LWT - Food Science and Technology 85 (2017) 158e169

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LWT - Food Science and Technology

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

Comparison and optimization of conventional and ultrasound assisted

extraction for bioactive compounds and antioxidant activity from
agro-industrial acerola (Malpighia emarginata DC) residue
Yara Rafaella Ribeiro Santos Rezende, Juliete Pedreira Nogueira, Narendra Narain*
~o Cristo
Laboratory of Flavor and Chromatographic Analysis, PROCTA, Federal University of Sergipe, 49100-000, Sa ~o, SE, Brazil
va

a r t i c l e i n f o a b s t r a c t

Article history: The acerola (Malpighia emarginata DC) residue resulting from the processing of pulp and juice possesses
Received 6 June 2017 high concentrations of bioactive compounds. The use of this residue could increase its commercial value
Received in revised form and hence result in the profitability of processing. This study was aimed to compare the conventional
10 July 2017
(CE) and ultrasound assisted extraction (UAE) for bioactive compounds and antioxidant activity from
Accepted 11 July 2017
acerola residue and to optimize the best extraction method using the response surface methodology
Available online 12 July 2017
(RSM) and desirability function. The selection of the extraction method was based on designing exper-
iments. A central composite rotatable design (CCRD) was employed to investigate the effects of eight
Keywords:
Acerola residue
independent variables. The extracts obtained by UAE contained higher concentrations of bioactive
Ultrasound assisted extraction compounds and antioxidant activity. Ethanol concentration (X1), ethanol-residue ratio (X2) and extrac-
Bioactive compounds tion time (X3) significantly affect response variables. The quadratic model was fitted for most responses.
Antioxidant activity The ethanol concentration of 46.49%, ethanol-residue ratio of 8.66 mL/g and extraction time of 49.30 min
Response surface methodology were standardized as optimal conditions. The optimized conditions model showed a good correlation
between the predicted and experimental values. Overall, these results demonstrate the efficacy and
better utility of agro-industrial acerola residue in the form of extraction of bioactive compounds with
better antioxidant activity.
© 2017 Elsevier Ltd. All rights reserved.

1. Introduction
Acerola (Malpighia emarginata DC) is a fruit native to Central
America and Northern South America, with some of its largest
plantings in Brazil (Malegori et al., 2017). Of late, Brazil has
expanded its areas of cultivation of acerola fruit, which is largely
marketed as fresh fruits, pulp and juice (Rodriguez-Amaya, 2016).
The demand for this fruit has grown, mainly due to its rich contents
of ascorbic acid (AA), carotenoids (CA) and phenolic compounds
(PC), besides its high antioxidant activity (Jaeschke, Marczak, &
Mercali, 2016; Mezadri, Villan ~ o, Fernandez-Pacho n, García-
Parrilla, & Troncoso, 2008).
The production of acerola juice or pulp leads to the generation of
a deep red residue which is about 40% of the fruit volume and is
often discarded, generating large volumes of organic residue
(Marques, Prado, & Freire, 2009). The high contents of the total
* Corresponding author.
E-mail address: narendra.narain@gmail.com (N. Narain).
anthocyanins (TA), total flavonoids (TF) and PC present in the some
fruit residue indicate that the residue could be better utilized (Silva
et al., 2014). The extraction and processing of compounds of in-
terest present in the acerola residue could increase the commercial
value of the raw material processing and its profitability.
Extraction represents the first step to obtain bioactive materials
from a plant and several methods are known to obtain these
compounds from peel wastes (Garcia-Castello et al., 2015). These
include conventional extraction (CE), ultrasound assisted extrac-
tion (UAE), microwave assisted extraction, pressurized liquid
extraction and supercritical fluid extraction (Barba, Zhu, Koubaa,
Sant’Ana, & Orlien, 2016).
Most of these methods have some limitations, such as use of
toxic solvents (health-related risks), high temperatures and long
extraction times (degradation of compounds of interest) (Garcia-
Castello et al., 2015). UAE is a potential alternative to CE of me-
tabolites from plants, and is largely applied since it reduces use of
toxic compounds and requires lower temperatures and time pe-
riods. Therefore, this method as one of the most efficient, cheap,
simple, fast and with greater reproducibility (Chemat, Zill-e-Huma,

http://dx.doi.org/10.1016/j.lwt.2017.07.020
0023-6438/© 2017 Elsevier Ltd. All rights reserved.
 Y.R.R.S. Rezende et al. / LWT - Food Science and Technology 85 (2017) 158e169
& Khan, 2011). For the extraction of bioactive compounds, in
particular PC from different plant materials using UAE, the ethanol
is considered to be the most suitable solvent as it is classified as
generally recognized as safe for application in food systems (Chen,
Zhao, & Yu, 2015).
Some studies on acerola have been published which report on
antioxidant compounds (Mezadri et al., 2008), different ripening
stages (Oliveira, Moura, De Brito, Mamede, & De Miranda, 2012),
pulp dehydration (Marques et al., 2009) and effects of ohmic
heating (Jaeschke et al., 2016). However, there is no work yet
published on extraction of the bioactive compounds and determi-
nation of antioxidant activity from acerola residue. Thus, the
objective of this study was to compare CE and UAE of bioactive
compounds and antioxidant activity from acerola residue and to
determine the optimum extraction conditions using response sur-
face methodology (RSM) and desirability function.
2. Materials and methods
2.1. Residue and pulp samples
The acerola (Malphigia emarginata DC) pulp and residue were
obtained from a fruit pulp processing factory, located in the city of
Aracaju, Brazil. The residue (seed and peel) was taken to the Lab-
oratory of Flavor and Chromatographic Analysis of the Federal
University of Sergipe, where it was mixed in a 1:1 (seed:peel, m/m)
proportion in a Mixer/Grinder (Morphy Richards Divo, India). The
pulp (integral and pasteurized) and triturated residue were packed
in polyethylene bags and stored in a freezer maintained at
18  C.
2.2. Chemicals and reagents
Ascorbic acid, 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic
acid) (ABTS), 2,2- diphenyl-1-picrylhydrazyl (DPPH), Folin-
Ciocalteu phenol reagent, gallic acid, quercetin, 2,4,6-tripyridyl-s-
triazine (TPTZ) and 6-hydroxy-2,5,7,8-tetramethylchroman-2-
carboxylic acid (Trolox) were obtained from Sigma-Aldrich (St.
Louis, Missouri, United States). 2,6-Dichlorophenol-indophenol
(DCPIP), ethanol, ferric chloride, hydrochloric acid and oxalic acid
were obtained from Vetec Química Fina Ltda (Duque de Caxias, Rio
de Janeiro, Brazil). All chemicals were of analytical grade or of HPLC
purity standard.
2.3. Preliminary analysis
In order to optimize the extraction conditions, initially pre-
liminary extractions were carried out using CE and UAE. The ex-
tractions were performed at specified conditions, 65% ethanol
acidified to pH 2 by using hydrochloric acid (2 M) and ethanol-
residue ratio of 5 mL/g. The extract was filtered using Whatman
N 2 filter paper, concentrated in a rotary evaporator (Quimis,
Q344B) at 55  C until removal of approximately 80% of the
extraction solvent occurred. The extracts were stored in amber
bottle which were kept in a freezer (
18  C) until analysis.
2.3.1. Conventional extraction (CE)
2.3.1.1. Agitation in shaker (AGS). The extracts were prepared using
a shaker (SL 222/Solab) maintained at 30  C for 3 h with an agita-
tion at 150 rpm.
2.3.1.2. Maintenance in refrigerator (MRG). The extraction was
carried out by maintaining the samples in a refrigerator at 8  C for
16 h, without light.
159
2.3.2. Ultrasound assisted extraction (UAE)
The extracts were prepared by dissolution in ultrasound (Ecel/3L
Alpha Plus) at 30  C for 30 min, working at 50 kHz of frequency and
250 VA of power.
2.4. Experimental design
Optimization experiments for extraction of bioactive com-
pounds and evaluation of antioxidant activity from acerola residue
were carried out and results were analyzed by using RSM. A central
composite rotatable design (CCRD) consisting of 17 experimental
runs (8 factorial points, 6 axial points and 3 central points). The
extraction variables were ethanol concentration (X1, %), ethanol-
residue ratio (X2, mL/g), extraction time (X3, min) as presented in
Table 1. Values for the extraction variables were established based
on previous information found in the revised literature about
extraction of bioactive compounds.
2.5. Extraction procedure
The acerola residue was mixed with ethanol (0e99.5% acidified
to pH 2 by using hydrochloric acid, 2 M) in different concentrations
(1e10 mL/g) and extracted on ultrasound for different times
(10e60 min). The extract obtained was filtered through Whatman
N 2 filter paper and later the liquid was concentrated on a rotary
evaporator (Quimis, Q344B) at 55  C until removal of approxi-
mately 95% of the extraction solvent occurred. The dried extract
was resuspended in water and acidified to pH 2 by using hydro-
chloric acid (2 M). The extracts were conditioned in amber bottle
which were stored in a freezer (
18  C).
2.6. Analysis of bioactive compounds
2.6.1. Total anthocyanins (TA) content
The extracts obtained by the methods described earlier (pre-
liminary analysis and extraction procedure) were analyzed. The
absorbance was measured in a spectrophotometer (Jenway 6705
UV/Vis) at 535 nm (Moo-Huchin et al., 2015). The result was
expressed as mg total anthocyanins (TA)/100 g of residue.
2.6.2. Carotenoids (CA) content
The CA content was determined according to the method
described by Lichtenthaler (1987). The absorbance was measured
in a spectrophotometer (Jenway 6705 UV/Vis) at 454 nm and the
result was expressed in mg b-carotene equivalent (b-CE)/g of
residue.
2.6.3. Ascorbic acid (AA) content
The AA content was determined by the methodology described
by Benassi and Antunes (1988) which uses 2% oxalic acid as
extraction solution and DCPIP as titrant solution. The result was
expressed in mg ascorbic acid (AA)/100 g of residue.
2.6.4. Phenolic compounds (PC) content
The PC content were determined by using Folin-Ciocalteu re-
agent according to the method described by Singleton and Rossi
(1965). The PC content was calculated using a standard curve pre-
pared from the aqueous solutions of gallic acid (0.1e1 mg/mL) and
expressed in mg gallic acid equivalent (GAE)/100 g of residue.
2.6.5. Total flavonoids (TF) content
The TF content was determined according to the methods
described by Gonza lez-Aguilar, Villegas-Ochoa, Martínez-Te
llez,
Gardea, and Ayala-Zavala (2007). The absorbance was measured at
415 nm, using a spectrophotometer (Jenway 6705 UV/Vis). The TF
Table 1

160
A central composite rotatable design for three-variables and five-levels and observed responses under different experimental conditions on bioactive compounds and antioxidant activity of acerola residue.

Experiment No. Extraction conditions Bioactive compounds Antioxidant activity

X1 X2 X3 TA CA AA PC TF ABTS DPPH FRAP

(%) (mL/g) (min) (mg TA/100 g) (mg b-CE/g) (mg AA/100 g) (mg GAE/100 g) (mg QE/100 g) (mM TE/g) (mM TE/g) (mM TE/g)

