Journal of Food Engineering 154 (2015) 1724

Contents lists available at ScienceDirect

Journal of Food Engineering

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

Protein adsorption onto alginate-pectin microparticles and lms

produced by ionic gelation
Karla Crdova Aguilar a, Fernando Tello b, Andrea C.K. Bierhalz c, Ma.G. Garnica Romo d,
Hctor E. Martnez Flores e, Carlos R.F. Grosso b,
a

Programa Institucional de Maestra en Ciencias Biolgicas de la Universidad Michoacana de San Nicols de Hidalgo, Morelia, Michoacn, Mexico
Department of Foods and Nutrition, Faculty of Food Engineering, University of Campinas, Campinas, So Paulo 13083-862, Brazil
School of Chemical Engineering, University of Campinas, Campinas, So Paulo 13083-852, Brazil
d
Facultad de Ingeniera Civil, Universidad Michoacana de San Nicols de Hidalgo, Morelia, Michoacn, Mexico
e
Facultad de Qumico Farmacobiologa, Universidad Michoacana de San Nicols de Hidalgo, Morelia, Michoacn, Mexico
b
c

a r t i c l e

i n f o

Article history:
Received 6 May 2014
Received in revised form 2 December 2014
Accepted 26 December 2014
Available online 2 January 2015
Keywords:
Ionic gelation
Electrostatic adsorption
Microparticles
Edible lms
Pectin
Alginate

a b s t r a c t
Microparticles and lms containing sunower oil were produced by ionic gelation using a 1:1 alginate:pectin mixture and were electrostatically coated with whey and egg white proteins. Emulsions of
the polysaccharide mixture and the protein solutions were evaluated in terms of their zeta potentials.
The microparticles were characterized based on their mean size, size distribution, moisture content, calcium content, adsorbed protein content, encapsulation efciency and morphology. The lms were characterized with respect to their thickness, moisture content, calcium content, adsorbed protein content,
mechanical properties, water vapor permeability and morphology. High encapsulation efciency
(87.6% at pH 3.5 and 90.8% at pH 3.75) was obtained for the microparticles produced by ionic gelation.
The calcium content after ionic gelation was signicantly higher in the lms (on average, 3.0 lmol/
mg db) than in the microparticles (on average, 1.62 lmol/mg db). For the microparticles, an increase in
the protein content in solution yielded an increase in the protein content adsorbed, independent of the
type of protein used. When 4% protein in solution was used, protein adsorption onto the microparticles
(59.2% for whey protein and 45.5% for egg white protein) was signicantly higher than that onto the lms
(25.3% for whey protein and 24.1% for egg white protein) likely due to the smaller amount of calcium
present on the microparticles and the larger surface area of the particles relative to that of the lms.
Although the process of producing lms by ionic gelation and later coating them with proteins was
straightforward, homogeneous drying of the lms was difcult.
2015 Elsevier Ltd. All rights reserved.

1. Introduction
External ionic gelation is a simple process used to obtain gels in
which, an anionic polysaccharide solution is dripped over an ionic
solution at appropriate concentrations, making it possible to obtain
gels of different shapes and sizes that form three-dimensional
structures with a high water content (Gombotz and Wee, 1998;
Smrdel et al., 2008).
Among polysaccharides, alginate, pectin or a mixture of the two
may be used to produce matrices in the form of microparticles or
lms. The cation concentration, the ionic strength and the pH
determine the kinetics of gel formation as well the volume and stability of gel beads (Mestdagh and Axelos, 1998).
Corresponding author at: Depan, Fea, Unicamp. Tel.: +55 19 3521 4079.
E-mail address: grosso@fea.unicamp.br (C.R.F. Grosso).
http://dx.doi.org/10.1016/j.jfoodeng.2014.12.020
0260-8774/ 2015 Elsevier Ltd. All rights reserved.

Alginate has a linear structure and a high molar mass and comprises two types of uronic acids, b-D mannuronic acid (M) and a-L
guluronic acid (G), with either homopolymeric or heteropolymeric
blocks in which the G units form crosslinks with divalent ions, to
produce egg-box model gels (Grant et al., 1973). Alginate has
pKa values between 3.20 and 3.38 (Martinsen et al., 1992).
The characteristic structure of pectin is a linear chain composed
of a (1 ? 4)-D-galacturonic acids that are partially esteried with
methoxyl groups and neutral sugars, such as galactose, glucose,
rhamnose, arabinose and xylose. Pectin has a pKa value of 2.9
(Ralet et al., 2001).
Although ionic gelation is a simple and mild technique, the gels
produced are porous, which can accelerate oxygen permeation
through the matrix or allow for the release of active compounds
with low molar mass that are inserted into the gels (Sezer and
Akbuga, 1999). The size of the pores varied greatly, ranging from

18

K.C. Aguilar et al. / Journal of Food Engineering 154 (2015) 1724

5 to 200 nm (Mestdagh and Axelos, 1998; Smidsrod, 1974). To circumvent this limitation and improve the functionality of the gels in
lm or microparticle form, the literature proposes a mixing with
other biopolymers (Devi and Kakati, 2013) or coating with a layer
of polyelectrolytes with the opposite charge by electrostatic complexation (Gbassi et al., 2011; Hbrard et al., 2010).
Complexes between proteins and polysaccharides can form
spontaneously in an aqueous solution due to the electrostatic
interactions between groups with opposite charges (Tolstoguzov,
2003). The formation and stability of the complexes are affected
by the pH, ionic strength, protein: polysaccharide ratio, and biopolymer charge density as well as production conditions such as
temperature, shearing and agitation time (Ye, 2008). The formation
of protein and polysaccharide complexes generally occurs between
the pKa values of the anionic groups of the polysaccharide and the
isoelectric point (pI) of the protein (De Kruif et al., 2004).
Coating pectin and alginate microparticles with polycations
(such as chitosan and whey proteins) has been observed to result
in the formation of a membrane on the microparticlessurface
which retard the release of the active encapsulated compounds
(Humblet-Hua et al., 2011). The electrostatic interactions in the
microparticles can be controlled by varying the charge of the biopolymer surface. Recently, the adsorption of whey proteins onto
alginate or pectin microparticles was investigated (Gbassi et al.,
2011).
Egg white and whey proteins are two sources of globular proteins used in food products. The major egg white protein is ovalbumin (54%, 44.5 kDa) with a reported isoelectric point of 4.8
(Oakenfull et al., 1997). Whey proteins contain a mixture of b-lactoglobulin (82%, 18.5 KDa) and a-lactalbumin (15%, 14.5 KDa) with
isoelectric points ranging from 4.4 to 5.2 (Damodaran, 2008).
The preparation of microparticles via external ionic gelation has
been widely studied and demonstrated to involve a high ease of
production (De Vos et al., 2010; Sriamornsak and Kennedy,
2011). However, considering that the external ionic gelation process is very rapid and non-homogeneous, optimization of the lm
production process remains a challenge.
The objectives of this study were to produce microparticles containing sunower oil by ionic gelation using a mixture of alginate
and pectin (1:1, w/w). The microparticles were covered with egg
white and whey proteins in solution (1.7%, 3.0% and 4.0% protein
in solution). The zeta potentials of the proteins and polysaccharide
mixture solutions/emulsions were then evaluated at various pH
values. The microparticles were characterized in terms of their
morphology, size, size distribution and quantity of adsorbed
protein. Subsequently, lms (FP) were produced from the alginate-pectin mixture by ionic gelation. Then, the moist lms were
covered with proteins at the highest levels of protein adsorptions
obtained with the microparticles. The amounts of calcium bound
to the microparticles and to the lms produced by ionic gelation
were determined. The mechanical properties (tensile strength
and elongation at break), morphology, permeability to water vapor
and quantity of adsorbed protein of the lms were evaluated.

2. Materials and methods

2.1. Materials
High molecular weight sodium alginate (ALG) with a high
guluronic acid content (lot G3512301 MANUGEL DMB; FMC
Biopolymer, Campinas, So Paulo, Brazil, 0.5 0.0% moisture content, AOAC, 2006), citrus pectin (PEC) GENU with a low amidation
and low methoxyl content [CP Kelco, Limeira, So Paulo, Brazil;
81.3 1.2% galacturonic acid content, 30.4 1.6% degree of esterication and 10.1 1.0% degree of amidation, determined according

to FAO (2009) and 0.4 0.0% moisture content, AOAC, 2006], whey
protein concentrate (WPC) [Lacprodan lot Lac804U17601, 76,
Arla Foods Ingredients, Portea, Province of Crdoba, Argentina;
7.2 0.3% moisture content, 80.5 0.3% protein content and
3.9 0.0% ash content, determined according to AOAC (2006),
and 7.6 0.3% lipid content, determined according to Bligh and
Dyer (1959)], egg white proteins (OVA) (Saltos Alimentos LTDA,
Distrito Industrial, Parque do Lago Salto, So Paulo, Brazil;
6.7 0.5% moisture content, 90.5 0.8% protein content,
0.4 0.1% lipid content and 5.0 0.1% ash content), anhydrous calcium chloride (Dinmica, Diadema SP, Brazil), sodium hydroxide
(Nuclear, Diadema SP, Brazil), hydrochloric acid and glycerol
(Merck, So Paulo SP, Brazil) and sulfuric acid (Synth, Diadema
SP, Brazil), commercial sunower oil, and all other reagents used
were of analytical grade. All aqueous solution/emulsions were prepared with destilled and deionized water.
2.2. Zeta potential of the biopolymers
The zeta potential was determined for protein solutions (OVA,
OVA + Glycerol, WPC and WPC + Glycerol) and for alginate-pectin
mixture solution and emulsion, at concentrations of 0.2% (w/w).
To prepare the polysaccharide mixture solution, pectin and alginate were weighed as dry powders and mixed, deionized water
added to adjust the volume. All solutions were stirred overnight
at room temperature before measurements. The alginate-pectin
mixture solutions (2% w/w, moist weight basis) were emulsied
with 1.0% (w/w, moist weight basis) sunower oil, at room temperature, in a Turrax agitator (IKA, Works do Brasil, Rio de Janeiro RJ,
Brazil) at 14,000 rpm for 3 min. The resulting emulsion was diluted
to a concentration of 0.2% (v/v), and their zeta potentials were
measured, at varying the pH values ranging from 3.0 to 7.0, at room
temperature. Before the readings, the pH of the solutions/emulsions was adjusted manually with HCl (0.1 N) or NaOH (0.1 N).
The measurements were performed using a Zetasizer Nano-Z (Malvern, Worcestershire, U.K.). At least ve measurements were
recorded at each pH.
2.3. Production of microparticles
The emulsion (alginate-pectin-sunower oil) was sprayed over
a 2% (w/w) calcium chloride solution (pH adjusted to 3.5 or 3.75)
at room temperature using a double uid atomizer (/, 1 mm) with
a distance of 12 cm between the tip of the atomizer and the surface
of the calcium chloride solution, an air pressure of 0.125 kgf/cm2
and a spraying rate of 555 mL/h. During spraying, the emulsion
was constantly agitated at room temperature. After spraying, the
microparticles were kept in calcium chloride solution for 30 min.
Next, the microparticles were washed three times with deionized
water (pH 3.5 or 3.75) and separated by sieves (/ 125 lm). The
microparticles obtained by ionic gelation were transferred to egg
white (pH 3.5) and whey (pH 3.75) protein solutions at concentrations of 1.7%, 3% and 4% (w/w), using 50 g of moist particles and
200 mL of protein solution. The microparticles were kept in the
protein solutions for 30 min at room temperature under agitation
(500 rpm). Then, the microparticles were washed three times with
deionized water (pH 3.5 or 3.75) and separated by sieves (/
125 lm) to remove any protein that was not adsorbed onto the
microparticles. Three sets of microparticles were produced independently for each evaluated concentration. A fraction of the moist
microparticles was frozen and lyophilized (Mod. 501, Edwards
Pirani, Crawley, West Sussex, UK) at 40 C and 0.1 mmHg pressure for a total cycle time of 48 h. The dry microparticles were
packed into asks with lids and kept refrigerated. The protein
and dry material contents of the microparticles were determined
according to the AOAC (2006) guidelines using a conversion factors

K.C. Aguilar et al. / Journal of Food Engineering 154 (2015) 1724

of 6.68 and 6.38 for the amounts of nitrogen in OVA and WPC,
respectively. The nitrogen content of the alginate-pectin microparticles was determined and used to correct the total nitrogen content of the microparticles containing adsorbed protein. The
protein content was expressed as the weight percentage (%) of protein/weight of total solids on a dry weight basis.
2.4. Production of the alginate-pectin lms
The emulsion (pectin-alginate sunower oil) was prepared as
described above. The lms were fabricated in two steps; crosslinking and hardening, as in the production of microparticles. In
the rst step, the lm-forming emulsion (50 mL) was transferred
to a square acrylic plate (225 cm2) and sprayed for 1 h with a 2%
(w/w) calcium chloride solution with the pH adjusted (3.5 or
3.75) using a sprayer, with an air pressure of 1.0 kgf/cm2. During
spraying, the plate was kept inside a closed plastic chamber. After
spraying, the plate was removed from the chamber and kept at
room temperature for 15 min to stabilize the lm. In the second
step (hardening time), the lm was manually inverted and
100 mL of 2% (w/w) calcium chloride solution was added for 1 h
to complete the cross-linking and produce polysaccharide lms
(FP3.50 and FP3.75). The moist lms were cut into small discs (
29 mm, 0.5 g), and one disc was transferred to egg white (pH
3.5) and to whey (pH 3.75) protein solutions (10 mL) containing
glycerol (2:1, protein:glycerol, w/w) at a 4% protein concentration
and kept for 30 min under agitation at room temperature to produce protein-coated polysaccharide lms (FP + OVA3.50 and FP +
WPC3.75). The lms were then washed three times with deionized
water (pH 3.5 or 3.75) to remove any protein that was not
adsorbed onto the lms. Three sets of lms were independently
produced. The moist washed lms were weighed to determine
the total solids and adsorbed protein following the same procedure
described previously for the microparticles. The lms were dried
for 36 h at room temperature (25 3 C) and conditioned into desiccators (5054% RH) for three days before physical and mechanical characterization. The preparation of the lms is shown in the
Supporting Materials (video data).
2.5. Characterization of the microparticles and lms
2.5.1. Morphology of the microparticles and lms
The morphologies of the moist microparticles with and without
protein coatings were examined using an optical microscope
(JENAVAL, Carl Zeiss, Toronto, Canada) with 12.5 and 25 objectives and 1 and 1.25 optovar. Image capture was performed
using the EDN-2 Microscopy Image Processing System software
program. The lyophilized microparticles and dry lms with and
without protein coatings were examined with a scanning electron
microscope (model JMS T300 Jeol, Tokyo, Japan). The samples
were xed onto stubs with two-sided copper metallic tape and
covered with a ne layer of gold (180 s and 40 mA current) using
a Baltzer evaporator (Baltec SCD50, Liechtenstein). The analysis
conditions included voltage accelerations of 15 and 20 kV.
2.5.2. Determination of calcium present in the microparticles and lms
after ionic gelation
The calcium content in the alginate-pectin microparticles and
lms after ionic gelation was determined using an atomic absorption spectrophotometer (Analytik Jena AG-NOVAA300, Jena,
Germany) and a standard calcium solution (1000 lg/mL, SCP
Science, lot S120221015, Quebec, Canada) in absorption mode with
an air-acetylene ame detector. Samples of moist microparticles
and lms (400 mg) were dissolved in 25 mL of 3% (w/w) sodium
citrate solution. Preliminary tests revealed that the sodium citrate
solution did not affect the determination of the calcium content. To

19

avoid interference, microparticles and lms were produced without the addition of sunower oil to evaluate the calcium content.
2.5.3. Average size, size distribution, moisture content of
microparticles with and without protein and efciency of
encapsulation
The average size (d 0.5, lm) and the size distribution (d 0.1; d
0.9 and polydispersity index) of the moist microparticles were
determined using a Mastersizer 2000 apparatus (Malvern, Worcestershire, WR, UK) with a Hydro 2000S sampling unit (Malvern,
Worcestershire, WR, UK). Deionized water (at pH 3.5 and 3.75)
was used as the dispersant material to read the samples. The moisture content was gravimetrically determined by drying the samples at 105 C in a vacuum oven (Lab-Line, Squaroid, USA) for
24 h. After this period, the samples were weighed at 1-h intervals
using an analytical balance (Analytical Plus, Ohaus) until a constant weight was reached. The encapsulation efciency was only
determined after ionic gelation. Sodium citrate was added to 5 g
of moist microparticles at a concentration of 3% (w/w) to release
the oil, which was then quantied (Bligh and Dyer, 1959). The
encapsulation efciency was determined on dry basis using the following equation:

EE%

Total oil of the particles g=total solids g

 100
Initial oil g=total solids g

2.6. Functional characterization of the alginate-pectin lms

The thickness of the lms was measured with a digital micrometer (0.001 mm, model MDS-25S, Mitutoyo, Japan) at ten random
positions. The moisture content of the lm was gravimetrically
determined as previously described. The Tensile strength (TS)
and elongation at break (E) of the preconditioned lms were determined at room temperature using a TA.XT2 (Stable Microsystems
SMD, Surrey, UK) according to the ASTM standard method D882
(1995a) and the measurements were repeated at least ve times.
The water vapor permeability coefcient (WVP) through the lms
was determined gravimetrically at 25 C (1 C) according to ASTM
method E96-95 (1995b).
2.7. Statistical analysis
Signicant differences between the means obtained were evaluated using ANOVA and Tukeys test with the aid of the SAS program at a 5% signicance level. Each type of microparticle or lm
was produced in three independent lots, and the determinations
were performed in triplicate for each lot.
3. Results and discussion
3.1. Zeta potential of the biopolymers
The 1:1 alginate-pectin mixture solution and emulsion had negative zeta potential values within the pH range tested (Fig. 1). The
electronegativity of the solution and emulsion increased with
increasing pH, from 31.9 4.3 mV and 30.7 4.5 mV (pH 3.5)
to 63.2 4.7 mV and 60.5 4.8 mV (pH 7), respectively. The
solution and emulsion showed similar values that were not significantly different, indicating that the addition of the oil did not alter
the net charge of the alginate-pectin mixture. Similar behavior was
previously reported for an alginate solution whose the zeta potential decreased from 8.7 to 68.4 mV when the pH was increased
from 2 to 8 (Harnsilawat et al., 2006). The zeta potential of the
WPC solution varied from 22.5 3.7 mV (pH 3) to 27.2 4.2 mV
(pH 7), whereas the zeta potential of the OVA solution varied from

20

K.C. Aguilar et al. / Journal of Food Engineering 154 (2015) 1724

SOL ALG:PEC
EMULALG:PEC
SOL WPC+GLYCEROL
SOL OVA+GLYCEROL

40

Zeta Potential (mV)

20
0

2.5

3.0

3.5

4.0

4.5

5.0

5.5

6.0

6.5

7.0

7.5

-20
-40

Table 1
Size distribution of the alginate-pectin microparticles with and without protein.
Microparticle

d 0.1 (lm)

d 0.9 (lm)

Polydispersity index (span)

MP + WPC3.75
MP + OVA3.50
MP3.75
MP3.50

139
156
100
120

568
631
600
590

1.5
1.6
2.0
1.8

pH

-60
-80
Fig. 1. Zeta potential of the biopolymers (SOLWPC+GLYCEROL, SOLOVA+GLYCEROL,
SOLALG:PEC, EMULALG:PEC) at a concentration of 0.2% (w/w) at pH values ranging
from 3 to 7.

28.5 3.7 mV (pH 3) to 27.9 5.3 mV (pH 7). The addition of

glycerol did not signicantly alter the zeta potential of the proteins. When WPC and WPC with glycerol solutions were evaluated,
the isoelectric point observed for both was 4.3. The egg white proteins solutions with and without glycerol both had an isoelectric
point of 4.5, with no signicant difference between the two.
After ionic interaction between carboxyl groups from the
polysaccharide mixture and calcium ions, the zeta potential of
the particles or lms is expected to be less electronegative than
the zeta potential of the polysaccharide mixture solution before
the addition of calcium ions. Opanasopit et al. (2008) obtained a
zeta potential of 30 mV for a 0.1% pectin solution (low-ester amidated pectin) at pH 4.0 and a zeta potential of 10.4 mV for pectin
microparticles (particle size <10 lm). Unfortunately, in the present
work, the zeta potential of the particles was not measured because
the Zeta Nano-Z apparatus could not be used to this due to the sedimentation of the microparticles.
Because the amount of charge carried by biopolymers is important for complex formation (pKa < pH < pI, De Kruif et al., 2004) the
zeta potentials of the protein solutions and polysaccharide emulsion (Fig. 1) were analyzed to choose the pH values for protein
adsorption evaluation. For the remainder of the study, the pH of
the egg white protein solution was adjusted to 3.5 and that of
the whey protein solution was adjusted to 3.75. The polysaccharide
emulsion was also adjusted to each of these pH values. At these pH
values, the OVA and WPC solutions had zeta potentials of
22.4 3.2 mV and 11.5 3.8 mV respectively, and the alginate-pectin emulsions had values of 30.7 4.5 mV and 33.3 4.6 mV
respectively. In recent studies, the interactions between WPC
emulsions and pectin (Santipanichwong et al., 2008) or WPC and
pectin microparticles (Souza et al., 2012) were adjusted to pH 4.0.
3.2. Average size, size distribution, moisture content of microparticles
with and without protein and efciency of encapsulation
The microparticles produced at pH 3.5 without protein coatings
measured 261 19 lm and the microparticles coated with egg
white proteins measured 287 10 lm. With respect to microparticles produced at pH 3.75, those that were uncoated measured
259 22 lm and that were coated with whey proteins measured
283 6 lm. No signicant difference in size of protein-coated particle was observed.
The size distribution of all samples were unimodal and normal
and the average values of d 0.1, d 0.9 and the polydispersity index
(span) are shown in Table 1. These values indicated a large range of
sizes for all samples, which is attributed to the use of a double uid
atomizer to produce the microparticles.
The moisture content of the microparticles varied from
95.2 0.2% to 88.6 0.3% for microparticles without and with egg

