Materials and methods
Materials
Frozen blackberry pulp (De Marchi Ltd., Jundiaı´,
Brazil) was stored in a freezing chamber at )18 C
and thawed in a refrigerator (4–5 C) for 18 h, according
to the amount required for each experiment. Table 1
presents its physicochemical properties. The carrier
agents used were maltodextrin Maltogill 20 (Cargill,
Uberlaˆ ndia, Brazil) with 20 DE and gum arabic
Instantgum (Colloides Naturels, Sa˜o Paulo, Brazil).Sample preparation and spray drying process
The carrier agent – maltodextrin, gum arabic or a blend
(1:1 w⁄ w) of both carrier agents – was added to the pulp
in a final concentration of 7 g ⁄ 100 g fresh juice (w ⁄ w),
which corresponds to 48 g ⁄ 100 g total solids. Then, the
pulp and the carrier agent were homogenised in a colloid
mill (Meteor, Sa˜o Paulo, Brazil) until complete dissolution.
About 2 kg of blackberry pulp was used for each
experiment.
The process was performed using a laboratory-scale
spray dryer (model B290; Bu¨ chi, Flawil, Switzerland) at
a drying rate of 1.0 kg of water h)1. The mixture was fed
into the drying chamber at room temperature (25 C)
through a peristaltic pump with the flow rate adjusted to
0.49 kg h)1. The inlet air temperature was 145 C and
the outlet air temperature varied from 75 to 80 C.
Spray drying was carried out with a concurrent regime,
using a two-fluid nozzle atomiser (0.7 mm diameter), a
drying air flow rate of 0.36 m3 h)1 and an aspirator flow
rate of 35 m3 h)1 (100% of its maximum capacity).
These conditions have been established in a previous
work (Ferrari et al., 2011). The different powders
produced were placed in hermetic containers and stored
in desiccators containing silica gel until utilisation.
Analytical methods
Moisture content
The moisture content of the powder was determined
gravimetrically. Samples were weighed and dried in a
vacuum oven at 70 C for 24 h (AOAC, 2006).
Anthocyanin content
The anthocyanin content of the samples was determined
according to the spectrophotometric pH differential
method (AOAC, 2006), which is based on the anthocyanin
structural transformation that occurs with a change
in pH (coloured at pH 1.0 and colourless at pH 4.5).
Two dilutions of each sample were prepared with
potassium chloride (0.025 m) and sodium acetate
(0.4 m), which were used as buffer solutions at pH 1.0
and 4.5, respectively. Anthocyanins were extracted with
an acetone solution (70%), according to the methodology
described by Falca˜o et al. (2007), with some
modifications. These extracts were used for both anthocyanin
and antioxidant activity determinations. Absorbance
was measured in a spectrophotometer (model
700Plus; Femto, Sa˜o Paulo, Brazil) at 520 and 700 nm.
Total anthocyanin content was calculated using the
molar extinction coefficient of 26 900 L cm)1 mol for
cyanidin-3-glucoside (cyd-3-glu), which is the predominant
anthocyanin found in blackberry pulp (Koca &
Karadeniz, 2009). Results were expressed as mg cyd-
3-glu ⁄ 100 g dried juice (excluding the mass of carrier
agents). Total anthocyanin content in the mixture fed into the spray dryer was also determined. Anthocyanin
retention (AR) after the process was calculated according
to eqn 1.
AR ¼
TAC in spray dried powder
TAC in feed solution
100 ð1Þ
where AR is anthocyanin retention (%) and TAC is the
total anthocyanin content (mg ⁄ 100 g dried juice).
Antioxidant activity
Antioxidant activity was determined by Oxygen Radical
Absorbance Capacity (ORAC) method (Ou et al.,
2001), using a microplate fluorescence reader containing
a 96-well plate (NOVOStar; BMG Labtech, Ortenberg,
Germany). ORAC is a kinetic assay that measures the
loss of fluorescein fluorescence over time because of the
peroxyl-radical formation by the breakdown of 2,2¢-
azobis(2-amidino-propane) dihydrochloride (AAPH) at
37 C. Trolox, a water soluble vitamin E analogue, acts
as a positive control inhibiting fluorescein decay. Fluorescence
is continuously monitored for 80 min, when it
remained constant, with an excitation wavelength of
485 nm and an emission wavelength of 520 nm. AAPH
solution (153 mm) was used as free radical initiator and
fluorescein solution (8.16 · 10)5 mm) as oxidisable substrate.
Both solutions were prepared in 75 mm phosphate
buffer (pH 7.4) and kept at 4 C in dark
conditions. The extraction was also performed using
an acetone solution (70%). Each well was filled with
20 lL of blackberry extract (at different dilutions),
120 lL of fluorescein solution and 60 lL of AAPH
solution. The same analysis was done to Trolox methanolic
solutions, in some dilutions ranging from 0 to
700 lm, which allowed the construction of a standard
Trolox curve. Results were expressed as lmol Trolox
Equivalent (TE) per gram of dried juice (excluding the
mass of carrier agents).
Colour
The colour of the blackberry powder was measured
using a colorimeter (model CR400; Konica Minolta,
Osaka, Japan), with a CIELab scale (L*, a* and b*),
D65 as an illuminant and a 10 observer angle as a
reference system. The colour measurements were
expressed in terms of lightness L* (L* = 0 for black
andL* = 100 for white) and the chromaticity parameters
a* [green ()) to red (+)] and b* [blue ()) to yellow (+)].
C* (chroma) and H* (hue angle) were calculated from
these parameters, as the following equations (eqns 2 and
3). Chroma indicates colour intensity, whereas hue angle
values vary from 0 (pure red colour), 90 (pure yellow
colour), 180 (pure green colour) to 270 (pure blue
colour). The measurements were performed in triplicate
and three reading were done for each replicate.Bulk density, absolute density and porosity
The bulk density (qbulk) of the powders was measured
by weighing 1 g of sample and placing it in a 10 mL
graduated cylinder. The cylinder was tapped by hand
five times from a height of 10 cm and the bulk density
was calculated as the ratio between the mass of
powder contained in the cylinder and the volume
occupied (Goula & Adamopoulos, 2004). The absolute
density (qabs) was determined in a picnometer, using
99% ethanol as the immiscible liquid (Souza et al.,
2009). Porosity (e) was calculated as follows (Lewis,
1987):
e ¼ 1
qbulk
qabs
ð4Þ
Wettability
The wettability was evaluated according to the method
described by Vissotto et al. (2010), considering the time
required for 1.0 g of powder deposited on liquid surface
to become completely submersed in 400 g of distilled
water at 25 C.
Particle size distribution
The particle size was determined using a laser light
diffraction instrument (Mastersizer S, model MAM
5005; Malvern Instruments, Malvern, UK). A small
amount of powder was dispersed in 99% isopropanol
under magnetic agitation, and the distribution of
particle size monitored during three successive measurements.
The particle size was expressed as D[4,3] (De
Brouckere mean diameter), the mean diameter over the
volume distribution, which is generally used to characterise
a particle.
Scanning electron microscopy (SEM)
The microstructure of the particles was evaluated using
SEM. Powders were attached to SEM stubs using a
double-sided adhesive tape and coated with gold ⁄ palladium
under vacuum in a Sputter Coater Polaron
(model SC7620; VG Microtech, Ringmer, UK) at a
coating rate of 0.51 A ˚ s)1, 3–5 mA, 1 V and 0.08–
0.09 mbar for 180 s. The coated samples were observed
with a LEO440i scanning electron microscope (LEICA
Electron Microscopy Ltd., Oxford, UK). SEM was
operated at 20 kV and 150 pA with magnification of 1000X. Sorption isotherms
Sorption isotherms were determined by the gravimetric
method. Approximately 1 g of each spray dried
blackberry powder was weighed in aluminium vials
and equilibrated over some saturated salt solutions in
dessicators at 25 C (LiCl, CH3COOK, MgCl2, K2CO3,
Mg(NO3)2, KI, NaCl and KCl) to provide relative
humidity values of 11.3%, 22.6%, 32.8%, 43.2%,
52.9%, 68.9%, 75.3% and 84.3%, respectively. The
sample weights were controlled until a constant value
(<±0.001 g) was reached (around 4 weeks), where the
equilibrium was assumed. The equilibrium moisture
content was determined from the initial water content
data and the change in the registered weight until
equilibrium. These values were used to construct the
sorption isotherms (Mosquera et al., 2011).
GAB model was used to predict the water sorption
behaviour of spray dried samples, according to eqn 5.
We ¼
XmCGABKGABaw
½ð1KGABawÞð1KGABaw þCGABKGABawÞ
ð5Þ
where We is the equilibrium moisture content (g water ⁄ g
dry matter), Xm is the monolayer moisture content (g
water ⁄ g dry matter), aw is the water activity, CGAB and
KGAB are constants.
A non-linear regression analysis was carried out using
the software Statistica 8.0 (StatSoft, Inc., Tulsa, OK,
USA) to obtain the model parameters.
Statistical analysis
The experiments were carried out in duplicate, and all
the analyses were done in triplicate. Results were
presented as mean values with standard deviations.
Different mean values were statistically evaluated by
analysis of variance (anova), using the software
Statistica 8.0 (StatSoft, Inc., Tulsa, OK, USA). Mean separation was determined using the Tukey test at
P £ 0.05.
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