1 79.4 (1) 8.18 (1) 50 (1) 23.28 ± 0.17a 7.29 ± 0.08 d 443.02 ± 45.14e 1183.70 ± 7.07b 556.47 ± 3.67b 109.90 ± 4.39e 180.28 ± 1.29a 275.00 ± 2.10c
2 20.1 (
1) 8.18 (1) 50 (1) 3.56 ± 0.03g 0.86 ± 0.07l 450.83 ± 45.93e 966.96 ± 5.89f 360.40 ± 1.92g 134.20 ± 0.33c 128.06 ± 0.82e 236.93 ± 0.00f
3 79.4 (1) 2.82 (
1) 50 (1) 5.30 ± 0.06f 4.90 ± 0.27f 440.49 ± 54.50e 775.77 ± 7.82h 317.26 ± 0.48h 119.30 ± 1.42d 93.71 ± 0.70h 193.87 ± 2.31h
4 20.1 (
1) 2.82 (
1) 50 (1) 2.50 ± 0.02 i 5.40 ± 0.13e 422.24 ± 45.71e 723.23 ± 5.91i 241.26 ± 2.30jl 80.59 ± 0.17h 106.27 ± 0.79g 184.28 ± 0.00hi
5 79.4 (1) 8.18 (1) 20 (
1) 12.87 ± 0.12c 1.67 ± 0.03ij 451.75 ± 46.03e 1279.70 ± 29.48a 408.29 ± 7.35i 103.17 ± 4.57f 173.22 ± 1.95b 255.10 ± 6.43d
6 20.1 (
1) 8.18 (1) 20 (
1) 3.11 ± 0.02h 1.51 ± 0.03j 437.68 ± 44.59e 906.43 ± 0.94g 246.83 ± 0.55j 100.75 ± 1.76f 121.96 ± 1.07f 610.21 ± 4.31a
7 79.4 (1) 2.82 (
1) 20 (
1) 7.35 ± 0.04e 11.02 ± 0.07c 621.36 ± 77.67cd 700.85 ± 7.04i 223.05 ± 4.71m 88.88 ± 1.43g 129.84 ± 0.32e 213.35 ± 3.25g
8 20.1 (
1) 2.82 (
1) 20 (
1) 1.43 ± 0.01l 12.32 ± 0.12b 374.45 ± 46.33e 550.80 ± 0.49j 160.97 ± 1.60 70.38 ± 0.85h 124.05 ± 1.16f 112.41 ± 0.33l
9 99.5 (1.68) 5.5 (0) 35 (0) 7.60 ± 0.04e 3.92 ± 0.07g 618.32 ± 46.56cd 902.28 ± 8.32g 282.97 ± 1.98i 82.47 ± 1.66i 102.79 ± 1.73g 161.26 ± 2.66j
10 0 (
1.68) 5.5 (0) 35 (0) 2.77 ± 0.02i 1.82 ± 0.02i 785.92 ± 78.59ab 777.68 ± 3.29h 227.61 ± 6.50lm 82.51 ± 0.51h 154.77 ± 1.41c 175.93 ± 3.54i
11 49.75 (0) 10 (1.68) 35 (0) 18.81 ± 0.23b 0.89 ± 0.03l 104.53 ± 45.26f 1119.78 ± 14.42c 582.86 ± 5.92a 155.63 ± 2.06a 181.78 ± 2.34a 282.89 ± 5.97c
12 49.75 (0) 1 (
1.68) 35 (0) 1.94 ± 0.00j 18.27 ± 0.17a 809.27 ± 80.93a 469.73 ± 5.17l 181.70 ± 0.61n 63.56 ± 0.66j 51.78 ± 0.05i 156.81 ± 1.45j
13 49.75 (0) 5.5 (0) 60 (1.68) 13.01 ± 0.07c 1.76 ± 0.03ij 644.66 ± 50.75bcd 1046.39 ± 11.58e 518.21 ± 5.95c 143.85 ± 2.06b 147.04 ± 0.54d 307.70 ± 1.60b
14 49.75 (0) 5.5 (0) 10 (
1.68) 5.56 ± 0.04f 3.27 ± 0.00h 507.71 ± 46.28de 968.16 ± 9.89f 385.11 ± 5.96f 115.99 ± 1.54de 146.90 ± 1.13d 242.65 ± 4.16ef
15 49.75 (0) 5.5 (0) 35 (0) 10.75 ± 0.08d 3.04 ± 0.07h 686.88 ± 45.76abc 1084.50 ± 11.59d 493.30 ± 5.81d 132.31 ± 1.58c 148.07 ± 1.80d 247.42 ± 2.89de
16 49.75 (0) 5.5 (0) 35 (0) 10.96 ± 0.10d 3.09 ± 0.04h 655.96 ± 45.45abcd 1104.94 ± 3.36cd 497.41 ± 5.39d 135.91 ± 0.44c 146.93 ± 0.72d 253.50 ± 3.50d
17 49.75 (0) 5.5 (0) 35 (0) 10.98 ± 0.05d 3.02 ± 0.03h 663.03 ± 45.94abcd 1076.71 ± 8.32de 514.49 ± 7.12c 132.79 ± 1.76c 148.51 ± 1.70d 252.49 ± 3.50de

X1 ¼ Ethanol concentration, X2 ¼ Ethanol-residue ratio, X3 ¼ Extraction time, TA ¼ Total anthocyanins, CA ¼ Carotenoids, AA ¼ Ascorbic acid, PC ¼ Phenolic compounds, TF ¼ Total flavonoids, ABTS ¼ ABTSþ scavenging activity,
DPPH ¼ DPPH radical scavenging activity, FRAP ¼ Ferric reducing antioxidant power.Values are expressed as mean ± standard deviation. Means followed by the same lower case letters on the columns do not differ significantly
by Tukey's test at 5% probability.

acerola pulp.
2.7.1. ABTS assay

2.7.3. FRAP assay

2.7.2. DPPH assay
(QE)/100 g of residue.

in mM TE/g of residue.

2.9. Statistical analysis

2.7. Antioxidant activity
Y.R.R.S. Rezende et al. / LWT - Food Science and Technology 85 (2017) 158e169

2.8. Validation of the model

and the independent variables.

Trolox equivalent (TE)/g of residue.

(0e0.3 mg/mL) and result expressed in mM TE/g of residue.

(R2) of the model. The response variables were fitted to the

response variables were calculated. The significance of the
interaction effects of the three variables considered on the
standard curve prepared of Trolox (0e0.15 mg/mL) and expressed
(Jenway 6705 UV/Vis). Antioxidant activity was calculated from a
ity was calculated using a standard curve prepared of Trolox
measured from t ¼ 0 min to t ¼ 30 min of reaction, using a
to the methodology described by Brand-Williams, Cuvelier, and
The DPPH radical scavenging activity was performed according
UV/Vis). The antioxidant activity was calculated using a standard

which was able to describe relationship between the responses

All the bioactive compounds and antioxidant activity analyses
The ABTS radical cation scavenging activity was conducted
cetin (0.05e0.5 mg/mL) and expressed in mg quercetin equivalent

value (p  0.05) and the value of the coefficient of determination

generated models was tested by analysis of variance (ANOVA), F
mental data of CCRD were submitted to RSM using the software
any difference between the means of the treatments. Experi-
extraction time). The experimental values were compared with
tained using the desirability function (Derringer & Suich, 1980).
Boonprakob, Crosby, Cisneros-Zevallos, and Byrne (2006). The
ducted according to the methodology reported by Thaipong,

following second order polynomial model equation (Equation (1))

for extraction of acerola residue were also used for extraction of
idity of the model. Moreover the optimized identical conditions
All the responses were determined under optimized conditions of
antioxidant activity (DPPH, ABTS, FRAP) based on the values ob-
The ferric reducing antioxidant power (FRAP) assay was con-
measured at 734 nm, using a spectrophotometer (Jenway 6705
content was calculated using a standard curve prepared of quer-

Statistica 12.0 (StatSoft Inc., Tulsa, USA). Linear, quadratic and

were analyzed in triplicate and results were expressed as
predicted values based on CV% in order to authenticate the val-
The optimized conditions were validated for the maximum

the extraction (ethanol concentration, ethanol-residue ratio and

extraction of bioactive compounds (TA, CA, AA, PC and TF) and
Berset (1995). The decrease in absorbance at 515 nm was
according to the methodology described by Miller, Rice-Evans,

curve prepared of Trolox (0.05e0.35 mg/mL) and expressed in mM

Davies, Gopinathan, and Milner (1993). The absorbance was

mean ± standard deviation. The Tukey's test was performed to test

spectrophotometer (Jenway 6705 UV/Vis). The antioxidant activ-

absorbance was measured at 593 nm, using a spectrophotometer

 Y.R.R.S. Rezende et al. / LWT - Food Science and Technology 85 (2017) 158e169
X
k X
k X
k
Y ¼ b0 þ bi Xi þ bii Xi2 þ bij Xi Xj
i¼1 i¼1 isj¼1
where, Y is the response variable, Xi and Xj are the independent
variables, and k is the number of tested variables (k ¼ 3). Regression
coefficient is defined as b0 for intercept, bi for linear, bii for quadratic
and bij for interaction.
Multiple response optimization was performed through the use
of the desirability function (D) (Derringer & Suich, 1980) by using
the Statistica 12.0 software, in order to find the extraction condi-
tions leading to compromise levels of the compounds analyzed.
Desirability was obtained by using Equation (2).
D ¼ ðd1 ðY1 Þ:d2 ðY2 Þ…di ðYi ÞÞ1=i
where di (Yi) are the normalized values (from 0 to 1) of each of the
studied responses.
3. Results and discussion
3.1. Preliminary experiments - extraction method selection
Selection of the best extraction method was based on the values
of bioactive compounds and antioxidant activity of the extracts.
Among all extraction methods used (AGS, MRG and UAE), the UAE
showed better results in TA content (20.3 ± 0.1 mg TA/100 g), CA
(0.54 ± 0.03 mg b-CE/100 g), AA (489 ± 26 mg AA/100 g), PC
(1034 ± 12 mg GAE/100 g), TF (405 ± 14 mg QE/100 g), and the
highest antioxidant activity by assays of ABTS (179.8 ± 0.6 mM TE/g),
DPPH (155 ± 3 mM TE/g) and FRAP (334 ± 3 mM TE/g) compared
with other extraction methods (Fig. 1).
The strong influence of the UAE on the high contents of bioac-
tive compounds and antioxidant activity of acerola residue extracts
can be attributed to disruption of cell walls, reducing particle size
and improving the mass transfer from the cell to the solvent
resulting in the collapse of the bubble produced by cavitation which
Fig. 1. Effect of different extraction methods: agitation in shaker (AGS), maintenance in
refrigerator (MRG) and ultrasound assisted extraction (UAE) of acerola residue on
concentrations of bioactive compounds (TA, CA, AA, PC, TF) and antioxidant activity
measured by assays of ABTS, DPPH and FRAP.
TA ¼ Total anthocyanins, CA ¼ Carotenoids, AA ¼ Ascorbic acid, PC ¼ Phenolic com-
pounds, TF ¼ Total flavonoids, ABTS ¼ ABTSþ scavenging activity, DPPH ¼ DPPH
radical scavenging activity, FRAP ¼ Ferric reducing antioxidant power. Means followed
by the same letter on the bars of equal colors do not differ significantly by Tukey's test
at 5% probability. (For interpretation of the references to colour in this figure legend,
the reader is referred to the web version of this article.)
161
consequently results in an accelerated release of target compounds
(Xu et al., 2017).
(1)
Song et al. (2014), Garcia-Castello et al. (2015) and He et al.
(2016) also reported in their studies the efficacy of UAE which
was more effective in extraction of TF from pine (Larix olgensis), TA
and PC from grapefruit (Citrus paradisi L.) and TA and PC from
blueberry (Vaccinium ashei) residue, respectively, as compared to
other CE.
3.2. Determination of process parameters on extraction and fitting
of the model
Three parameters viz. ethanol concentration (X1), ethanol-
residue ratio (X2) and extraction time (X3) were chosen to vary
their conditions in UAE and their effects evaluated on extraction of
(2)
bioactive compounds and antioxidant activity. The results are
presented in Table 1. It could be observed that the TA content varied
between 1.4 and 23.3 mg TA/100 g, CA between 0.9 and 18.3 mg b-
CE/g, AA between 104 and 809 mg AA/100 g, PC between 470 and
1280 mg GAE/100 g and TF between 161 and 583 mg QE/100 g, as
well as the antioxidant activity determined by the ABTS between 64
and 156 mM TE/g, DPPH between 52 and 424 mM TE/g and FRAP
between 112 and 610 mM TE/g.
The three data on central points (ethanol concentration of
49.75%, ethanol-residue ratio of 5.5 mL/g and extraction time of
35 min) showed no significant difference (p  0.05) in their re-
sponses which indicate a good repeatability of the extraction
process.
The significance probabilities of the analyzed effects are detailed
in Table 2, which describe that the variables used in the extraction
process influenced the extraction of bioactive compounds, as well
as the antioxidant activity of these extracts. The parameters were
considered significant with p-value less than 5% (p  0.05), but
because of the large variability of the extraction process, some
specific cases such as linear effect X3 (PC), quadratic effect X21 (TA),
X22 (ABTS) and interactions X1X2 (TA and ABTS) and X1X3, X2X3 (PC)
were considered significant between 0.05  p  0.1. It is noted that
the linear effect (L) of ethanol-residue ratio (X2) was significant at
p  0.05 for the compounds tested, except for AA, in which no
variable interfered statistically. Meanwhile, for the variables
ethanol concentration (X1) and extraction time (X3), the linear ef-
fect was significant (p  0.05) for TA, PC and TF; and TF and ABTS,
respectively. Higher interaction was observed between the effects
of ethanol concentration and the ethanol-residue ratio (X1X2), and
ethanol-residue ratio with the extraction time (X2X3) for PC and CA,
respectively.
The multiple regression coefficients were calculated for all re-
sponses variables using the method of least square (MLS). The
regression coefficients (b) of the model for each response variable
are presented in Table 2. These values were recalculated after
exclusion of coefficients of non-significant variables to getting the
adjusted model coefficients.
Table 3 also presents the R2 of the models, F values and signif-
icance probabilities (pF) of the lack-of-fit test for each adjusted
model. The optimization of the extraction process was determined
by applying the polynomial equations of second order (Equations
(3)e(9)), which were used to generate response surfaces, as shown
in Fig. 2.
3.2.1. Effect of extraction variables on bioactive compounds
3.2.1.1. TA content. The variables that significantly influenced the
TA were: ethanol concentration (X1, X21), ethanol-residue ratio (X2),
and the interaction between them (X1X2), as shown in Table 2.
However, only the terms X1 (p  0.01) and X1X2 (p  0.1) showed a
positive effect on TA extraction, as confirmed by the adjusted model
162 Y.R.R.S. Rezende et al. / LWT - Food Science and Technology 85 (2017) 158e169

