Journal of Food Engineering 97 (2010) 228–234

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Journal of Food Engineering

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

Effect of calcium and sodium caseinates on physical characteristics

of soy protein isolate–lipid films
F. María Monedero, Maria José Fabra, Pau Talens *, Amparo Chiralt
Departamento de Tecnología de Alimentos, Instituto de Ingeniería de Alimentos para el Desarrollo, Universidad Politécnica de Valencia, Camino de Vera, s/n 46022 Valencia, Spain

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

Article history: The effect of addition of caseinates to soy protein isolated (SPI) based films containing lipids (33% of oleic
Received 4 June 2009 acid or 85:15 oleic acid (OA)–beeswax blend (BW)) on water vapour permeability (WVP), mechanical and
Received in revised form 6 October 2009 optical properties was evaluated. SPI–lipids was combined with caseinates (sodium or calcium) in differ-
Accepted 10 October 2009
ent SPI:caseinate ratios with the aim of improving water vapour barrier, mechanical and optical proper-
Available online 20 October 2009
ties of SPI films containing lipids. Caseinate incorporation to SPI based films provoked an increase of
elastic modulus and tensile strength at break, mainly for calcium caseinate. Both caseinates contributed
Keywords:
to increase the water vapour barrier properties of soy protein-based films. Caseinates also provoked an
Soy protein
Caseinate
increase of transparency of SPI based films and colour softening. The most effective combination was
Oleic acid 1:1 sodium caseinate:SPI ratio, when film contains 85:15 oleic acid:beeswax ratio.
Beeswax Ó 2009 Elsevier Ltd. All rights reserved.
Tensile properties
Water vapour permeability
Optical properties

1. Introduction
The good film-forming ability of soy protein has been widely
demonstrated containing (Monedero et al., 2009; Rhim et al.,
1999) or not lipids (Gennadios and Weller, 1991; Gennadios
et al., 1998; Kim et al., 2002; Park et al., 2001; Rhim et al., 2000).
Soy protein isolated (SPI) films present adequate properties to be
used as edible packaging, such as very low aroma and oxygen per-
meability (Gennadios et al., 1993, 1997; Ghorpade et al., 1995),
which makes it useful for applying to sensitive-to-oxygen products
or for preserving flavours. Nevertheless, its high water vapour per-
meability is an obstacle to its use in food coatings (Rhim et al.,
2000). To improve the water barrier ability of SPI films, composite
films consisting of SPI and lipids have been produced. Rhim et al.
(1999) combined SPI with lauric, palmitic, stearic and oleic acid,
finding out that the incorporation of fatty acids decreased water
vapour permeability of the films. Monedero et al. (2009) found a
decrease of almost 50% in water vapour permeability of SPI when
the protein was mixed with beeswax and oleic acid. In this work,
a plasticizing effect of oleic acid was observed that lead to an indi-
rect improvement in the film mechanical properties. Other at-
tempts to improve water vapour permeability of SPI films have
been made, through chemical, physical and enzymatic treatments
(Stuchell and Krochta, 1994; Ghorpade et al., 1995; Gennadios
* Corresponding author. Tel.: +34 963879836; fax: +34 963877369.
E-mail address: pautalens@tal.upv.es (P. Talens).
et al., 1998; Rhim et al., 2000), but few studies have been carried
out by combining SPI with other more hydrophobic proteins. In
this sense, Pol et al. (2002) produced soy protein films thermally
laminated with either single or double coats of corn zein, where
the hydrophobic nature of the corn zein improved the water va-
pour barrier properties of the laminates, while the superior oxygen
barrier characteristics remained unaffected, as compared with the
SPI film. Tensile properties indicated ductile behaviour of the soy
films, but brittle behaviour for the double-coat laminates. Were
et al. (1999) obtained SPI/wheat gluten composite films (with
and without cysteine incorporation) evaluating also the effect of
the pH (3, 7 and 9). Gluten addition and pH 3.0 provoked a de-
crease in water vapour permeability. The best compromise be-
tween barrier and mechanical properties was observed for the
cysteine-added soy:gluten (4:1) film at pH 7.0. These films could
find application as primary packaging for low moisture foods.
Cao et al. (2007) produced gelatin–SPI composite films, where, by
increasing gelatin ratio in composite films, tensile strength, elonga-
tion at break and elastic modulus increased at the same time as
films became more transparent and easier to handle.
Briefly, the highly hydrophilic nature of SPI based films limits
their ability to provide a significant moisture barrier. Lipids or
more hydrophobic proteins can be used in composite films to im-
prove the film’s ability to control water transport. In this sense,
some studies reveal that films from sodium and calcium caseinates
showed better water barrier properties although their films are
less flexible and stretchable (Fabra et al., 2008), especially those

0260-8774/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.jfoodeng.2009.10.014
 F.M. Monedero et al. / Journal of Food Engineering 97 (2010) 228–234
made with calcium caseinate (Ho, 1992; Fabra et al., 2010a). The
mix of caseinates with SPI in composite films, containing or not lip-
ids, could be an interesting alternative to improve SPI film
properties.
In this work, SPI based films containing sodium caseinate (Na-
Cas) or calcium caseinate (CaCas), with and without lipids (oleic
acid (OA) and beeswax (BW)) were obtained. The lipid phase was
incorporated in 1:0.5 protein–lipid ratio and was made up of
100:0 or 85:15 OA:BW ratio. Mechanical and optical properties
and water vapour permeability were measured in the obtained
films to analyze the effect of caseinate incorporation to SPI films,
with and without lipids.
2. Materials and methods
2.1. Materials
Soy protein isolate (SUPRO 760 IP Non-GM, The Solae Company,
Le Grand-Saconnex, Switzerland), Alanate 110 sodium caseinate
and Alanate-310 calcium caseinate (Llorella, S.A. Barcerlona, Spain)
were used as film-forming component of the hydrophilic continu-
ous phase for emulsion-based edible films. Beeswax (Brillocera,
S.A., Valencia) and oleic acid (Panreac Química, S.A. Castellar Del
Vallés, Barcelona, Spain) were used as the hydrophobic dispersed
phase. Glycerol (Panreac Química, S.A. Castellar Del Vallés, Barce-
lona, Spain) was used as plasticizer.
2.2. Solution preparation method
Films with only one protein, SPI, sodium caseinate or calcium
caseinate were formulated containing lipids in 1:0.5 protein:lipid
ratio. The lipid phase consisted of oleic acid–beeswax mixtures in
the ratios 100:0 and 85:15. The lipid ratios in the blend was se-
lected on the basis of previous studies where the best mechanical
and water barrier properties were obtained for these ratios in pure
sodium caseinate (Fabra et al., 2008) and SPI films (Monedero et al.,
2009). Likewise, films with different soy protein isolate:caseinate
(sodium or calcium) mixtures were prepared (in the ratios 4:1,
3:1, 2:1, 1:1) containing the above-mentioned lipid fractions.
Film-forming aqueous dispersions without lipids, containing 8%
(w/w) of caseinate (calcium or sodium) and the amount of plasti-
cizer required to obtain a protein:plasticizer ratio of 1:0.3, were
homogenized for 1 min at 13,500 rpm, followed by 3 min at
20,500 rpm, using an Ultra-Turrax homogenizer (Ultraturax T25,
Janke & Kunkel, Germany). The homogenization took place at
85 °C. For soy protein films, the procedure was the aforementioned
but aqueous dispersions were heated at 90 °C for 30 min, to denat-
uralize the protein and afterwards stirred for about 20 h, prior to
homogenization to favor the solution of the protein. For films con-
taining BW, the amount of beeswax required was melted and
incorporated to the hot solution and was then homogenized for
1 min at 13,500 rpm, followed by 1 min at 20,500 rpm. The tem-
perature of homogenization was 85 °C. The emulsions were cooled
at room temperature and oleic acid was added in the amount re-
quired for film composition. Each emulsion was homogenized
again for 2 min at 20,500 rpm. The film-forming dispersions were
degasified at room temperature with a vacuum pump.
For SPI/caseinate films, film-forming dispersions were prepared
with 8% total protein in the aqueous dispersion. Firstly, SPI was dis-
persed as described above; afterwards, sodium or calcium casei-
nate was incorporated and finally the lipids were added, all of
which was performed in the same way mentioned above. In all ser-
ies the pH of dispersions were adjusted to 7 before the lipid
addition.
229
2.3. Film formation method
Films were prepared by weighing the amount of the degasified
film-forming dispersion that would provide 2 g of total solids on a
150 mm diameter Teflon coated casting plate resting on a level sur-
face (113.2 g solids m2). The solution was spread evenly over the
whole surface area and films were dried for approximately 24 h at
45% RH and 20 °C. Afterwards, dry films were peeled from the cast-
ing surface.
2.4. Tensile properties
A universal test Machine (TA.XTplus model, Stable Micro Sys-
tems, Haslemere, England) was used to determine film elastic
modulus (EM) tensile strength (TS), and elongation (E) at break,
according to ASTM standard method D882 (ASTM, 2002). EM, TS
and E properties were determined from stress–strain curves, esti-
mated from force–distance data. After drying, seven samples of
each obtained film were selected for the tensile property measure-
ments. At least ten replicates of each film formulation were tested.
The cut film samples had a rectangular section of 2.5 cm wide and
10 cm long. Before testing, all samples were equilibrated for four
days at 54% RH in a cabinet using over-saturated magnesium ni-
trate-6-hydrate saturated solution (Panreac Química, S.A. Castellar
Del Vallés, Barcelona, Spain) at 20 °C. Equilibrated film specimens
were mounted in the film-extension grips of the testing machine
and stretched at a rate of 50 mm min1 until breaking. The relative
humidity and temperature of the testing environment was con-
trolled before and after each testing session with a thermohygrom-
eter (testo 60-H2, Testo GmbH & Co., Lenzkirch, Germany) and was
held constant at 54 ± 2% RH and 20 ± 1 °C, respectively.
2.5. Water vapour permeability measurements
A modification of the ASTM E96-92 (McHugh et al., 1993) gravi-
metric method for measuring water vapour permeability (WVP) of
hydrophilic films was employed for all films with Payne perme-
ability cups (Elcometer SPRL, Hermelle/s Argenteau, Belgium). Nine
films were cast from each solution. Upon drying, six films were
chosen for WVP testing based on a lack of physical defects such
as cracks, bubbles or pinholes. Distilled water (15 mL) was placed
in each test cup to expose the film to high relative humidity on
one side through a circular opening of 6 cm diameter. The film side
in contact with the casting plate surface was exposed to the condi-
tions inside the test cups. Once the films were secured, the cups
were placed in a preequilibrated cabinet fitted with a variable-
speed fan to provide a strong driving force across the film for water
vapour diffusion. Thus, the free film surface during film formation
was exposed to the RH of the cabinet. The environment within the
cabinet was held constant at 58% RH using over-saturated magne-
sium nitrate-6-hydrate. The cabinets were placed at controlled
temperature of 5 °C. After the steady state was reached, the cups
were weighed periodically using an analytical balance
(±0.0001 g). Water vapour transmission (WVTR) (g s1 m2) was
determined as the ratio between the slope obtained from the
regression analysis of weight loss data as a function of time, once
the steady state was reached, and film area.
From WVTR data, the values of the vapour pressure on the film’s
inner surface (p2) were obtained taking into account the method
proposed by McHugh et al. 1993 (Eq. (1)) to correct the effect of
concentration gradients established in the stagnant air gap inside
the cup.
P  D  Ln½ðP  p2 Þ=ðP  p1 Þ
WVTR ¼ ð1Þ
R  T  Dz
230 F.M. Monedero et al. / Journal of Food Engineering 97 (2010) 228–234

where, P, total pressure (Pa); D, diffusivity of water through air a*, b*, chrome (C*ab) and hue angle (h*ab) were calculated. Chrome
at 5 °C (2.3  105m2 s1); R, gas law constant (8.314 Pa m3 g1 and hue angle were calculated from Eqs. (3) and (4).
mol1 K1), T, absolute temperature; Dz, mean stagnant air gap qffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi

height, considering the initial and final z value; p1, water vapour C ¼ ða Þ2 þ ðb Þ2 ð3Þ
pressure on the solution surface; p2, corrected water vapour pres-
sure on the film’s inner surface in the cup.  b


Water vapour permeance (g m2 s1 Pa1) was calculated using h ¼ arctg ð4Þ
a
Eq. (2) as a function of the vapour pressure on the inner surface of
the film (p2) and on the film’s outer surface in the cabinet (p3). Per- Total colour differences (DT) with respect to the control film
meability (g m1 s1 Pa1) was obtained by multiplying the perme- and whiteness index (WI) were also determined using Eqs. (5)
ance by the average film thickness. and (6) (Francis and Clydesdale, 1975; Rhim et al., 1999):
qffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi

Permeance ¼
WVTR
ð2Þ DE ¼ ðDa Þ2 þ ðDb Þ2 þ ðDL Þ2 ð5Þ
ðp2  p3 Þ
qffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi
2
WI ¼ 100  ð100  L Þ2 þ a2 þ b ð6Þ
2.6. Film thickness measurements

Film thickness was measured with a Palmer digital micrometer
to the nearest 0.0025 mm at six random positions around the film
used for WVP measurements and at four positions along the strips
used for the mechanical properties. Average values were used in all
WVP and tensile property determinations. Average thickness of
films was 0.100 ± 0.009 mm.
2.8. Statistical analysis
Statistical analysis of data was performed through analysis of
variance (ANOVA) using Statgraphics Plus for Windows 5.1 (Manu-
gistics Corp., Rockville, MD). Fisheŕs least significant difference
(LSD) procedure was used, considering a confidence level of 95%.

2.7. Optical properties 3. Results and discussion

The optical parameters of films were measured with a MINOLTA
spectro-colorimeter (model CM-3600d (Minolta Co., Tokyo, Japan).
Film samples were placed on a white standard plate (white calibra-
tion standard plate 78271018) and reflection spectra were mea-
sured. With these values and standard light source D65 and
standard observer 10° CIE-Lab, colour parameters luminosity (L*),
3.1. Effect of caseinates on tensile properties of SPI–lipid films
Values of tensile parameters (EM, TS and E) of SPI–lipid–casei-
nate films were plotted in Fig. 1 as a function of the SPI–caseinate
ratio for the different lipid and caseinate types. The values of pure
SPI, NaCas and CaCas, previously published (Monedero et al., 2009;

800 14
700 12
600
10
500
EM (MPa)

TS (MPa)

8
400
6
300
200 4

100 2
0 0
1:0 4:1 3:1 2:1 1:1 0:1 1:0 4:1 3:1 2:1 1:1 0:1
SPI:caseinate SPI:caseinate

45
40
SPI/NaCas 85:15
35 SPI/NaCas 100:0
30 SPI/CaCas 85:15
25 SPI/CaCas 100:0
E (%)

SPI
20
NaCas
15 CaCas
10
5
0
1:0 4:1 3:1 2:1 1:1 0:1
SPI:caseinate

Fig. 1. Mean values (and LSD intervals) of elastic modulus (EM), tensile strength (TS) and elongation at break (E) as a function of SPI:caseinate ratio. Values of pure protein
films are shown.
 F.M. Monedero et al. / Journal of Food Engineering 97 (2010) 228–234
Fabra et al., 2008) are also reflected as a reference. As expected,
elastic modulus (EM) and tensile strength (TS) were higher
(p < 0.05) for calcium caseinate films than for NaCas and SPI films,
while elongation at break (E) was lower (p < 0.05), indicating that
SPI and NaCas provide more flexible structures, in contrast to Ca-
Cas which gives rise to stiffer and less flexible films. This effect
has been related to the different electrostatic interactions and cal-
cium cross-linking between polymer chains, which strengthen the
matrix structure, thus increasing the film stiffness (Ho, 1992; Fabra
et al., 2010a). Without lipids, SPI films are slightly stiffer than Na-
Cas films, although they show similar behaviour at break. Never-
theless, they behave differently when lipids are incorporated.
Lipid presence significantly affected (p < 0.05) all tensile parame-
ters analyzed. In general, lipids produced a decrease in EM and
TS values and an increase in E values, to a different extent depend-
ing on the protein matrix. This has been related with the develop-
ment of a heterogeneous structure, where lipids lead to
discontinuities in the protein matrix (Fabra et al., 2009) which
makes it less resistant to fracture, but more deformable. The effect
of lipid type differed according to the protein matrix. In SPI films,
no significant differences (p > 0.05) were found in any of the
mechanical parameters for the two lipid combinations used. This
means that the partial substitution of OA by BW did not modify
mechanical behaviour of SPI films. Nevertheless, in NaCas based
films, significant differences were observed (p < 0.05) for all the
mechanical parameters, depending on the lipid type. While pure
OA induced a great decrease of EM and TS and a great increase in
E, the incorporation of BW led to a recovery of the values. In CaCas
based films, the lipid type provoked no differences in EM and TS,
although films with pure OA were significantly more deformable
than those prepared using BW, which in turn were less deformable
than those which were lipid free. Interactions between OA and Na-
Cas have been demonstrated through DSC analysis and lead to a
plasticization effect of the lipid on NaCas matrix when equilibrated
with a relative humidity higher than 52% (Fabra et al., 2010b DSC).
This effect is responsible for the great decrease in EM and TS and
the increase in the deformation capacity, which was not observed
to the same extent in CaCas and SPI matrices where probably the
same lipid–protein interactions did not occur.
Fig. 1 reflects that the incorporation of caseinates (sodium or
calcium) to SPI–lipid films provoked a significant increase
(p < 0.05) of EM and TS with respect to the SPI based films, this ef-
fect being more noticeable for calcium caseinate. CaCas contributes
to the modification of the film matrix mechanical response to the
greatest extent, giving rise to the stiffest and the least flexible
and stretchable films. A progressive increase in the stiffness and
TS occurred when the ratio of caseinate (Na or Ca) increased in
the protein matrix when lipid was pure OA, whereas these values
did not vary significantly with the caseinate ratio when the lipid
present was 85:15 OA:BW. Only one exception can be mentioned
which was the film made up of 1:1 SPI:NaCas and 85:15 OA:BW,
which showed much higher values of EM and TS, thus indicating
particular interactions among the components that give rise to
reinforcements of the entanglements in the polymer matrix. The
increase in mechanical resistance of SPI films by the incorporation
of sodium caseinate, above the values of pure proteins, seems to
indicate that both proteins are thermodynamically compatible
and a mixte network was obtained where the interactions forces
of proteins are stronger than in the pure protein network. It is
remarkable that the intense plasticizing effect observed for OA in
NaCas matrix seems not to occur when this caseinate is mixed with
SPI and the values of EM and TS of the composite film tend towards
that shown by pure NaCas when this component increases in the
mixture. It is also remarkable that, contrary to that observed in Na-
Cas films, pure OA in the matrix gives rise to films which are stiffer
and more resistant to break than the OA:BW mixture, especially
231
when caseinate increases in the matrix. This is coherent with the
establishing of protein–protein interactions that can inhibit the
OA–caseinte interactions responsible of the plasticizing effect. In
SPI–CaCas films protein–protein interactions in the mixte network
difficult the establishment of Ca cross-linking and mechanical
resistance of these films is lower than those of CaCas matrix, espe-
cially when BW is present en the lipid blend, since OA contributes
to increase the mechanical resistance of the film.
Film deformability decreased when small ratios of CaCas were
incorporated in the mixture, but practically recovered the values
of the SPI films when the CaCas ratio increased. Small and fluctuat-
ing changes occurred in this parameter when NaCas were incorpo-
rated in the SPI matrix. Lipid type did not affect film deformability
of SPI–CaCas films, except for 1:1 CaCas:SPI film, where the films
with pure OA appeared more deformable than those containing
BW, as occurs in CaCas films. This influence was slightly more pro-
nounced in SPI:NaCas films where, in general, those containing
pure OA were more deformable.
3.2. Effect of caseinates on water vapour permeability of SPI–lipid films
Fig. 2 shows the values for the water vapour permeability of the
composite films as a function of SPI:caseinate ratio for the two
types of caseinates and lipids. Values of the pure protein films
are indicated as a reference. As expected, WVP of calcium caseinate
films were significantly (p < 0.05) lower than those corresponding
values of SPI and NaCas based films. Divalent ions, such as calcium,
promotes intermolecular cross-linking, which tightens the protein
matrix by limiting biopolymer segmental mobility, which produces
films with reduced WVP (Ho, 1992; Banerjee and Chen, 1995;
Fabra et al., 2010a). The highest values of WVP observed for SPI
based films can be attributed to the higher number of polar amino
acids present in this protein (McHugh et al., 1993), which makes
the matrix more hydrophilic, thus showing less resistance to water
transport. As has been previously reported, water barrier efficiency
increases with lipid addition, mainly when beeswax is used, be-
cause it is more efficient at controlling moisture migration than
OA (Fabra et al., 2008; Monedero et al., 2009). This effect is partic-
ularly intense for NaCas and less relevant for CaCas probably due to
the fact that the values in this protein matrix are already low.
The kind of lipid and the SPI:caseinate ratio were observed to
cause significant differences (p < 0.05) in WVP, but not the casei-
nate type (p > 0.05). Similar behaviour was observed for SPI:NaCas
and SPI:CaCas films when these contain the same lipid. When
beeswax was added, a statistically significant decrease (p < 0.05)
of WVP was observed, in agreement with that observed for each
isolated protein and which can be attributed to its non-polar char-
acter, which makes it more efficient at controlling moisture migra-
tion than OA. The effect of caseinate incorporation in the SPI matrix
was similar for both lipids, showing a decrease of WVP as caseinate
(sodium or calcium) content increased, although a change of ten-
dency was observed for CaCas films containing 85:15 OA:BW from
2:1 SPI–caseinate ratio. This could be attributed to an increase in
the film structural heterogeneity, associated with aggregate forma-
tion when CaCas increased. Some studies reveal both the greater
ability of calcium caseinate to form aggregates in the emulsion
(Srinivasan et al., 1999) and also the loss of the emulsifying capa-
bility of CaCas (Khwaldia et al., 2004). As a result, lipid could not be
dispersed effectively in the protein matrix, decreasing the film tor-
tuosity which further increases water diffusion and thus WVP
(Debeaufort and Voilley, 1995). The decreasing tendency of the
WVP of the film when the caseinate ratio increased in the mixture
was particularly marked for NaCas when the lipid was 85:15
OA:BW mixture and when protein ratio was 1:1. This confirms
the hypothesis mentioned from the mechanical behaviour analysis
of particularly intense component interactions in this case, which
232 F.M. Monedero et al. / Journal of Food Engineering 97 (2010) 228–234
2.0
1.5
WVP (g·m·Pa-1·s-1·m -2)·109
1.0
0.5
0.0
1:0 4:1
Fig. 2. Mean values (and LSD intervals) of WVP as a function of SPI:caseinate ratio. Values of pure protein films are shown.
reinforce the tightening of the matrix and makes mass transfer
more difficult.
In order to improve water vapour barrier of soy protein films,
other authors combined SPI with different proteins. Were et al.
(1999) obtained soy protein (SPI)–wheat gluten (G) films with
SPI:G ratios 4:1, 3:1 and 2:1, with or without cysteine addition at
pH 3, pH 7 and pH 9. They found that cysteine addition had no ef-
fect on the WVP decrease but SPI–G films showed lower WVP than
control films without wheat gluten. The lowest WVP values were
0.6  109 g m1 s1 Pa1 and they were obtained for films SPI:G
ratios 4:1 and 2:1, at pH 7. WVP of the control film, at the same
pH, were 2.5  109 g m1 s1 Pa1 with cysteine and 2.7  109
g m1 s1 Pa1 without cysteine addition. Pol et al. (2002) obtained
soy protein isolate–corn zein laminated films by using the thermal
compaction technique. They laminated soy based films with one or
two layers of corn zein (both sides of the soy film), thus reaching a
significant decrease in WVP for laminated films as compared to
control film. They obtained a 20% of reduction for single coated
laminate films (1.61  109 g m1 s1 Pa1) and a 50% of reduction
for double coated laminate films (0.9  109 g m1 s1 Pa1) re-
lated to soy based film (2.02  109 g m1 s1 Pa1).
3.3. Effect of caseinates on optical properties of SPI–lipid films
Fig. 3 shows the reflectance spectral distributions of SPI–casei-
nate films, showing the effect of NaCas and CaCas on SPI–lipid
films. Since spectral measurements were taken upon the white
standard plate, the more similar and nearer to the white standard
spectrum, the more transparent the films. SPI based films were
slightly yellow due to the greater light absorption in the blue
wavelength range. In contrast, sodium and calcium caseinate based
films showed a more similar spectral distribution which was
nearer to the white standard, which gave rise to more colourless,
transparent films than SPI. As can be observed in Fig. 3, lipid addi-
tion modified film spectra in different ways. For SPI films, the oleic
acid addition provoked an increase in reflection as compared with
the film containing beeswax in the lipid blend, which indicates that
OA slightly increases the film transparency. In caseinate based
films, both lipid types provoked a decrease in transparency and
SPI/NaCas
OA:BW 85:15
SPI/CaCas
OA:BW 85:15
SPI/NaCas
OA:BW 100:0
SPI/CaCas
OA:BW 100:0
SPI
NaCas
CaCas
3:1 2:1 1:1 0:1
SPI:caseinate
yellowness promotion. This occurs to a similar extent for NaCas
based films with both lipids, but to a lesser extent for OA–BW
blends in CaCas films. Molecular interactions developed in the ma-
trix and the heterogeneity in the refractive index of continuous and
dispersed phases are responsible for these slight absorption
changes. In general, the incorporation of caseinate (sodium or cal-
cium) to the SPI–lipid films lead to an increase in transparency and
a decrease of colour intensity, especially when the ratio of casei-
nate increase in the mixture (1:1).
To better understand optical properties of SPI/caseinate films,
colour coordinates L*, Cab* and hab* and WI were analyzed and col-
our difference (DE) due to lipid addition or protein mixture was
determined. Table 1 shows the values for these optical parameters
for all studied films. As can be observed, all films show high lumi-
nosity, the values being greater for caseinate films, as expected
from the reflection spectrum. Adding lipids to caseinate films
slightly decreased luminosity, but the luminosity of SPI films in-
creased when OA was added and decreased with OA:BW blend.
The hue angle values (around 93) and higher chroma values reflect
the greater yellowness of SPI films as compared with caseinates.
The yellowness was scarcely affected by lipid addition in any pro-
tein film. Since spectra were measured upon a standard white
plate, the whiteness index can be used as a measurement of the
films’ transparency. In this sense, caseinates provide films which
are more transparent and colourless than SPI ones. The whiteness
index (and so film transparency) was not affected by lipid incorpo-
ration in NaCas films, but it slightly decreased in CaCas films for
both lipid types and increased in SPI films when pure OA was
added. Concerning colour differences induced by lipid incorpora-
tion to protein matrix, it produces very small DE values (in the hu-
man perception threshold) and slightly higher in CaCas.
As shown in Table 1, incorporation of caseinates modified very
slightly luminosity of SPI–lipid films when the caseinate ratio in-
creases. When increase caseinate ratio in the matrix, a very slight
tendency to increase in Hue angle (hab*) and a tendency to decrease
in colour saturation was observed, which means that yellowness of
SPI films is reduced when mixed with caseinates, mainly at the ra-
tio 1:1 SPI:caseinate. This effect is slightly lower in NaCas than in
CaCas and quite similar for OA and OA:BW mixture. So, in general,
 F.M. Monedero et al. / Journal of Food Engineering 97 (2010) 228–234 233

100 100

90 90

80 80

%R
%R

70 SPI:NaCas 4:1; OA:BW 100:0 70

SPI:NaCas 4:1; OA:BW 85:15
SPI:NaCas 3:1; OA:BW 100:0 SPI:NaCas 3:1; OA:BW 85:15
60 SPI:NaCas 2:1; OA:BW 100:0 60
SPI:NaCas 2:1; OA:BW 85:15
SPI:NaCas 1:1; OA:BW 100:0 SPI:NaCas 1:1; OA:BW 85:15
50 SPI:NaCas 1:0; OA:BW 100:0 50 SPI:NaCas 1:0; OA:BW 85:15
SPI:NaCas 0:1; OA:BW 100:0 SPI:NaCas 0:1; OA:BW 85:15
40 40
400 450 500 550 600 650 700 400 450 500 550 600 650 700
λ (nm) λ (nm)

100 100

90 90

80 80
%R

%R
70 SPI:CaCas 4:1; OA:BW 100:0 70
SPI:CaCas 4:1; OA:BW 85:15
SPI:CaCas 3:1; OA:BW 100:0 SPI:CaCas 3:1; OA:BW 85:15
60 SPI:CaCas 2:1; OA:BW 100:0 60 SPI:CaCas 2:1; OA:BW 85:15
SPI:CaCas 1:1; OA:BW 100:0 SPI:CaCas 1:1; OA:BW 85:15
50 SPI:CaCas 1:0; OA:BW 100:0 50 SPI:CaCas 1:0; OA:BW 85:15
SPI:CaCas 0:1; OA:BW 100:0 SPI:CaCas 0:1; OA:BW 85:15
40 40
400 450 500 550 600 650 700 400 450 500 550 600 650 700
λ (nm) λ (nm)
Fig. 3. Reflectance spectral distribution (measured on a white standard plate) of SPI:caseinate (Na and Ca) films with 1:0 and 85:15 OA:BW ratio.

Table 1
Optical parameters of SPI–lipid–caseinate composite films.

Protein SPI:caseinate OA:BW L* C* h*ab DT WI

j a
NaCas 0:1 0:0 97.5(0.1) 3.0(0.1) 103.2(0.3)i – 96.1(0.1)k
100:0 96.6(0.6)h,i 3.2(0.3)a,b 104.7(0.9)j 1.0(0.4)$ 95.3(0.3)j,k
85:15 96.4(0.5)h 2.9(0.3)a 105.7(0.3)j 1.1(0.5)$ 95.0(0.6)i,j,k
CaCas 0:1 0:0 97.4(0.1)j 2.7(0.3)a 101.7(0.8)i – 96.2(0.3)k
100:0 97.2(0.1)i,j 5.4(0.2)b,c,d 102.3(0.1)i 2.6(0.2)$ 93.9(0.2)i
85:15 97.2(0.1)i,j 4.4(0.4)b,c 102.6(0.3)i 1.7(0.4)$ 94.8(0.4)i,j
SPI 1:0 0:0 94.0(0.1)b 10.1(0.2)j,k,l 93.9(0.3)c,d,e – 88.2(0.3)b,c
100:0 95.0(0.1)c,d,e,f 9.4(0.2)h,i,j,k 94.9(0.2)e,f,g 1.3(0.3)$ 89.4(0.3)d
85:15 93.9(0.1)b 11.5(0.3)l,m 92.9(0.3)c 1.4(0.1)$ 87(0)a,b
SPI/NaCas 4:1 100:0 94.4(0.1)b,c 9.6(0.5)i,j,k 93.6(0.4)c,d 0.8(0.2) 88.9(0.5)c,d
3:1 94.5(0.7)b,c,d 9.5(1.3)i,j,k 92.9(0.9)c 1.3(0.3) 89.1(1.5)c,d
2:1 95.2(0.3)d,e,f 8.0(0.7)f,g 94.0(0.7)c,d,e 1.4(0.7) 90.7(0.8)e,f,g
1:1 95.7(0.2)f,g 6.5(0.6)c,e 96.0(0.5)g,h 2.9(0.6) 92.2(0.6)h
SPI/NaCas 4:1 85:15 94.6(0.6)b,c,d 9.5(0.7)i,j,k 94.9(0.5)e,f,g 2.1(0.8) 89.0(0.9)c,d
3:1 94.5(0.5)b,c,d 10(1)j,k 94.7(0.5)d,e,f 1.8(1.2) 88.8(1.2)c,d
2:1 93.0(0.7)a 8.5(2.1)g,h,i 95.3(1.9)f,g 3.2(2.1) 89.0(1.7)c,d
1:1 95.5(0.6)d,f,g 7.0(0.6)e,f 95.8(0.2)g,h 4.8(0.7) 91.7(0.8)g,h
SPI/CaCas 4:1 100:0 94.0(0.4)b 12.3(1.5)m 95.0(0.7)b 3.2(1.4) 86.3(1.4)a
3:1 95.4(0.1)d,f 9.3(0.2)h,i,j,k 95.7(0.1)a 0.4(0.1) 89.6(0.1)d,e
2:1 95.0(0.4)c,d,e 10.1(1.1)j,k,l 95.5(0.8)a 1.1(0.8) 89(1)c,d
1:1 95.4(0.7)d,f 9(1)g,h,i,j 96.4(1.2)a 1.1(0.2) 89.7(0.3)d,e,f
SPI/CaCas 4:1 85:15 94.5(0.4)b,c,d 10.5(0.6)k,l 95.0(0.3)e,f,g 1.2(0.7) 88.1(0.7)b,c
3:1 94.9(0.3)c,d,e 9.9(0.5)j,k 96.0(0.3)g,h 2.0(0.6) 88.9(0.6)c,d
2:1 94.6(0.4)b,c,d 10.0(0.3)j,k,l 95.8(0.1)f,g,h 1.7(0.3) 88.6(0.3)c,d
1:1 96.1(0.1)g,h 8.2(0.3)f,g,h 97.0(0.2)h 4.0(0.3) 90.9(0.3)f,g,h

DE without symbols are referred to the corresponding SPI–lipid film with 100:0 or 85:15 OA:BW ratio, respectively. Same letter a, b, . . . in a column indicates homogeneous
groups (p < 0.05).
$
DE values with respect to the corresponding film without lipid.

all studied SPI/caseinate composite films showed high luminosity incorporation in SPI–lipid films were, in general, higher than 1 an
and less yellowness than SPI films. DE values induced by caseinate increased when caseinate concentration increased in the films.
234 F.M. Monedero et al. / Journal of Food Engineering 97 (2010) 228–234
This increase in colour difference agrees with the colour softening
of SPI by incorporation of caseinates (more clearly for NaCas)
which is coherent with the increase of whiteness index.
4. Conclusions
The combination of SPI with caseinates to obtain edible films
with lipids (OA and 85:15 OA:BW blend) provoked an increase in
mechanical properties: elastic modulus and tensile strength at
break, mainly if this was CaCas. Film stretchability decreased with
CaCas, but oscillated around the SPI values for NaCas, depending on
the caseinate ratio and lipid composition. Water vapour permeabil-
ity decreased by caseinate addition, mainly when OA:BW blend
(85:15 OA:BW) was added. In this case, a clear increase in water
vapour barrier was observed for 1:1 NaCas:SPI ratio, at the same
time as the film became stiffer and more resistant to break. White-
ness index and transparency of the SPI films increased when case-
inates increased in the matrix, for both OA–SPI films and OA:BW–
SPI films. So, caseinate incorporation (especially NaCas) to SPI
based films has two advantages: a statistically significant reduc-
tion in WVP, which is the most important handicap of SPI based
films, and colour softening. In this sense, 1:1 SPI:NaCas with
85:15 OA:BW ratio should be recommended.
Acknowledgments
The authors acknowledge the financial support from the Span-
ish Ministerio de Educación y Ciencia through the project
AGL2007-65503/ALI, from the Conselleria de Educación de la
Comunidad Valenciana through the Project GVPRE/2008/355 and
from Vice-rectorate for Research from UPV through the project
PAID-06-08-3242.
References
ASTM, 2002. Standard test methods for tensile properties of plastics. Standard
designation: D882-02. In: ASTM International. United States.
Banerjee, R., Chen, H., 1995. Functional properties of edible films using whey
protein concentrate. Journal of Dairy Science 78 (8), 1673–1683.
Cao, N., Fu, Y., He, J., 2007. Preparation and physical properties of soy protein isolate
and gelatin composite films. Food Hydrocolloids 21 (7), 1153–1162.
Debeaufort, F., Voilley, A., 1995. Effect of surfactants and drying rate on barrier
properties of emulsified edible films. International Journal of Food Science and
Technology 30, 183–190.
Fabra, M.J., Talens, P., Chiralt, A., 2008. Tensile properties and water vapor
permeability of sodium caseinate films containing oleic acid–beeswax
mixtures. Journal of Food Engineering 85, 393–400.
Fabra, M.J., Talens, P., Chiralt, A., 2009. Microstructure and optical properties of
sodium caseinate films containing oleic acid–beeswax mixtures. Food
Hydrocolloids 23, 676–683.
Fabra, M.J., Talens, P., Chiralt, A., 2010a. Influence of calcium on tensile, optical and
water vapour permeability properties of sodium caseinate edible films. Journal
of Food Engineering 96, 356–364.
Fabra, M.J., Talens, P., Chiralt, A., 2010b. Water sorption isotherms and phase
transitions of sodium caseinate–lipid films as affected by lipid interactions.
Food Hydrocolloid, in press.
Francis, F.J., Clydesdale, F.M., 1975. Food Colorimetry: Theory and Applications. The
AVI Publishing Company, Westport.
Gennadios, A., Weller, C.L., 1991. Edible films and coatings from soymilk and soy
protein. Cereals Foods World 36 (12), 1004–1009.
Gennadios, A., Weller, C.L., Testin, R.F., 1993. Temperature effect on oxygen
permeability of edible protein-based films. Journal of Food Science 58 (1),
212–214, 219.
Gennadios, A., Weller, C.L., Hanna, M.A., 1997. Soy protein/fatty acid films and
coatings. Inform 8 (6), 622–624.
Gennadios, A., Rhim, J.W., Handa, A., Weller, C.L., Hanna, M.A., 1998. Ultraviolet
radiation affects physical and molecular properties of soy protein films. Journal
of Food Science 63 (2), 225–228.
Ghorpade, V.M., Li, H., Gennadios, A., Hanna, M.A., 1995. Chemically modified soy
protein films. Trans ASAE 38, 1805–1808.
Ho, B., 1992. Water Vapor Permeabilities and Structural Characteristics of Casein
Films and Casein–Lipid Emulsion Films. M.S. thesis, University of California,
Davis.
Khwaldia, K., Banon, S., Desobry, S., Hardy, J., 2004. Mechanical and barrier
properties of sodium caseinate–anhydrous milk fat edible films. International
Journal of Food Science and Technology 39, 403–411.
Kim, K.M., Weller, C.L., Hanna, M.A., Gennadios, A., 2002. Heat curing of soy protein
films at selected temperatures and pressures. Lebensmittel Wissenschaft and
Technologie 35 (2), 140–145.
McHugh, T.H., Avena-Bustillos, R., Krochta, J.M., 1993. Hydrophilic edible films:
modified procedure for water vapor permeability and explanation of thickness
effects. Journal of Food Science 58 (4), 899–903.
Monedero, F.M., Fabra, M.J., Talens, P., Chiralt, A., 2009. Effect of oleic acid–beeswax
mixtures on mechanical, optical and water barrier properties of soy protein
isolate based films. Journal of Food Engineering 91 (4), 509–515.
Park, S.K., Rhee, C.O., Bae, D.H., Hettiarachchy, N.S., 2001. Mechanical properties and
water-vapor permeability of soy-protein films affected by calcium salts and
glucono-d-lactone. Journal of Agricultural and Food Chemistry 49, 2308–2312.
Pol, H., Dawson, P., Acton, J., Ogale, A., 2002. Soy protein isolate/corn zein laminated
films: transport and mechanical properties. Journal of Food Science 67 (1), 212–
217.
Rhim, J., Wu, Y., Weller, C.L., Schnepf, M., 1999. Physical characteristics of emulsified
soy protein–fatty acid composite films. Sciences des Aliments 19, 57–71.
Rhim, J.W., Gennadios, A., Handa, A., Weller, C.L., Hanna, M.A., 2000. Solubility,
tensile, and color properties of modified soy protein isolate films. Journal of
Agricultural and Food Chemistry 48 (10), 4937–4940.
Srinivasan, M., Singh, H., Munro, P.A., 1999. Adsorption behaviour of sodium and
calcium caseinates in oil-in-water emulsions. International Dairy Journal 9,
337–341.
Stuchell, Y.M., Krochta, J.M., 1994. