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Dried camu-camu (Myrciaria dubia H.B.K. McVaugh) industrial residue: A

bioactive-rich Amazonian powder with functional attributes

Article  in  Food Research International · August 2014

DOI: 10.1016/j.foodres.2014.05.018

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 Food Research International 62 (2014) 934–940

Contents lists available at ScienceDirect

Food Research International

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

Dried camu-camu (Myrciaria dubia H.B.K. McVaugh) industrial residue:

A bioactive-rich Amazonian powder with functional attributes
Juliana Chrís Silva de Azevêdo a, Alice Fujita b, Edson Leandro de Oliveira a,
Maria Inês Genovese b, Roberta Targino Pinto Correia a,⁎
a
Laboratory of Food Bioactive Compounds and Dairy Technology, Chemical Engineering Department, Federal University of Rio Grande do Norte, Campus Lagoa Nova, 59075-180 Natal, RN, Brazil
b
Laboratory of Food Bioactive Compounds, Food and Experimental Nutrition Department, FCF, University of São Paulo, 05508-900 São Paulo, SP, Brazil

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

Article history: Camu-camu is a tropical Amazonian fruit, extensively processed into fruit pulp and other derivatives. This work
Received 10 February 2014 investigates the fresh and dried (hot air dried and freeze dried) camu-camu depulping residue in regard to its
Accepted 3 May 2014 physicochemical characteristics, bioactive content, in vitro antidiabetic potential and antimicrobial activities.
Available online 10 May 2014
The phenolic content of fresh residue (FR), hot air dried residue at 50 °C (HAD50), hot air dried residue at
80 °C (HAD80) and freeze dried residue (FD) were 3738.0, 1843.6, 1349.4 and 2160.2 mg GAE/100 g DW, respec-
Keywords:
Industrial residue
tively. Important flavonoids were identified (quercetin, myricetin, and catechin), besides high amount of ellagic
Amazonia acid (18.9 mg/100 g DW for the fresh residue), and for the first time in literature, the presence of syringic acid
Drying (3.1 to 7.0 mg/100 g DW) in the camu-camu residue was shown. The Minimum Inhibitory Concentration against
Flavonoids Staphylococcus aureus ranged from 0.3125 to 0.625 mg/mL for freeze dried and hot air dried camu-camu residues.
Antimicrobial Moderate in vitro alpha-amylase and potent alpha-glucosidase inhibitory activities were also observed for all
Antienzymatic extracts. This paper presents the dried camu-camu residue as a natural powder with bioactive and functional
properties.
© 2014 Elsevier Ltd. All rights reserved.

Introduction
Camu-camu is a Brazilian native plant found throughout the Amazon
rainforest. The fruits are small red-colored berries, round in shape,
belonging to the Myrtaceae family (Rodrigues, Menezes, Cabral, Dornier,
& Reynes, 2001). There is an increased interest in camu-camu products
in the health-oriented market, since they are known as one of the greatest
natural sources of vitamin C (Chirinos, Galarza, Betalleluz-Pallardel,
Pedreschi, & Campos, 2010; Rufino et al., 2010). Studies have also identi-
fied health-relevant phenolic compounds and carotenoids (Fracassetti,
Costa, Moulay, & Tomás-Barberan, 2013; Fujita, Borges, Correia, Franco,
& Genovese, 2013; Zanatta & Mercadante, 2007) and important func-
tional and biological effects have been demonstrated (Akter, Oh, Eun,
& Ahmed, 2011).
Nevertheless, the fruit's acidic taste discourages in natura consump-
tion. Consequently, camu-camu fruits are processed into several deriva-
tives such as fruit pulp, extract and juice which are mainly exported to
Japan and Europe, or as an ingredient in the production of several food
products (Akter et al., 2011; Rodrigues et al., 2001). The seeds and
skin represent nearly 40% of the fruit weight and the yield of refined
⁎ Corresponding author. Tel.: +55 84 33422285x801.
E-mail address: roberta@eq.ufrn.br (R.T.P. Correia).
pulp is low (Rodrigues et al., 2001). Previous studies have demonstrated
the bioactive relevance of fruit peels and residues, including some scien-
tific reports that show similar or higher concentration of bioactive
compounds in the fruit by-products when compared to the fruit pulp
(Balasundram, Sundram, & Samman, 2006).
The aim of this work was to evaluate the fresh and dried camu-camu
industrial residue, in order to reveal their bioactive compounds and
in vitro functional attributes such as radical scavenging capacity, inhibi-
tion of diabetes-related enzymes and antimicrobial activity. For this,
two different dehydration processes were employed: hot air drying
and freeze drying. The impact of these processes is discussed, along
with the bioactive and technological values of freeze dried and hot air
dried camu-camu residues.
Material and methods
Material
The depulping residue of camu-camu, consisting mainly of seeds and
peels, was collected from a fruit processing industry in Amazonas,
Brazil. Each 100 g of residue consisted of 65.6 g of seeds and 34.4 g of
peels and residual skins. The industrial residue was kept frozen until
drying and the camu-camu residue before drying was identified as the
fresh residue (FR).

http://dx.doi.org/10.1016/j.foodres.2014.05.018
0963-9969/© 2014 Elsevier Ltd. All rights reserved.
 J.C.S. de Azevêdo et al. / Food Research International 62 (2014) 934–940
Experimental drying
The camu-camu residue was submitted to hot air drying (HAD) and
freeze drying (FD) processes. Two HAD experimental groups were
investigated: the camu-camu residue dried at 50 °C with air velocity
of 4 m/s (HAD50) and the camu-camu residue dried at 80 °C with air
velocity of 6 m/s (HAD80). These experimental groups were selected
based on preliminary experiments (data not shown). The hot air dryer
equipment consists of a 5.5 hp and 3490 rpm centrifugal blower
(model 112 M, WEG, Jaraguá do Sul, Brazil). The drying experiments
were conducted by spreading the previously thawed camu-camu resi-
due in 10–15 mm thick layers on a perforated tray. The airflow crossed
perpendicularly through the tray placed at the center of the drying
chamber. Digital thermocouples were placed along the drying chamber
in order to monitor the drying temperature. The freeze drying operation
was conducted in a freeze dyer equipment model L101 (Liobras, Campi-
nas, Brazil). The freeze dried samples (FD) were prepared under the
following conditions: temperature of − 40 °C, inferior vacuum of
0.5 mm Hg, constant lyophilization speed of 1 mm/h and final pressure
of 0.050 mm Hg. The freeze dried samples were identified as FD
samples.
Sample preparation
The ground samples (mill TE-631/2, Tecnal, Piracicaba, Brazil) were
kept frozen (−18 °C) until further analysis. The analysis of carotenoids
and ascorbic acid was made directly from the obtained powder. For the
phenolic compounds and antioxidant activity determinations, aqueous
extracts were prepared by sequential extraction in three steps according
to Nóbrega, Oliveira, Genovese, and Correia (2014).
Physicochemical characterization
Camu-camu samples were analyzed for pH (Hanna Instruments,
Woonsocket, RI, USA), water activity (Aqualab Decagon, Pullman, WA,
USA) and moisture (934.06, AOAC, 1995). Powder solubility and hygro-
scopicity were determined according to Cano-Chauca, Stringheta,
Ramos, and Cal-Vidal (2005), and Tonon, Brabet, and Hubinger (2009),
respectively.
Surface color measurement
Color measurements (L*, a* and b*) of fresh, freeze dried and hot air
dried camu-camu residues were carried out using a Color Quest XE
colorimeter (HunterLab, Reston, VA, USA). The parameters C* (chroma)
and h* (hue angle) were calculated according to C* = (a*2 − b*2)1/2 and
h* = arctan (a*/b*), respectively. In addition, the total color difference
(ΔE) was calculated according to Pathare, Opara, and Al-Said (2013).
Determination of ascorbic and dehydroascorbic acids
Ascorbic and dehydroascorbic acids were determined according to
Pasternak, Potters, and Caubergs (2005), with some modifications.
Ascorbic acid (AA) was extracted (1:100 w/v) with metaphosphoric
acid (6% w/v) and analyzed by reversed-phase HPLC (Hewlett-Packard
1100, Palo Alto, CA, USA) with an autosampler and a quaternary pump
coupled to a diode array detector (DAD). The column used was
150 mm × 3.6 mm i.d., HP®, NucleoSil 100C18 and elution (flow rate
of 0.8 mL/min) was performed in isocratic condition with a 2 mM
potassium chloride buffer (pH 2.5), monitored at 245 nm. Total ascorbic
acid was estimated after reduction of dehydroascorbic acid (DHA) with
10 mM dithiotreitol. The dehydroascorbic acid content was calculated
by the difference between total and reduced ascorbic acid. The results
were expressed as mg/100 g sample dry weight (DW).
935
Total monomeric anthocyanins
Total monomeric anthocyanins were measured by the pH dif-
ferential method (Giusti & Wrolstad, 2001) using two buffer solutions
(potassium chloride 0.025 M, pH = 1 and sodium acetate 0.4 M, pH
= 4.5). The values were expressed as mg eq. cyanidin 3-glycoside/g
of dry weight, taking into account the dilutions made in each
determination.
Total proanthocyanidin content
The determination of total proanthocyanidin content was made ac-
cording to Porter, Hrstich, and Chan (1986). An aliquot (0.25 mL) of
the extract obtained in 1% HCl in methanol was added to 2.5 mL of
Porter's reagent (154 mg of FeSO4·7H2O/L of 3:2 n-butanol:chloridric
acid), mixed thoroughly, and heated for 30 min in a water bath at
95 °C. The absorbance was measured at 550 nm using a UV/VIS spectro-
photometer (model U1 100, Hitachi, Tokyo, Japan). The results were
expressed as mg of quebracho tannin equivalents (QTE)/100 g powder.
Folin–Ciocalteau (FC) reducing capacity
The analysis of Folin–Ciocalteau (FC) reducing capacity was per-
formed according to Cheplick, Kwon, Bhowmik, and Shetty (2010).
The sample absorbance was measured (Genesys 10S UV–VIS Spectro-
photometer, Thermo Scientific, Waltham, MA, USA) at 725 nm against
blank consisting of a solution of 95% ethanol. A calibration curve of
different concentrations of gallic acid was used in order to express the
results as mg GAE/100 g DW.
Total phenolics
Total phenolics were calculated according to Fujita et al. (2013) by
subtracting the ascorbic acid content from the value of FC reducing
capacity, using a standard curve.
Total carotenoids
The analysis of total carotenoids was performed according to
Lichtenthaler and Buschmann (2001). The carotenoid extraction was
conducted by mixing the samples (2 g) with 18 mL of acetone (VETEC,
Brazil) for 20 min in the absence of light at room temperature (25 °C).
The results were expressed as micrograms per 100 g of dry weight
(μg/100 g DW).
2,2-Diphenyl-1-pricrylhydrazil DPPH scavenging activity
It was determined according to Nóbrega et al. (2014). Aliquots of
200 μL of a DPPH (Sigma-Aldrich, St. Louis, MO, USA) methanolic solu-
tion (20 mg/mL) with absorbance between 0.6 and 0.7 at 517 nm
were mixed with 40 μL of sample extracts (or methanol for the control).
After 30 min at 25 °C, the absorbance was read at 517 nm (Thermoplate
Reader, Bio-Rad Laboratories, Hercules, CA, USA). The results were
expressed as μmol Trolox equivalents (TE)/g DW.
HPLC-DAD analysis of flavonoids and free ellagic acid
HPLC-DAD analysis of flavonoids and free ellagic acid was performed
according to Arabbi, Genovese, and Lajolo (2004) with modifications.
Samples of 0.5 g of camu-camu residue and 200 mL of distilled water
were paper filtered (n.6, Whatman Intl Ltd., Maidstone, UK) and
concentrated in a rotary evaporator (Rotavapor® R-215, Büchi,
Switzerland) at 72 °C. The samples were resuspended in 10 mL of
distilled water and a 4 mL-aliquot was passed (1 g/6 mL) through a
pre-conditioned polyamide column (CC6, Macherey-Nagel, Düren,
Germany). After this, the columns were washed with 20 mL of water
936 J.C.S. de Azevêdo et al. / Food Research International 62 (2014) 934–940
and the elution of the flavonoids was done with 50 mL of methanol
followed by 50 mL methanol:ammonia (99.5:0.5) solution, using mani-
fold Visiprep 24DL (Supelco, Bellefonte, PA, USA). Each eluate was evap-
orated until dryness and subjected to hydrolysis with 5 mL HCl 2 N:
methanol (1:1) at 100 °C for 1 h. The material was resuspended in
1 mL HPLC grade methanol and filtered through polyethylene filters
(PFTE membranes, 0.22 μm, Millipore Ltda., Bedford, MA, USA) prior to
HPLC analysis. The identification and quantification of flavonoids and
ellagic acid were conducted using a Prodigy 5μ ODS 3 reversed-phase
silica 250 × 4.60 mm i.d. column (Phenomenex Ltda, Torrance, CA,
USA). Solvent gradient consisted of solutions A (water:tetrahydrofu-
ran:trifluoroacetic acid 98:2:0.1) and B (acetonitrile). Initially, solution
B was used in the proportion of 17% for 2 min increasing to 25% after
over 8 min and 50% after 5 more minutes. The identification and quan-
tification of flavonoids and free ellagic acid were done using a Hewlett-
Packard 1100 system (automatic sample injector, quaternary pump and
diode array detector (DAD) controlled by the ChemStation software
(Hewlett-Packard, Palo Alto, CA, USA)). The samples were injected in
duplicate and the phenolic compounds were identified by comparing
the retention and spectra time with appropriate standards (myricetin,
cyanidin, luteolin, apigenin, kaempferol, quercetin, catechin, epicate-
chin, ellagic acid and syringic acid obtained from Sigma-Aldrich,
St. Louis, MO, USA and Extrasynthèse, Genay, France). The quantifica-
tion was based on external calibration.
Antimicrobial activity and determination of the Minimum Inhibitory
Concentration (MIC)
The aqueous extracts were used for the preparation of two
polyphenolic-rich fractions for antimicrobial activity determination:
polyphenolic-rich fraction 1 (PP1) obtained from polyamide CC6
(Macherey-Nagel, Düren, Germany) and polyphenolic-rich fraction 2
(PP2) prepared through C18 solid-phase extraction (SPE, Macherey-
Nagel, Düren, Germany) columns (1 g/6 mL). The antimicrobial activity
of camu-camu extracts was tested for Staphylococcus aureus (ATCC
29213), according to CSLI (2009). For determination of the MIC, the
microdilution method was used (Clinical and Laboratory Standards
Institute (CSLI), 2010). The lowest concentration able to inhibit visible
microorganism growth (MIC) was determined after incubating the
microplates at 37 °C for 24 h. Ampicillin was used as the positive control.
Enzymatic inhibition assays
The alpha-amylase (EC 3.2.1.1) inhibition assay was determined ac-
cording to the non-pre-incubation method described by Ali, Houghton,
and Soumyanath (2006). Absorbance was measured at 540 nm
(Genesys 10S UV–VIS Spectrophotometer, Thermo Scientific, Waltham,
MA, USA) and results were expressed as % alpha-amylase inhibition =
100 − (% reaction after 3 min). The % reaction was obtained by % reac-
tion = (% maltose sample / % maltose control) × 100.
The alpha-glucosidase (EC 3.2.1.20) inhibition test was assayed
according to Cheplick et al. (2010). The absorbance was measured
at 405 nm using a microplate reader (BioChrom ASYS UVM340,
Cambridge, UK). Results were calculated as % alpha-glucosidase inhibi-
tion = [((ACt5 − ACto) − (ASt5 − ASto)) / (ACt5 − ACto)] × 100,
where Sto, Cto, St5, Ct5 are the absorbance of sample (S) and control (C)
at the beginning and after 5 min of reaction, respectively.
Statistical analysis
Three drying batches were performed for each drying process and all
analyses were performed at least in triplicate (N = 9), unless noted. The
results were expressed as mean ± standard deviation and the differ-
ences between means were first analyzed by ANOVA test and then
Tukey's test (p b 0.05) was applied (Statistica 7.0 software, StatSoft,
Tulsa, OK, USA).
Results and discussion
Physicochemical characterization
Both drying processes led to a significant 10-fold reduction in the
moisture levels (Table 1). The results of water activity of all groups
differed significantly from each other (p b 0.05) and the freeze dried
sample reached the lowest value of all. The low water activity of
dehydrated residues (HAD50, HAD80 and FD) leads to extended shelf
life and fewer tendencies to deteriorate caused by microbiological reac-
tions (Bonazzi & Dumoulin, 2011).
Acid pH (b4.5) was observed for all experimental groups and the
dried camu-camu residue, either freeze dried and hot air dried, showed
lower pH (p b 0.05) when compared to the fresh residue. The pH of
camu-camu residue was remarkably higher than the pH of the frozen
pulp (2.62) and camu-camu fruits (2.44) evaluated by Fujita et al.
(2013) and Akter et al. (2011), respectively. This is a desirable result,
since the extreme natural acidity of camu-camu pulp is recognized as
a negative sensory attribute that hampers in natura consumption.
Even though the camu-camu residue can still be considered acidic, it
has a pH comparable to acerola (3.28, Malpighia emarginata) and blue-
berry pulps (3.18) (Mercali, Sarkis, Jaeschke, Tessaro, & Marczak, 2011).
The solubility of freeze dried and hot air dried camu-camu samples
was four times higher when compared to fresh residue. However, it is
lower when compared to mango powder obtained by spray-drying
and adding modified starch (31%) (Cano-Chauca et al., 2005). In this
research, drying carriers, which may increase the solubility of the ob-
tained powders, were not added. Both freeze dried and hot air dried
camu-camu samples presented higher hygroscopicity when compared
to microencapsulated jabuticaba extracts, another tropical fruit from
the Myrciaria family (Silva, Stringheta, Teófilo, & Oliveira, 2013).
Color parameters
Table 1 also shows that FD samples had higher L* (p b 0.05) than
HAD50 and HAD80. A similar tendency was reported for camu-camu
pulp dried in spouted bed dryer (Fujita et al., 2013). The experimental
chroma results of hot air dried samples are higher than the fresh and
freeze dried residues (p b 0.05). This significant difference may indicate
that higher temperatures are able to promote some color intensification
on camu-camu powders.
ΔE indicates the magnitude of color difference between fresh and
dried samples and they can be analytically classified as very distinct
(ΔE N 3), distinct (1.5 b ΔE b 3) and slightly different (ΔE b 1.5)
(Pathare et al., 2013). According to this classification, the HAD and FD
processes led to drastic color changes to camu-camu residue, since
dried samples reached ΔE greater than 3.
Ascorbic acid and dehydroascorbic acid
Humans are among the few species that are not able to synthesize
vitamin C, and therefore, a dietary vitamin C intake is necessary (Jacob
& Sotoudeh, 2002). Our results (Table 2) reveal that fresh camu-camu
residue is also a remarkable ascorbic acid source, along with camu-
camu fruits, already pointed out as the richest natural ascorbic acid
source known so far (Akter et al., 2011; Fujita et al., 2013; Genovese,
Pinto, Gonçalves, & Lajolo, 2008; Rufino et al., 2010).
The FD and HAD residues presented lower ascorbic acid when com-
pared to spray dried camu-camu pulp with or without maltodextrin
(Fujita et al., 2013), which may indicate that this water soluble nutrient
is concentrated at the fruit pulp. FD samples presented ascorbic acid
worth more than double the results of HAD80. The latter was submitted
to the higher drying temperature used in this study, thus, this finding
demonstrates the thermolability of the ascorbic acid, already described
elsewhere (Mrad, Boudhrioua, Kechaou, Courtois, & Bonazzi, 2012).
 J.C.S. de Azevêdo et al. / Food Research International 62 (2014) 934–940 937