Table 2
Regression coefficient (b) of the predicted second order polynomial models for bioactive compounds and antioxidant activity of acerola residue.

Regression coefficient (b)

TA AA CA PC TF ABTS DPPH FRAP

Intercept
X0 1.0882***
87.9760ns 39.4206***
416.2945***
337.5969***
73.6823*** 116.8803***
72.2658**
Linear
X1 0.1136** 6.2960ns
0.1282 ns 11.1259*** 9.9533* 3.0048ns
0.0566ns 2.4385ns
X2
0.9013** 125.5244ns
7.7480*** 226.7585*** 87.3598*** 27.5759** 10.1463** 104.8979*
X3 0.0368ns 19,9454ns
0.4379ns 17.7880fcs 9.3463* 1.4684*
1.1698ns
3.6746ns
Quadratic
X21
0.0025fcs
0.0163ns 0.0001ns
0.0955***
0.1093***
0.0218**
0.0071ns
0.0229ns
X22
0.0473ns
14.0604ns 0.3385**
13.9175***
7.1273*
1.3346fcs
1.4557ns
0.2805ns
X23
0.0032ns
0.2614ns
0.0003ns
0.1103ns
0.1185ns
0.0107 ns 0.0010 ns 0.0780ns
Interaction
X1X2 0.0327fcs
0.4073ns 0.0132ns 0.6094* 0.3452ns
0.1244fcs 0.1734ns
0.6726ns
X1X3 0.0019ns
0.0704ns 0.0020ns
0.0714fcs 0.0136ns
0.0018ns
0.0049ns 0.0848ns
X2X3 0.0368 ns 0.4275ns 0.0560*
0.8794fcs 0.2713ns
0.0014ns 002086ns
1.2617ns

X1 ¼ Ethanol concentration, X2 ¼ Ethanol-residue ratio, X3 ¼ Extraction time, TA ¼ Total anthocyanins, CA ¼ Carotenoids, AA ¼ Ascorbic acid, PC ¼ Phenolic compounds,
TF ¼ Total flavonoids, ABTS ¼ ABTSþ scavenging activity, DPPH ¼ DPPH radical scavenging activity, FRAP ¼ Ferric reducing antioxidant power. Significance
level ¼ ***p  0.001, **p  0.01, *p  0.05, 0.05 fcsp0.1 (factor considered significant (Rodrigues & Iemma, 2009)), nsp>0.1 (not significant).

