.30
b93.86±2.70a– –
CEP 2.35±0.45
A
10.32±0.45
B
2.19±0.00
B
49.11±0.21
C– –
Total 4.36±0.45 32.45±0.69 4.58±0.30 142.97±2.4
9
3.39±0.26
a
45.28±1.97
A
Semi-ripe
CES 1.65±0.10
b
29.26±3.66
a
2.49±0.06
b77.25±1.05b– –
CEP 1.97±0.04
A
13.46±0.70
A
2.30±0.00
A
61.27±0.02
A– –
Total 3.62±0.12 42.72±4.23 4.79±0.06 138.52±1.03 3.17±0.00a 38.99±0.94
B
Immature
CES 2.92±0.04
a
75.19±0.52
b– –
CEP 2.29±0.02
A
60.09±0.45
B– –
Total 5.21±0.02
135.28±0.0
7
0.84±0.00
b3.87±0.29C
mature by the authors) the anthocyanin content ranged
from 14.06 to 50.98 mg/100g.
Acerola skin, which is generally rejected in the processing
stage of the fruit, has a high content of anthocyanin and
this could be exploited as a great potential source of pig-
ment (VENDRAMI; TRUGO, 2004). The index of total phe-
nols (ITP) decreased with the advancement of fruit ripen-
ing, and the green fruits showed the highest levels in
parts (skin and pulp) and in total. No significant differ-
ence was observed between the ripe and semi-ripe fruit.
As reported by de Lima et al. (2005), this phytochemical
content also decreased in a non-linear manner during
the maturation period of acerola fruits.
The results of antioxidant activity (FRAP) as well as the
total phenols were found in the skin, in agreement with
TABLE 1. RESULTS OF COLOUR HUE (H) AND COLOUR INTENSITY (CI) OF THE EXTRACTS
OF ANTHOCYANINS AND PHENOLICS OF DIFFERENT STAGES OF MATURATION OF
ACEROLA FRUITS
Note: H= hue (A420nm/A520nm); CI= colour intensity (A420nm+A520nm+A620nm).
CES=crude extract of skin; CEP=crude extract of pulp. Values in the columns with same letters
are not significantly different (p<0.05). Letters subscribed (Lower case letters refer to CES, and
capital letters refer to CEP) and letters not subscribed refer to juices.
226 November/December 2013
RAW MATERIAL
Wolfstädter (2009). The highest levels were found in
the skin, with the immature fruit having the
highest activity (340.51±45.15mMol/g) and the pulp
(237.13±0.84mMol/g). The content of phenolic com-
pounds and antioxidant activity are particularly higher in
the skin than the pulp of many fruits (Lima et al., 2005;
Mezadri et al., 2008).
Thaipong et al. (2006) estimated the antioxidant activity
of guava fruits and found that the method of determin-
ing the antioxidant activity by FRAP was the most repro-
ducible technique and was the one that showed a higher
correlation between ascorbic acid and phenolic com-
pounds because results of anthocyanin, total phenolic
compounds and antioxidant activity of acerola juice in
three different stages of maturation.
Table 3 shows the results of anthocyanins, total phenolic
compounds and antioxidant activity of acerola juice in
three different stages of maturation.
The highest level of anthocyanin found for the acerola
juice was for ripe fruit with a value of 20.67±2.74
g/100mL. This was probably in relation to the pulp and
the longer period of time that it was in contact with the
skin, allowing the release of anthocyanin in the middle of
the fruit, which is mainly found in the skin (MEZADRI et
al., 2008). As was the case for the fruits, the index values
of total phenols and antioxidant activity (FRAP) showed
the highest level for the juice of the unripe fruit, with the
values as high as 272.16±10.33 mg/100mL and
1097.09±57.61 mMol/mL, respectively.
CONCLUSION
The chromatic parameters confirmed the homogeneity of
the groups of samples from immature, semi-mature and
mature acerola, made by visual evaluation regarding the
skin and pulp.
The increasing degree of maturity, indicated by spec-
trophotometric characteristics, occurs simultaneously
with decreased
amounts of phyto-
chemicals agents
and the loss of an-
tioxidant activity.
The immature fruits
showed the highest
levels of total phe-
nolic compounds
and antioxidant ac-
tivities, but no dif-
ferences were found
between the mature
and semi-mature
Anthocyanin extracts Phenol extracts
Samples TMA ITP FRAP
Ripe
CES 16.39±0,74b118.52±3.26b276.37±16.46ab
CEP 1.69±0.38A102.44±2.71B126.58±1.69C
Total 18.08±0.78b 220.97±5.27b 402.95±14.77b
Semi-ripe
CES 23.03±3.91a122.24±0.84b246.84±15.61b
CEP 1.43±1.28A106.72±2.20B154.01±2.95B
Total 24.45±2.73ª228.96±2.24b 400.84±18.57b
Immature
CES nd 128.96±009a340.51±45.15a
CEP nd 123.21±0.31A237.13±0.84A
Total nd 252.16±0.32a 577.64±44.30a
Note: TMA= mg/100g; ITP= mg/100g; FRAP = mMol of TEAC/g of fruit, nd = not deter-
mined. CES=crude extract of skin; CEP=crude extract of pulp. Values in the columns with
same letters are not significantly different (p<0.05). Letters subscribed (Lower case letters
refer to CES, and capital letters refer to CEP) and letters not subscribed refer to total.
TABLE 2. RESULTS OF TOTAL MONOMERIC ANTHOCYANINS (TMA), INDEX OF
TOTAL PHENOLS (ITP) AND ANTIOXIDANT ACTIVITY (FRAP) OF DIFFERENT
MATURITY STAGES OF ACEROLA FRUIT
Anthocyanin extracts Phenol extracts
Fruit juices TMA ITP FRAP
Ripe 20.67±2.74a 147.58±1.08b 737.22±9.06c
Semi-ripe 9.23±0.77b 147.63±1.73b 828.16±18.45b
Immature 0.29±0.04c 272.16±10.33a 1097.09±57.61a
TABLE 3. RESULTS OF TOTAL MONOMERIC ANTHOCYANIN (TMA), INDEX OF
TOTAL PHENOLS (ITP) AND ANTIOXIDANT ACTIVITY (FRAP) OF DIFFERENT
MATURITY STAGES OF ACEROLA JUICE
Note: TMA= mg/100g; ITP= mg/100g; FRAP = mMol of TEAC/g of fruit. Values in the
columns with same letters are not significantly different (p<0.05).
Fig. 3: Juices of different stages of acerola ripening (Malpighia glabra L.).
November/December 2013 227
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samples. The semi-mature fruits showed the highest total
monomeric anthocyanin levels. The highest levels of an-
thocyanin were found in the juice of ripe fruits, a fact
linked to the process that causes the skin and the flesh to
remain longer in contact. The phenolic compounds and
antioxidant activity of acerola and its products can be
better used if they are consumed at a semi-mature stage.
ACKNOWLEDGEMENTS
The authors are deeply grateful to CNPq and CAPES for
the scholarships granted, and also to the University for
the use of the laboratory park, with scientific equipment
recently acquired. They also congratulate the University
for adopting a multi-user philosophy.
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RAW MATERIAL
AUTHORS
Suelen Avila, Acácio Antonio Ferreira Zielinski ,
Caroline Goltz, Alessandro Nogueira, Gilvan Wosiacki.
State University of Ponta Grossa – Brasil
wosiacki@uol.com.br
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This text is a comprehensive reference covering the chemistry, physiology, chemotaxonomy, biotechnology and food technology aspects of the anthocyanins. Topics discussed include types of anthocyanins, structural transformations, colour stabilization and intensification factors, biosynthesis and intensification factors, biosynthesis, analysis and functions of anthocyanins. An in-depth review of the literature discussing anthocyanins of fruits, cereals, legumes, roots, tubers, bulbs, cole crops, oilseeds, herbs, spices, and minor crops is included as well.
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