Table 1
Physicochemical characterization and color parameters of fresh (FR), hot air dried (HAD50 and HAD80) and freeze dried (FD) camu-camu residues.

FR HAD50 HAD80 FD

Aw 0.993 ± 0.000a 0.268 ± 0.001b 0.183 ± 0.003c 0.091 ± 0.002d

Moisture, % 86.0 ± 2.8a 5.9 ± 1.2b 5.9 ± 0.9b 5.9 ± 0.1b
pH 4.2 ± 0.1a 3.3 ± 0.1b 3.8 ± 0.1b 3.3 ± 0.1b
Solubility, % 3.5 ± 0.1a 12.0 ± 0.0b 14.2 ± 0.1b 13.3 ± 0.1b
Hygroscopicity, g/100 g 65.7 ± 0.2a 92.9 ± 0.6b 90.5 ± 0.1b 90.4 ± 0.1b
L* 51.8 ± 0.3a 65.3 ± 1.0b 58.9 ± 1.1c 70.3 ± 0.2d
Chroma* 22.7 ± 0.2a 26.0 ± 0.2b 27.2 ± 0.2b 23.6 ± 0.3a
ΔE ne 21.4 ± 1.1a 22.3 ± 0.8a 18.6 ± 0.2b

Results expressed as mean ± standard deviation (N = 9).

Aw: water activity; ne: not evaluated; ΔE: total color difference.
a–d: Different letters in the same line differ significantly by Tukey's test (p b 0.05).
HAD50 (hot air dried, 50 °C and 4 m/s), HAD80 (hot air dried, 80 °C and 6 m/s).

All dried residues reached ascorbic acid content significantly lower
than the fresh residue (p b 0.05) (Table 2), which confirms previous re-
ports showing that ascorbic acid is unstable at high temperatures and
in the presence of oxygen, conditions normally associated to drying
(Mrad et al., 2012). Despite the observed ascorbic acid losses and consid-
ering that the Recommended Dietary Allowance (RDA) for vitamin C is 75
mg for women and 90 mg for men (Jacob & Sotoudeh, 2002), the camu-
camu residue powders still represent a relevant source of vitamin C.
Anthocyanins, proanthocyanidins (PA) and total phenolics (TP)
The phenolic-related results are also presented in Table 2. The Folin–
Ciocalteau reagent used in the method is not specific for phenolics,
because some non-phenolic compounds such as ascorbic acid and sac-
charides may interfere and lead to false positive results. The high content
of AA in camu-camu justifies the experimental procedure adopted in this
study, which avoids phenolic overestimation (Gonçalves, Lajolo, &
Genovese, 2010).
The drying process causes significant reduction in camu-camu resi-
due, in the order of anthocyanins N total phenolics N proanthocyanidins.
Our results are in accordance with Rosso and Mercadante (2007) who
have shown that high ascorbic acid concentrations may lead to extensive
anthocyanin degradation. Similar to what was reported for acerola
(Nóbrega et al., 2014), the registered losses for both ascorbic acid and an-
thocyanins may be due to the direct condensation of the ascorbic acid on
carbon 4 of the camu-camu anthocyanins. Results also show that the dry-
ing impact followed a temperature-dependent behavior, since the HAD80
group presented the highest losses (100%, 63.9% and 46.7% for anthocya-
nins, TP and proanthocyanidins, respectively). On the other hand, FD
samples presented the lowest losses among the dried samples for all
the above components (53.6%, 42.2% and 15.8%, respectively).
This relative heat resistance of proanthocyanidins was also observed
by Fujita et al. (2013) in spray dried camu-camu pulp. Due to its signif-
icant amount of seeds, the camu-camu residue is expected to be an
important source of condensed tannins, which include several health-
relevant compounds such as catechin and its derivatives (Fracassetti
et al., 2013).
Different results for total phenolics of camu-camu and their deriva-
tives under the various extraction procedures were reported in the liter-
ature. Gonçalves et al. (2010) found 28.8 mg CE/100 g DW for camu-
camu fruits extracted in methanol/water/acetic acid, the pulp and peel
of camu-camu subjected to the extraction in methanol/water showed
11.6 mg GAE/100 g DW (Rufino et al., 2010) and the industrial residue
had the remarkable concentration of 116.0 mg GAE/100 g after metha-
nolic extraction (Myoda et al., 2010). TP results depend on the polarity
of the solvent used for extraction, but it is noteworthy that among these
reports, only Gonçalves et al. (2010) have considered the interference of
ascorbic acid on the total phenolic determination.
According to Bennett et al. (2011), the relative bioavailability of the
oxidized phenolics has not been sufficiently studied. Thus, it may not be
assumed that the oxidized phenolics, said as “lost”, are not bioavailable
or bioactive, but instead, they might have been chemically converted. In
addition, despite the decrease caused by drying, the TP results are either
higher or comparable to several other fruit sources (Rufino et al., 2010;
Silva et al., 2014). Based on the results, the freeze dried and hot air dried
residues of camu-camu is a phenolic-rich natural product that could be
offered all year long, in places with restricted access to the fresh
product.
Total carotenoids
Severe impact was observed for the carotenoid content, with losses
ranging from 74.2% (FD) to 86.8% (HAD80). Once again, the drying tem-
perature seems to be more important than the drying technique itself,
since FD and HAD50 samples are statistically similar (p N 0.05). The
carotenoid content of HAD80 is lower (p b 0.05), which might be a con-
sequence of combining higher temperature and higher drying speed.
According to Rodriguez-Amaya, Kimura, Godoy, and Amaya-Farfan

Table 2
Bioactive compounds and DPPH scavenging activity of fresh residue (FR), hot air dried (HAD50 and HAD80) and freeze dried (FD) camu-camu residues.

FR HAD50 HAD80 FD

Ascorbic acid, mg/g DW 458.2 ± 9.2a 8.2 ± 0.3b 5.1 ± 0.1b 11.8 ± 0.3b
Dehydroascorbic acid, mg/g DW 72.7 ± 2.3a 0.6 ± 0.4b 0.6 ± 0.1b 0.2 ± 0.1b
Total anthocyanins, mg eq. cyanidin 3-glycoside/g DW 4.1 ± 0.3a 0.5 ± 0.1c ND 1.9 ± 0.1b
Proanthocyanidins, mg QTE/g DW 33.5 ± 0.4a 23.8 ± 0.5b 17.9 ± 0.5c 28.2 ± 0.9b
Total phenolics, mg GAE/100 g DW 3738.0 ± 20.8a 1843.6 ± 1.9b 1349.4 ± 22.2b 2160.2 ± 54.4b
Total carotenoids, μg/100 g DW 5694.6 ± 2.9a 1082.4 ± 1.8b 748.7 ± 8.5c 1467.9 ± 1.6b
DPPH scavenging activity, μmol TE/g DW 166.6 ± 1.1a 34.5 ± 0.2b 28.3 ± 1.1b 32.4 ± 1.6b

Results expressed as mean ± standard deviation (N = 9); ND: not detected.

a–c: Different letters in the same line differ significantly by Tukey's test (p b 0.05).
HAD50 (hot air dried, 50 °C and 4 m/s), HAD80 (hot air dried, 80 °C and 6 m/s).
938 J.C.S. de Azevêdo et al. / Food Research International 62 (2014) 934–940

(2008), the main cause of carotenoid degradation is oxidation, either Table 3

enzymatically or non-enzymatically. Main phenolic compounds (mg/100 g DW) detected in fresh (FR), hot air dried (HAD50
and HAD80) and freeze dried (FD) camu-camu residues.
Total carotenoid levels between 354.8 and 1095.3 μg/100 g for
camu-camu fruits were reported, which are comparable to dried Ellagic acid Syringic acid Quercetin Myricetin Catechin
camu-camu residue. It was also demonstrated that this Amazonian FR 18.9 ± 0.3a 7.0 ± 0.4a 0.9 ± 0.1a 2.1 ± 0.5a 12.4 ± 1.0a
fruit is an important source of β-carotene and lutein (Zanatta & HAD50 5.8 ± 0.7c 3.1 ± 0.1b 0.8 ± 0.1a 0.7 ± 0.4c 1.1 ± 0.4b
Mercadante, 2007). Despite the impact observed, the dried camu- HAD80 7.6 ± 0.1bc 5.4 ± 0.1c 0.3 ± 0.1b 1.0 ± 0.4b 1.3 ± 0.8b
FD 9.6 ± 0.2b 4.1 ± 1.1bc 0.4 ± 0.1b 1.1 ± 0.2b 1.6 ± 0.4b
camu residue (both HAD and FD) has an expressive carotenoid concen-
tration, which is still higher than several other tropical fruits (Rufino Results expressed as mean ± standard deviation (N = 9).
et al., 2010). a–c: Different letters in the same column differ significantly by Tukey's test (p b 0.05).
HAD50 (hot air dried, 50 °C and 4 m/s), HAD80 (hot air dried, 80 °C and 6 m/s).

DPPH scavenging activity

Ramachandran, & Muruganathan, 2013). Ellagic acid, quercetin,
myricetin, and catechin were identified in high amounts in fresh camu-
The experimental results of the DPPH scavenging activity for the
camu residue, but in lower amounts in the HAD50, HAD80 and FD
fresh residue (Table 2) is similar to camu-camu fruits, which proved
samples, most likely as a consequence of the applied thermal treatment
to be 10 times higher than other Brazilian native fruits (Genovese
(Table 3).
et al., 2008). It is also close to camu-camu fruits in three different matu-
Gonçalves et al. (2010) have identified quercetin (42 mg/100 g
ration stages (Chirinos et al., 2010), but lower than what Fracassetti
DW), kaempferol (2.1 mg/100 g DW), cyanidin (306 mg/100 g DW)
et al. (2013) obtained for both camu-camu flour and pulp powder.
and ellagic acid (free, 16 mg/100 g DW and total, 490 mg/100 g FW)
Significant correlation values were observed between DPPH results
in camu-camu pulp. The authors did not detect catechin in camu-
and ascorbic acid (r2 = 0.999), total phenolics (r2 = 0.955), anthocya-
camu pulp, but Chirinos et al. (2010) found catechin contents of 1.3,
nins (r2 = 0.905), carotenoids (r2 = 0.994) and proanthocyanins
1.7 and 2.2 mg/100 g FW for green, reddish-green and red camu-
(r2 = 0.786). A similar correlation between DPPH and total phenolics
camu fruits, respectively. More recently, Fracassetti et al. (2013) found
was observed by Genovese et al. (2008), Chirinos et al. (2010) and
myricetin in dried camu-camu pulp (0.98 mg/100 g) and residue
Myoda et al. (2010).
(5.28 mg/100 g).
Both HAD and freeze dried samples had DPPH scavenging capacity
significantly lower compared to the fresh residue (p b 0.05) and pre-
sented antioxidant retention around 20%. The observed decrease
Antimicrobial activity
might be strongly associated with the discussed ascorbic acid losses,
since it represents a major contribution to the antioxidant capacity of
The inhibition zone of camu-camu residue against S. aureus
camu-camu fruits (Chirinos et al., 2010), but the role of phenolic com-
(Table 4), is higher than the antimicrobial effect of camu-camu peels
pounds, including anthocyanins and proanthocyanins, and carotenoids
and seeds shown by Myoda et al. (2010). The raw extracts of freeze
cannot be neglected.
dried and hot air dried samples exhibited lower MIC. This might be
the result of both concentrating the natural bioactive compounds
HPLC analysis of free phenolic acids and flavonoids by drying and also the effect of possible synergistic interactions be-
tween the phytochemicals found on water extracts, which are not
Fig. 1 shows a representative chromatogram of the HPLC analysis possible in the purified PP1 and PP2 fractions. Both phenolic frac-
performed in the camu-camu residue. For the first time in the literature, tions were obtained through solid-phase extraction and present similar
the presence of syringic acid was reported in fresh and dried (HAD and flavonoid composition. The main difference between these two frac-
FD) camu-camu residues (Table 3). This natural phenolic acid has tions is the absence of tannins in the fraction obtained using polyamide
proved to be an efficient in vivo antidiabetic agent, in addition to other (PP1) compared to C18 SPE (PP2), as these compounds bind irreversibly
health-relevant attributes (Muthukumaran, Srinivasan, Venkatesan, to polyamide.

Fig. 1. Representative HPLC chromatogram of the dried camu-camu residue.

 J.C.S. de Azevêdo et al. / Food Research International 62 (2014) 934–940 939

Table 4
Inhibition zones and Minimum Inhibitory Concentration (MIC) of extracts of fresh (FR), freeze dried (FD) and hot air dried (HAD50, HAD80) camu-camu residue against Staphylococcus
aureus ATCC 29213.

Raw PP1 PP2

Inhibition zone, mm MIC, mg/mL Inhibition zone, mm MIC, mg/mL Inhibition zone, mm MIC, mg/mL

FR 10 ± 1 2.5 13 ± 1 2.5 16 ± 1 2.5

HAD50 12 ± 1 0.625 13 ± 1 2.5 15 ± 1 2.5
HAD80 12 ± 1 0.3125 14 ± 1 2.5 15 ± 1 2.5
FD 13 ± 1 0.3125 14 ± 1 2.5 15 ± 1 2.5
Ampicillin ne 0.25

Results expressed as mean ± standard deviation (N = 9); ne: not evaluated.

PP1: polyphenolic-rich fraction 1; PP2: polyphenolic-rich fraction 2.
HAD50 (hot air dried, 50 °C and 4 m/s), HAD80 (hot air dried, 80 °C and 6 m/s).

Both freeze dried and hot air dried camu-camu residues have a sig-
nificant amount of bioactive compounds, including proanthocyanidins,
anthocyanins, flavonoids, and phenolic acids (Tables 3 and 4), which
may play a role in inhibiting S. aureus growth.
The camu-camu residue showed higher inhibition zones and lower
MIC against S. aureus than the jabuticaba (Myrciaria cauliflora) residue
(Silva et al., 2014), but lower inhibitory activity than spouted dried
camu-camu pulp (Fujita et al., 2013). Gram-positive bacteria such as
S. aureus have shown special susceptibility to fruit phenolics, which
act through several mechanisms that include destabilization of cellular
membrane, inhibition of key enzymes and strong metal binding capacity
(Caillet, Côté, Sylvain, & Lacroix, 2012).
Antienzymatic activity
et al., 2013). In addition, a possible glycosidic linkage would be implicated
in the observed enzymatic inhibition (Correia et al., 2012; Gonçalves et al.,
2010).
Similar (p N 0.05) and strong alpha-glucosidase inhibitory activity
was observed for all camu-camu samples (Fig. 2). Similar to what
Correia et al. (2012) previously demonstrated, no clear relationship be-
tween TPC or DPPH and alpha-glucosidase inhibition was found.
The combination of moderate alpha-amylase and potent alpha-
glucosidase inhibitory activities has been considered as the best possible
antienzymatic association, since it would prevent possible discomfort
caused by undigested carbohydrates and consequent stomach disten-
sion (Cheplick et al., 2010). All experimental groups, both fresh and
dried (FD and HAD), presented this desirable inhibitory combination,
which suggests their adequacy as natural antidiabetic dietary sources.

The inhibition of alpha-amylase and alpha-glucosidase has been Conclusions

investigated as means of modulating carbohydrate digestion and
retarding postprandial glycemia, which constitutes efficient ways to
manage the early stages of diabetes type 2 (Cheplick et al., 2010). All
extracts exhibited moderate in vitro alpha-amylase inhibition (Fig. 2)
and the higher amylolitic inhibition observed for FD extracts may be
justified by well preserved and concentrated active compounds found
in this sample. The alpha-amylase inhibition was not proportional to
the concentration of total phenolics (Table 2), which is in agreement
with previous reports showing that the anti-alpha-amylase activity
might be a structure-dependent attribute, and not directly connected
to the overall TP (Cheplick et al., 2010; Correia, Borges, Medeiros, &
Genovese, 2012). The presence of syringic acid (Table 3) may play a
role in the observed in vitro antidiabetic activity (Muthukumaran
The freeze dried and hot air dried camu-camu residue presents
significant residual total phenolics, anthocyanins, proanthocyanidins,
carotenoids and important flavonoids. Besides the identified health-
relevant compounds, our study also shows the multifunctionality of
camu-camu residue, which presents antioxidant, antimicrobial and
antienzymatic activities. The results support previous studies which
show that several natural phytochemical compounds are retained in
the solid wastes generated as co-products of the food industry. The
processing of fruit residues has the potential of becoming an important
segment of agribusiness, being used as an eco-friendly strategy to
minimize pollution and also a way to produce higher value products
from low-cost and abundant fruit materials.

110 Acknowledgments
100
Financial support and scholarship from CNPq (Ministry of Science,
90
Technology and Innovation, Brazil) is gratefully acknowledged. The
authors thank Jim Luippold for providing language assistance and proof-
Enzymatic inhibition, %

80

70
reading the article.
b
60
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