(Equation (3)), whose coefficients are significant.
YTA ¼
0:5087 þ 0:1534X1
0:1326X2
0:0022X12
þ 0:0327X1 X2
Since F-value (10.3042) is highly significant (p  0.00001) and
R2 of the model was 77.43%, it is concluded that the model fits well
with the experimental data (Table 3).
Through the response surface generated by the model (Fig. 2 a,
b, c), one can obtain the TA extraction conditions of acerola residue.
It is possible to observe from the surfaces that the ethanol-residue
ratio and its interaction with the ethanol concentration influenced
positively, with 99.5% and 10 mL/g the condition of maximum
extraction while the time factor was not significant. However, Celli,
Ghanem, and Brooks (2015) concluded that the extraction time
positively affected the extraction of TA from haskap berries (Loni-
cera caerulea L.).
The increase in ethanol-residue ratio facilitated access of the
solvent to the solute. He et al. (2016) also observed it in the TA
extraction optimization from blueberry (Vaccinium ashei) residue
using UAE. Furthermore, cavitation is known to produce a number
of mechanical effects, such as particle collisions and rupture of the
cell wall, which promotes the penetration of the solvent inside the
sample matrix and thus increases the TA transfer rates (Chemat
et al., 2011). The increase in ethanol-residue ratio may lead to a
more effective dissolution of constituents.
3.2.1.2. CA content. The following equation describes the CA con-
tent predicted by the model according to the encoded variables. The
model (Equation (4)) contains only statistically significant terms
that are ethanol-residue ratio (X2, X22) and their interaction with
time extraction (X2X3).
YCA ¼ 20:9771
5:0842X2 þ 0:3387X22
0:0014X2 X3
For the second order polynomial equation for CA ðYCA Þ only the
quadratic term of the variable ethanol-residue ratio (X22) showed
positive effect (p  0.01). Table 3 indicates R2 of the model was
85.49% and calculated F-value (25.5334) much higher than the
tabulated value (3.41), suggesting that this model is suitable for
evaluating the CA content with respect to the variation of the
ethanol-residue ratio.
The response surface graphs on effect of interacting variables on
CA contents are presented in Fig. 2 (d, e) where it is observed that
the ethanol concentration and the extraction time did not affect the
CA extraction, but variable ethanol-residue ratio had a negative
(3) influence, because it is reduction favored the extraction, that is,
1 mL/g was the concentration at which the highest CA content was
extracted.
3.2.1.3. PC content. Equation (5) describes the PC content predicted
by the model according to the encoded variables. The equation was
derived based only on statistically significant regression
coefficients.
YPC ¼
261:3368 þ 10:2960X1 þ 215:5276X2 þ 10:0651X3

0:0872X12
12:8965X22
þ0:6094X1 X2
0:0714X1 X3
0:8794X2 X3
(5)
According to the data presented in Table 2, all independent
variables in linear terms, quadratic and interaction, except X23, were
statistically significant for extraction of PC. However, only the terms
X1, X2 (p  0.001), X3 (p  0.1) and X1X2 (p  0.05) showed positive
effect on the extraction, as confirmed by the adjusted model
(Equation (5)). The second order polynomial equation for PC ðYPC Þ
fitted well with the experimental data well and it was highly sig-
nificant (p  0.0001) for calculated F-value of 37.9435 and the R2 of
the model was 97.33%. The surface response graphs are shown in
Fig. 2 (f, g, h).
It is possible to verify through the graphs that the ethanol-
residue ratio positively influenced the PC extraction, being
maximum in the range of 8e10 mL/g. When associated with the
ethanol concentration, the increase in time influenced the extrac-
tion of PC which was also observed by Dranca and Oroian (2016).
This effect is understandable because it is known that longer
extraction time increases the concentration of PC (Escribano-Bailon
(4)
& Santos-Buelga, 2003). However when associated with the
ethanol-residue ratio, this behavior was the opposite. It is therefore
concluded that the influence of time depends on the variable with
which it is correlated.
The increased ethanol concentration also influenced positively
in the extraction, a behavior similar to those reported by Luthria
and Mukhopadhyay (2006). However, concentrations that exceed
the optimum value could cause a slight decrease in the recovery of
PC (Dranca & Oroian, 2016).
 Y.R.R.S. Rezende et al. / LWT - Food Science and Technology 85 (2017) 158e169 163

3.2.1.4. TF content. Through experimental planning by CCRD it was

X1 ¼ Ethanol concentration, X2 ¼ Ethanol-residue ratio, X3 ¼ Extraction time, TA ¼ Total anthocyanins, CA ¼ Carotenoids, AA ¼ Ascorbic acid, PC ¼ Phenolic compounds, TF ¼ Total flavonoids, ABTS ¼ ABTSþ scavenging activity,
DPPH ¼ DPPH radical scavenging activity, FRAP ¼ Ferric reducing antioxidant power. R2 ¼ Coefficient of determination. Significance level ¼ ***p  0.001, **p  0.01, *p  0.05, nsp>0.1 (not significant). F-valuetabulated
0.00019***

0,00019***
possible to determine the significant regression coefficients, as

0.0011**
P-value
shown in Equation (6) for the TF content. The variables that
significantly influenced the TF model were: ethanol concentration

ns

ns

ns
ns
ns

ns
ns
ns
(X1, X21), ethanol-residue ratio (X2, X22), extraction time (X3), as
shown in Table 2. However, only the linear terms X1, X3 (p  0.05),

5400,812

919.039
F-value

6.7795

0.3113
X2 (p  0.001) showed positive effect on the extraction.
FRAP

ns

ns

ns
ns
ns

ns
ns
ns
YTF ¼
342:4990 þ 11:4759X1 þ 101:9420X2 þ 3:2223X3

0:1007X12
6:0283X22
0.00007***

0,00007***

(6)

0.0011**
P-value

The model proved to be highly significant (p  0.0001), since the

ns

ns

ns
ns
ns

ns
ns
ns
R2 of the model was 89.39%. The calculated F-value was 19.5784,
which satisfies the requisites for building of response surface
14,978,60

904.0795
graphs as shown in Fig. 2 (i, j, k).
19.1134
F-value

0.5310
DPPH

The maximum extraction of TF was 40e70% at ethanol-residue

ratio of 6.5e10 mL/g and extraction time of 60 min, indicating
ns

ns

ns
ns
ns

ns
ns
ns

that increase in contact time was required to facilitate diffusion of

ANOVA for response surface quadratic polynomial model for significant independent variables of bioactive compounds and antioxidant activity of acerola residue.

this compound (TF).