white protein coatings, respectively, and from 95.3 0.2% to

85.2 0.4% for microparticles without and with whey protein coatings respectively, these values were signicantly different
(p < 0.05).
The microparticles without protein produced with the alginatepectin mixture at pH 3.5 and 3.75 had lipid concentrations of
28.9 2.2% and 29.9 2.6% (w/w of total solids on dry weight
basis) and therefore encapsulation efciencies of 87.6% and 90.8%
respectively, and these differences were not signicant. High
encapsulation efciencies of 94% for thyme and clove oil and of
90% for cinnamon oil encapsulated by ionic gelation using alginate
have been previously reported (Soliman et al., 2013).
3.3. Calcium content in alginate-pectin microparticles and lms
The calcium contents in the microparticles produced by ionic
gelation without oil addition were 1.62 0.09 lmol/mg (pH 3.5)
and 1.63 0.06 lmol/mg on a dry weight basis (pH 3.75). For the
lms produced by ionic gelation without oil addition, the calcium
contents were 3.08 0.34 lmol/mg and 2.90 0.22 lmol/mg on a
dry weight basis at pH 3.5 and 3.75, respectively, with no signicant differences. However, signicant differences were observed
when the microparticles and lms were compared. The greater
amount of calcium ions in the alginate-pectin mixture lms may
have resulted from the longer contact time of the lms with calcium ions compared with the contact time used with microparticles. The calcium contents of the lms are similar to the values
reported in a previous study in which alginate and pectin lms
were observed to have calcium contents of 3.29 0.02 and
3.56 0.01 lmol/mg, respectively, on a dry weight basis (Da Silva
et al., 2009).
3.4. Morphology of the microparticles and alginate-pectin lms
Fig. 2AF illustrate the morphologies of the moist and lyophilized alginate-pectin microparticles produced by ionic gelation
with and without protein coatings. The optical micrographs demonstrate that the moist microparticles have a spherical shape and
a broad distribution of oil drops spread throughout the matrix;
on the other hand, the lyophilized microparticles show rough
wrinkled surfaces and do not retain their original spherical shape,
independent of the type of protein (Fig. 2C and F). The presence of
roughness on the surface may be associated with the protein layer
adhered to the microparticles. No visual differences were observed
between microparticles coated with different proteins.
The SEM image of the lm surface without the protein coating
(Fig. 3A) shows visible drops of the lipid material dispersed
throughout the polysaccharide matrix, which is characterized by
a discontinuous and heterogeneous surface. The cross-section
(Fig. 3B) exhibits a high degree of porosity.
The SEM image of the lm coated with whey protein (Fig. 3C)
exhibits a more continuous and homogeneous surface with less
visible lipid drops; however, some holes can be observed. The
cross-section (Fig. 3D) shows a reduction in porosity relative to
that of the uncoated lm (Fig. 3B), most likely due to protein
absorption by this lm. The image of the lm surface coated with

K.C. Aguilar et al. / Journal of Food Engineering 154 (2015) 1724

21

Fig. 2. Optical microscopy (columns 1 and 2) and scanning electron microscopy (column 3) images of the alginate/pectin particles produced by ionic gelation: (A) (pH 3.5)
and (D) (pH 3.75) without coating. (B and C) coated with OVA (pH 3.5) and (E and F) coated with WPC (pH 3.75) at a 4% protein concentration in solution. (A, B, D and E) OM
bar = 100 lm; (C and F) SEM bar = 10 lm.

egg white proteins (Fig. 3E) shows a surface similar to that of the
uncoated lm but with lipid drops that are more spread out. In
general, the inside of the lms with the protein coating (Fig. 3D
and F) showed a more dense and compact structure relative to that
of the lms without the protein coating (Fig. 3B).
3.5. Adsorption of proteins onto alginate-pectin microparticles and
lms
The positively charged groups in the proteins can electrostatically interact with negatively charged groups present in the polysaccharides and the amount of protein adsorbed onto the
microparticles or lms is maximized when the charges of the
adsorbed protein are neutralized. Aside from electrostatic interactions other interactions can occur, including Wan der Waals
forces, hydrogen bonds, hydrophobic effects and steric hindrance
and all can also be responsible for associations between protein
protein and protein surface interactions (Haynes and Norde,
1994).
The amount of protein adsorbed onto the coated microparticles
increased as the protein concentration in solution increased, independent of the type of protein. The amount adsorbed in the case of
whey proteins (pH 3.75) were 38.8 4.7%, 46.1 2.1% and
59.2 1.9%, whereas when the egg white proteins (pH 3.50) were
used, the amounts adsorbed were 25.7 1.4%, 33.1 2.2% and
45.5 2.8% at concentrations of 1.7%, 3% and 4% protein in solution,
respectively. Recently, Souza et al. (2012) studied whey proteins
adsorption onto pectin-based microcapsules produced by ionic
gelation and obtained 50% protein adsorption (w/w, db) when
working with a high protein concentration in solution (12%). The
adsorption of whey proteins and egg white proteins onto alginate
and pectin-based microparticles was previously evaluated (Tello
et al., 2015). The authors found that more protein was adsorbed
onto pectin microparticles (62% of protein adsorption egg white
proteins and 60% of protein adsorption whey proteins) than
onto alginate microparticles when 8% protein in solution was used.
Because the microparticles or lms produced by ionic gelation
are porous, it is possible that in addition to electrostatic interac-

tions, hydrogen bonds and hydrophobic protein-polysaccharide

interactions, protein molecules may have also penetrated the pores
of the microparticles or lms. In the case of microparticles, more
whey protein than egg white protein was adsorbed probably due
to the higher molar mass of ovalbumin (44.5 kDa), compared with
b-lactoglobulin (18.5 KDa). Stewart and Swaigood (1993) used
molecular exclusion chromatography and proteins of known Stoke
radii and found that a-lactalbumin and b-lactoglobulin easily penetrated the pores of alginate microparticles. In another recent
study, the authors found that shorter polyelectrolyte chains
adsorbed larger quantities than larger chains (Malinova and
Wandrey, 2007).
The extents of protein adsorption onto lms were 24.1 1.2%
and 25.3 1.0% for lms coated with egg white protein (pH 3.5)
and whey protein (3.75), respectively, when 4% protein in solution
was used. The amounts of whey proteins or egg white proteins
adsorbed onto the lms were similar to one another, however
the values were different from the results observed for the microparticles indicating that pore size could be an important factor for
the adsorption of small molecules. The amount of calcium bound to
the lms was higher than in the microparticles indicating that
stronger and less porous gels were produced in the case of the
lms. In a previous study in which lms were produced by ionic
gelation using pectin and alginate, a greater amount of calcium
and lower swelling were observed in the alginate lms due to
the more effective cross-linking of calcium and carboxylic groups
when alginate was used (Sriamornsak and Kennedy, 2008).
The adsorption of proteins onto the microparticles was greater
than that onto the lms, independent of the type of protein coating
used, at a concentration of 4% protein in solution. It is possible that
the smaller quantity of free carboxylic groups present after ionic
gelation resulted in a lower availability of carboxylic groups for
interaction with protein molecules in solution, therefore resulting
in lower quantities of adsorbed protein in the lms. Additionally,
the smaller surface area available for electrostatic interactions
compared with that of the microparticles represents another characteristic of the lms that may be associated with the lower
amount of adsorbed protein.

22

K.C. Aguilar et al. / Journal of Food Engineering 154 (2015) 1724

Fig. 3. SEM images of the alginate/pectin lms obtained by SEM (left column, surface; right column, cross section). (A and B) (pH 3.75) without protein coating, (C and D)
(coated with WPC, pH 3.75) and (E and F) (coated with OVA, pH 3.5) at a 4% protein concentration in solution.

Moisture contents of 96.2 0.1% and 96.3 0.0% were observed

for the lms produced at pH 3.75 and 3.5, respectively. The moisture contents of the protein-coated lms (containing glycerol)
decreased to 93.6 0.4% and 93.8 0.0% when coated with whey
and egg white proteins, respectively, these values were signicantly different (p < 0.05) compared with those obtained for the
lms without protein coatings.
3.6. Functional characterization of the alginate-pectin lms
Because gel formation is very rapid, at least at the gel surface,
the formation of homogeneous lms through casting and solvent
evaporation is difcult. Thus various alternatives have been tested
to improve the homogeneity of the gel lms. Da Silva et al. (2009)
and Bierhalz et al. (2012) produced lms by ionic gelation in two
steps. In the rst step, small amounts of calcium were added to
the lm-forming solution, which was subsequently homogenized
and dried. The lms were then removed from the supports and
placed in a calcium solution to reinforce cross-linking and produce
stronger lms. Using a different strategy, Sriamornsak and
Kennedy (2006) also used external ionic gelation, placing the polysaccharide solution over a dialysis membrane and placing the
membrane on the surface of a calcium solution. The membrane
allowed for the diffusion of calcium ions through the entire contact
area occupied by the lm-forming solution; this method, according

to the authors produced strong homogeneous lms. Another alternative for improving the homogeneity of the polysaccharide distribution during the ionic gelation proposed by Draget et al. (1991)
involved the use of internal ionic gelation, with the addition of
an initially insoluble calcium salt to the lm-forming solution. Subsequently, through pH adjustment, the salt dissociated and became
available for association with carboxylic groups of the polysaccharide present in the solution, thus initiating gelation and producing
a homogeneous distribution of the polysaccharide in the gel.
Recently, Benavides et al. (2012) used this alternative method to
produce alginate lms containing oregano essential oil for antimicrobial applications in foods.
In this study, an alternative strategy to produce such lms
was tested. The polysaccharide lm-forming solution was placed
on an acrylic support, and the support was placed in a closed
chamber. The calcium solution was then passed through sprayers
placed in the closed chamber, producing an environment saturated with micro-droplets of calcium solution over the lm-forming solution. In preliminary tests, various calcium atomization
times, lm-forming solution volumes and hardening times were
tested.
The results in Table 2 indicate that there was a signicant
increase (p < 0.05) in the thickness of the coated lms (FP +
OVA3.50 and FP + WPC3.75) relative to that of the uncoated lms
(FP3.50 and FP3.75).

23

K.C. Aguilar et al. / Journal of Food Engineering 154 (2015) 1724

Table 2
Mechanical characterization of the alginate-pectin lms with and without protein.
Film
FP + WPC3.75
FP + OVA3.50
FP3.75
FP3.50

Thickness (lm)
a

112.7 10.0
107.4 11.1a
81.1 7.7b
73.4 5.0b

TS

(MPa)
a

32.8 2.6
27.6 4.6a
28.2 5.5a
32.6 2.6a

WVP 3(g mm/m2 d kPa)

(%)
b

9.4 1.0
13.2 2.4a
5.9 1.5c
3.0 0.3d

6.7 0.2
8.9 0.4b
11.5 0.6a
11.8 1.3a

Moisture (g H2O/100 g lm)

10.8 0.3b
11.4 0.3b
17.1 1.4a
17.0 0.5a

Mean standard deviation of t replicates. Means with the same letter in the columns indicate that there is no signicant difference according to Tukeys test.
1
TS Tensile strength;
2
E Elongation at break.
3
WVP Water vapor permeability.

Relative to the moisture content of the dried alginate-pectin

lms (17% for the alginate-pectin lms and 1011% for the protein-coated lms), a reduction in the values was observed after
protein coating. The type of protein used for coating did not significantly alter the moisture content of the lms. Rhim (2004)
observed moisture content values between 15.7% and 17.7% for
alginate lms, whereas values between 13% and 18% were
observed for lms composed of alginate and pectin (1:1)
(Bierhalz et al., 2012; Da Silva et al., 2009).
The protein-coated lms showed water vapor permeability values that were signicantly (p < 0.05) lower than those of the
uncoated lms. This result may be associated with an additional
resistance to the migration of water vapor after protein coating.
The lms with whey protein coatings demonstrated lower WVP
values than the lms coated with egg white proteins. A similar
trend was reported by Parris et al. (1995), who observed a reduction in the WVP values of pectin or alginate lms after the addition
of proteins. According to Cuq et al. (1996), the barrier properties of
protein-based lms are generally better than those of polysaccharide lms because in contrast to polysaccharides, proteins are composed of approximately 20 different amino acids that can confer
various functional properties to lms. Whey protein lms exhibit
good mechanical properties and act as an excellent barrier to oxygen relative to lms produced with other proteins (maize zein,
wheat gluten, isolated soy protein) or lms produced with polysaccharides (amide, cellulose, carrageenan and pectin) (Ramos et al.,
2013).
The tensile strength (TS) and Elongation at break (E) values
obtained for the lms (Table 2) indicate that the incorporation of
protein into the lms did not signicantly alter the TS compared
with that of the uncoated. However, the signicant increase in
the elongation of the lms after coating with proteins was
apparent.
The results obtained for tensile strength indicated that after
drying, all lms were fragile and breakable. Films composed of
alginate and pectin cross linked with calcium showed TS values
in the range of 60 to 94 MPa and E values between 3.6% and 14%
(Bierhalz et al., 2012; Da Silva et al., 2009; Pranoto et al., 2005;
Rhim, 2004), some studies have reported alginate lms with TS
values as high as 112 MPa (Fontes et al., 2011) and 160 MPa
(Zactiti and Kieckbusch, 2006).
4. Conclusions
The electrostatic adsorption of proteins onto microparticles or
lms produced by ionic gelation was achieved; however, signicantly greater adsorption onto the microparticles was observed.
This nding was most likely due to the lower amount of calcium
present in the microparticles and, consequently, the greater
amount of remaining free carboxylic groups available to associate
with the positive charges in the protein, as well the larger surface
area of the microparticles compared with that of the lms. The
method used for lm production is practical and simple and may

represent an alternative to producing lms through casting. However, it was not possible to produce lms via a single step (by
spraying calcium micro-droplets onto a lm-forming emulsion).
A second step (hardening time) involving reinforcement with calcium was required, as was the case for microparticle production.
The moist lms were strong, easily handled, and easily removed
from the support on which they were produced. Furthermore, protein adsorption was rapid and simple. However, due to the large
amount of water present in the lms, maintaining lm integrity
during the drying step was difcult. Solutions to this problem
require further investigation.
Acknowledgments
The authors are grateful to CPKelco (Limeira, S.P., Brazil) for the
pectin, to FMC Biopolymer (Campinas, S.P., Brazil) for the alginate
DMB, to Saltos Alimentos Ltda (Salto, S.P., Brazil) for the egg white
proteins and to CAPES (PEC-PG) for the scholarship given to F. T.
and to CONACYT Mxico for the scholarship given to K. C. A.
Appendix A. Supplementary material
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.jfoodeng.2014.12.
020.
References
AOAC, 2006. In: Ofcial Methods of Analysis, 18th ed. Association of Ofcial
Methods Analytical Chemists, Gaithersburg, Maryland.
ASTM, 1995a. Standard test method for tensile properties of thin plastic sheeting.
D882-02, American Society for Testing and Materials, Philadelphia.
ASTM, 1995b. Standard test methods of water vapor transmission of material. E9695, American Society for Testing and Materials, Philadelphia.
Benavides, S., Villalobos-Carvajal, R., Reyes, J.E., 2012. Physical, mechanical and
antibacterial properties of alginate lm: effect of the crosslinking degree and
oregano essential oil concentration. J. Food Eng. 110, 232239.
Bierhalz, A.C.K., Da Silva, M.A., Kieckbusch, T.G., 2012. Natamycin release from
alginate/pectin lms for food packaging application. J. Food Eng. 110, 1825.
Bligh, E.G., Dyer, W.J., 1959. A rapid method of total lipid extraction and
purication. Cana. J. Bioch. Phys. 37 (8), 911917.
Cuq, B., Gontard, N., Cuq, J.L., Guilbert, S., 1996. Functional properties of myobrillar
protein-based biopackaging as affected by lm thickness. J. Food Sci. 61, 580
584.
Da Silva, M.A., Bierhalz, A.C.K., Kieckbusch, T.G., 2009. Alginate and pectin
composite lms crosslinked with Ca2+ ions: effect of the plasticizer
concentration. Carbohydr. Polym. 77, 736742.
Damodaran, S., 2008. Amino acids, peptides, and proteins. In: Fennema, O. (Ed.),
Food Chemistry. Marcel Dekker, New York, USA, pp. 179262.
De Kruif, C.G., Weinbreck, F., De Vries, R., 2004. Complex coacervation of proteins
and anionic polysaccharides. Curr. Opin. Coll. Interface, Sci. 9, 340349.
De Vos, P., Faas, M.M., Spasojevic, M., Sikkema, J., 2010. Encapsulation for
preservation of functionality and targeted delivery of bioactive food
components. Int. Dairy J. 20, 292302.
Devi, N., Kakati, D.K., 2013. Smart porous microparticles based on gelatin/sodium
alginate polyelectrolyte complex. J. Food Eng. 117, 193204.
Draget, K.I., stgaard, K., Smidsrod, O., 1991. Homogeneous alginate gels: a
technical approach. Carbohydr. Polym. 14, 159178.
FAO, 2009. Compendium of food additive specications, Seventy-rst Meeting: FAO
JECFA Monographs Ed.

24

K.C. Aguilar et al. / Journal of Food Engineering 154 (2015) 1724

Fontes, L.C.B., Ramos, K.K., Sivi, T.C., Queiroz, F.P.C., 2011. Biodegradable edible lms
from renewable sources-potential for their application in fried foods. Am. J.
Food Technol. 6, 555567.
Gbassi, G.K., Vandamme, T., Yolou, F.S., Marchioni, E., 2011. In vitro effects of pH,
bile salts and enzymes on the release and viability of encapsulated Lactobacillus
plantarum strains in a gastrointestinal tract model. Int. Dairy J. 21, 97102.
Gombotz, W.R., Wee, S.F., 1998. Protein release from alginate matrices. Adv. Drug
Del. Rev. 31, 267285.
Grant, G.T., Morris, E.R., Ress, D.A., Smith, P.J.C., Thom, D., 1973. Biological
interactions between polysaccharides and divalent cations: the egg-box
model. FEBS Lett. 32, 195198.
Harnsilawat, T., Pangsawatmanit, R., McClements, D.J., 2006. Inuence of pH and
ionic strength on formation and stability of emulsions containing oil droplets
coated by beta-lactoglobulin-alginate interfaces. Biomacromolecules 7, 2052
2058.
Haynes, C.A., Norde, W., 1994. Globular proteins at solid liquid interfaces. Colloid
Surfaces B: Biointerfaces 2, 517566.
Hbrard, G., Hoffart, V., Beyssac, E., Cardot, J.M., Alric, M., Subirade, M., 2010. Coated
whey protein/alginate microparticles as oral controlled delivery systems for
probiotic yeast. J. Microencapsul. 27, 292302.
Humblet-Hua, K.N.P., Scheltens, G., Van der Linden, E., Sagis, L.M.C., 2011.
Encapsulation system based on ovalbumin brils and high methoxyl pectin.
Food Hydrocolloids 25, 307314.
Malinova, V., Wandrey, C., 2007. Loading polyelectrolytes onto porous
microspheres: impact of molecular and electrochemical parameters. J. Phys.
Chem. B 111, 84948501.
Martinsen, A., Storr, I., Skjrk-Brk, G., 1992. Alginate as immobilization material:
III. Diffusional properties. Biotech. Bioeng. 39, 186194.
Mestdagh, M.M., Axelos, M.A.V., 1998. Physico-chemical properties of
polycarboxylate gel phase and their incidence on the retention/release of
solutes. Biopolymer Sci.: Food Non Food Appl. 91, 303314 (Les Colloques 91,
INRA Ed.).
Oakenfull, D., Pearce, J., Burley, R.W., 1997. Protein gelation. In: Damodaran, S.,
Paraf, A. (Eds.), Food Proteins and Their Applications. Marcel Dekker, New York,
USA, pp. 111142.
Opanasopit, P., Apirakaramwong, A., Ngawhirunpat, T., Rajanarata, T., Ruktanonchai,
N., 2008. Development and characterization of pectinate micro/nanoparticles
for gene delivery. AAPSPharmSciTech 9, 6774.
Parris, N., Cofn, D.R., Remon, F.J., Pessen, H., 1995. Composition factors affecting
the water vapor permeability and tensile properties of hydrophilic lms. J.
Agric. Food Chem. 43, 14321435.
Pranoto, Y., Rakshit, S.K., Salokhe, V.M., 2005. Enhancing antimicrobial activity of
chitosan lms by incorporating garlic oil, potassium sorbate and nisin. LWT
Food Sci. Technol. 38, 859865.
Ralet, M.-C., Dronnet, V., Buchholt, H.C., Thibault, J.-F., 2001. Enzymatically and
chemically de-esteried lime pectins: characterisation, polyelectrolyte
behaviour and calcium binding properties. Carbohydr. Res. 336, 117125.

Ramos, O.L., Reinas, I., Silva, S.I., Fernandes, J.C., Cerqueira, M.A., Pereira, R.N., 2013.
Effect of whey protein purity and glycerol content upon physical properties of
edible lms manufactured therefrom. Food Hydrocolloids 30, 110122.
Rhim, J.W., 2004. Physical and mechanical properties of water resistant sodium
alginate lms. LWT Food Sci. Technol. 37, 323330.
Santipanichwong, R., Suphantharika, M., Weiss, J., McClements, D.J., 2008. Core
Shell biopolymers nanoparticles produced by electrostatic deposition of beet
pectin onto heat-Denatured b-lactoglobulin aggregates. J. Food Sci. 73, N23
N30.
Sezer, A.D., Akbuga, J., 1999. Release characteristics of chitosan treated alginate
beads: II. Sustained release of a low molecular drug from chitosan treated
alginate beads. J. Microencapsulation 16, 687696.
Smidsrod, O., 1974. Molecular basis for some physical properties of alginate in gels
state. Faraday Disc. Chem. Soc. 57, 263274.
Smrdel, P., Bogataj, M., Zega, A., Planinsek, O., Mrhar, A., 2008. Shape optimization
and characterization of polysaccharide beads prepared by ionotropic gelation. J.
Microencapsul. 5, 90105.
Soliman, E.A., El-Moghazy, A.Y., El-Din, M.S., Massoud, M.A., 2013.
Microencapsulation of essential oils within alginate: formulation and in vitro
evaluation of antifungal activity. J. Enc. Adsorption Sci. 3, 4855.
Souza, F.N., Gebara, C., Ribeiro, M.C.E., Chaves, K.S., Gigante, M.L., Grosso, C.R.F.,
2012. Production and characterization of microparticles containing pectin and
whey protein. Food Res. Int. 49, 560566.
Sriamornsak, P., Kennedy, R.A., 2006. A novel gel formation method, microstructure
and mechanical properties of calcium polysaccharide gel lms. Int. J. Pharm.
323, 7280.
Sriamornsak, P., Kennedy, R.A., 2008. Swelling and diffusion studies of calcium
polysaccharide gels intended for lm coating. Int. J. Pharm. 358, 205213.
Sriamornsak, P., Kennedy, R.A., 2011. Effect of sodium uorescein on release
characteristics of a macromolecule from calcium alginate beads. Carbohydr.
Polym. 84, 12081212.
Stewart, W.W., Swaigood, H.E., 1993. Characterization of calcium alginate pore
diameter by size exclusion chromatography using protein standards. Enzyme
Microb. Technol. 15, 922927.
Tello, F., Falfan-Corts, R.N., Martinez-Bustos, F., Da Silva, V.M., Hubinger, M.D.,
Grosso, C., 2015. Alginate and pectin-based particles coated with globular
proteins: production, characterization and anti-oxidative properties. Food
Hydrocolloids 43, 670678.
Tolstoguzov, V.B., 2003. Some thermodynamic considerations in food formulation.
Food Hydrocolloids 17, 123.
Ye, A., 2008. Complexation between milk proteins and polysaccharides via
electrostatic interaction: principles and applications a review. Int. J. Food
Sci. Technol. 43, 406415.
Zactiti, E.M., Kieckbusch, T.G., 2006. Potassium sorbate permeability in
biodegradable alginate lms: effect of the antimicrobial agent concentration
and crosslinking degree. J. Food Eng. 77, 462467.