0.00007***

0,0009***

0,0009***
0,0032**

0,0039**

0,0048**

0.0231*
P-value

3.2.2. Effect of the extraction variables on antioxidant activity

ns

ns

ns
ns

3.2.2.1. ABTS assay. The following equation describes the antioxi-

dant activity determined by the method of ABTSþ radical cation
1139,493

1102,663
17.2030

312,663

254,964

204,824

42.6135
F-value

0.8803

scavenging predicted by the model (Equation (7)) according to

ABTS

coded and significant (p  0.05) variables.

ns

ns

ns
ns

YABTS ¼ 43:7391 þ 12:1046X2 þ 0:6232X3
0:0037X12

0.00004***

0,0010***
0,0050**

0,0039**

0,0013**
0,0054**

0.0332*
P-value

0:7518X22 þ 0:0568X1 X2 (7)

ns

ns
ns
ns

Table 2 reveals that the variable ethanol concentration (X21),

ethanol-residue ratio (X2, X22), extraction time (X3), and the inter-
200,9853
986,2066
252,6789

761,1437
183,5190

action between ethanol concentration and ethanol-residue ratio

19.5784

29.5496
F-value

0.8939

(X1X2) influenced the antioxidant activity. However, only the linear

TF

terms X2 (p  0.01) and X3 (p  0.05) and interaction X1X2 (p  0.1)

ns

ns
ns
ns

showed a positive effect on the antioxidant activity of the extract.

The R2 of the model for ABTS assay was equal to 88.03%. The
0.00001***

0,0004***

0.0577ns
0,0029**

0,0029**
0,0020**
0,0238*

0,0111*
0,0254*
0,0206*
P-value

calculated F-value was 17.2030, being much higher than the tabu-
lated value (3.20), and thus it is highly significant (p  0.0001) with
ns

the model to describe the results by response surface analysis. It is

further verified from Fig. 2 (l), that higher ethanol concentration
(p  0.05) ¼ TA (3.26), CA (3.41), PC (3.44), TF and ABTS (3.20), DPPH and FRAP (4.54).
2471,611
37.9435

345,765

341,082
498,252

16.6676
F-value

and ethanol-residue ratio being 55e99.5% and 9e10 mL/g,

40,607

88,208
37,926
47,006

0.9733

respectively result in higher antioxidant activity.

PC

ns

Fig. 2 (m and n) show the effects of the extraction time on the

ethanol concentration and on the ethanol-residue ratio, respec-
0.00070***

0,0003***

0,0008***

tively. The antioxidant activity was greater at maximum extraction

0,0015**
0.0129*
P-value

time (60 min) when ethanol concentration varied from 20 to 65%

and ethanol-residue ratio from 9 to 10 mL/g.
ns

ns

ns

ns

ns
ns

3.2.2.2. DPPH assay. The increase in antioxidant activity by DPPH

3105,455

1231,252
25.5334

76.7139
647,339
F-value

0.8549

radical scavenging was due to the increase in ethanol-residue ratio

CA

(X2), which was the only variable that showed significant (p  0.01)
ns

ns

ns

ns

ns
ns

and positive effect, as described by the second order polynomial

equation for DPPH assay ðYDPPH Þ (Equation (8)).
0.00001***

0,00007***
0,0001***

0,0003***

0,0003***

0.0012**
P-value

YDPPH ¼ 79:1254 þ 10:0623X2 (8)

ns

ns
ns

ns
ns

2
From the coded variables, it can be observed that the R of the
model was 53.10%, reflecting a lower quality of model fit ðYDPPH Þ.
13,452,88
10.3042

9703,46

3013,71

3318,62
F-value

858.14
0.7743

Fig. 2 (o, p) show the effect of ethanol-residue ratio on the anti-

oxidant activity of the extract by DPPH radical scavenging, which
TA

ns

ns
ns

ns
ns

was larger at the ethanol-residue ratio of 10 mL/g.

Interaction

Lack of fit
Quadratic

3.2.2.3. FRAP assay. The following equation describes the antioxi-

Model
Linear
Table 3

Term

X1X2
X1X3
X2X3

dant activity determined by method of the ferric reducing which is

X1
X2
X3

X12
X22
X23

R2

predicted by the model (Equation (9)) according to coded and

164 Y.R.R.S. Rezende et al. / LWT - Food Science and Technology 85 (2017) 158e169

Fig. 2. Effect of interaction of the extracting variables on TA (a, b, c), CA (d, e), PC (f, g, h), TF (i, j, k); and the antioxidant activity by assays of ABTS (l, m, n), DPPH (o, p) and FRAP (q, r).
X1 ¼ Ethanol concentration, X2 ¼ Ethanol-residue ratio, X3 ¼ Extraction time, TA ¼ Total anthocyanins, CA ¼ Carotenoids, AA ¼ Ascorbic acid, PC ¼ Phenolic compounds, TF ¼ Total
flavonoids, ABTS ¼ ABTSþ scavenging activity, DPPH ¼ DPPH radical scavenging activity, FRAP ¼ Ferric reducing antioxidant power.
Y.R.R.S. Rezende et al. / LWT - Food Science and Technology 85 (2017) 158e169 165

Fig. 2. (continued).
166 Y.R.R.S. Rezende et al. / LWT - Food Science and Technology 85 (2017) 158e169

significant variables (p  0.05).

YFRAP ¼ 111:7665 þ 24:1902X2 (9)

Fig. 2. (continued).
 Y.R.R.S. Rezende et al. / LWT - Food Science and Technology 85 (2017) 158e169 167

Fig. 3. Desirability function for the abundance of bioactive compounds and antioxidant activity of acerola residue extracts as a function of ethanol concentration (%), ethanol-
residue ratio (mL/g) and extraction time (min); and predicted values of concentrations of TA, AA, CA, PC, TF, ABTS, DPPH and FRAP.
X1 ¼ Ethanol concentration, X2 ¼ Ethanol-residue ratio, X3 ¼ Extraction time, TA ¼ Total anthocyanins, CA ¼ Carotenoids, AA ¼ Ascorbic acid, PC ¼ Phenolic compounds, TF ¼ Total
flavonoids, ABTS ¼ ABTSþ scavenging activity, DPPH ¼ DPPH radical scavenging activity, FRAP ¼ Ferric reducing antioxidant power.

Table 4 activity was in the FRAP assay at ethanol-residue ratio of 10 mL/g,

Experimental data on the validation of predicted values at optimized extraction behavior being similar to the DPPH assay.
conditions.

Dependent variables Predicted value Experimental value % Difference (CV)

3.2.3. Desirability function
TA (mg TA/100 g) 16.9 16.6 ± 0.1 2.29
The response surface graphs shown in Fig. 2 (a, c, d, e) indicate
AA (mg AA/100 g) 408 489 ± 19 16.54
CA (mg b-CE/g) 3.4 4.0 ± 0.3 15.61 that the increase in ethanol-residue ratio positively influences the
PC (mg GAE/100 g) 1130 1068 ± 3 5.79 TA extraction, however, it decreases the CA extraction. This phe-
TF (mg QE/100 g) 570 559 ± 6. 1.88 nomenon limits interpretation of the results, creating the need to
ABTS (mM TE/g) 149 147 ± 2 1.50 use additional statistical technique in the analysis of such data,
DPPH (mM TE/g) 170 195 ± 3 12.65
which in this case, was selected to be desirability function proposed
FRAP (mM TE/g) 272 294 ± 3 7.40
by Derringer and Suich (1980).
TA ¼ Total anthocyanins, CA ¼ Carotenoids, AA ¼ Ascorbic acid, PC ¼ Phenolic
The desirability function is related to the numerical optimiza-
compounds, TF ¼ Total flavonoids, ABTS ¼ ABTSþ scavenging activity,
DPPH ¼ DPPH radical scavenging activity, FRAP ¼ Ferric reducing antioxidant po- tion, which is based on the idea that the quality of a product or
wer. CV ¼ Coefficient of variation. Values are expressed as mean ± standard process that has multiple responses need to find the process vari-
deviation. ations which satisfy all the restrictions applicable to the final
product or in other words, combining the models individually
adjusted for each response in a single univariate response. With the
The R2 of the model being 31.13% was not satisfactory for this results of the optimization, the ranges of most influential variables
type of process. It is observed that the model could not explain for process control were established.
68.87% of the observed values. Therefore, this model was consid- Fig. 3 shows the overall desirability function data showing the
ered inadequate to describe the response surface graphs (Fig. 2 q, r) operational conditions which lead to the optimization of the
since it did not fit the data. The maximum value of antioxidant extraction process of the bioactive compounds and antioxidant
168 Y.R.R.S. Rezende et al. / LWT - Food Science and Technology 85 (2017) 158e169
Fig. 4. Concentrations of bioactive compounds and antioxidant activity of extracts of
acerola pulp and residue.
EAP ¼ Extract of acerola pulp, EAR ¼ Extract of acerola residue, TA ¼ Total anthocy-
anins, CA ¼ Carotenoids, AA ¼ Ascorbic acid, PC ¼ Phenolic compounds, TF ¼ Total
flavonoids, ABTS ¼ ABTSþ scavenging activity, DPPH ¼ DPPH radical scavenging ac-
tivity, FRAP ¼ Ferric reducing antioxidant power. Means followed by the same letter on
the columns do not differ significantly by Tukey's test at 5% probability.
activity present in the acerola residue extracts. This analysis shows
that the maximum global desirability function, D of 0.7211 is
reached when the optimized process conditions are employed, viz.
the ethanol concentration of 46.49%, ethanol-residue ratio of
8.66 mL/g and extraction time of 49.30 min. Thus, the use of such
experimental conditions will result in an extract with desired
concentrations of TA (16.9 mg TA/100 g), AA (408 mg AA/100 g), CA
(3.4 mg b-CE/g), PC (1130 mg GAE 100/g), TF (570 mg QE/100 g),
having antioxidant activity by ABTS (149 mM TE/g), DPPH
(170 mM TE/g) and FRAP (272 mM TE/g).
3.3. Determination and experimental validation of optimized
conditions
Table 4 presents the data on predicted and experimentally ob-
tained values in validation testing of optimum conditions as pre-
dicted by the desirability function (Derringer & Suich, 1980).
For all the response variables, the experimental results were
similar to the predicted results. The coefficients of variation varied
from 1.50 to 15.61%, and these values were low in the desired region
where the responses are maximized. These results demonstrate the
utility and validity of using an experimental planning, RSM and
desirability function to optimize the extracting conditions of
bioactive compounds and antioxidant activity of acerola residue
extracts, or optimizing multiple responses while establishing con-
ditions which ensure higher extraction of bioactive compounds
with higher antioxidant activity.
The data on extracts of acerola pulp and residue obtained at
optimized conditions for the maximum extraction of bioactive
compounds (TA, CA, AA, PC and TF) and antioxidant activity (DPPH,
ABTS, FRAP) are shown in Fig. 4. The residue extract presented
higher levels of TA, CA, PC and TF, but the results showed that the
pulp presented greater antioxidant activity, differing significantly
(p  0.05) with respect to the residue. This behavior may be related
to AA, since it is the only compound, most abundant in the pulp
extract.
4. Conclusion
Ultrasound assisted extraction of bioactive compounds and
antioxidant activity from acerola residue was found to be very
effective when compared with the conventional extraction,
allowing higher extraction yields at lower extraction time. The
optimization of the extraction process was successfully examined
using the RSM and the desirability function. Ethanol concentration
of 46.49%, ethanol-residue ratio of 8.66 mL/g and extraction per-
formed for 49.30 min were the operating conditions that led to the
optimization of the extraction process having maximum yields of
bioactive compounds. For all response variables, the experimental
results resembled well with the predicted results, indicating that
the experimental design was satisfactory in the optimization of
several responses simultaneously. The extraction process optimi-
zation proved to be a viable option for obtaining phenolic com-
pounds, total flavonoids and total anthocyanins from acerola
residue.
Notes
All authors declare that there is no conflict of interest.
Acknowledgments
This study was funded by Conselho Nacional de Desenvolvimento
Científico e Tecnolo gico (CNPq) vide research project Instituto
Nacional de Ci^ encia e Tecnologia de Frutos Tropicais (Process
57573781/2008-7). The authors (Y.R.R.S.R. and J.P.N.) thank CAPES,
Brazil for their fellowships.
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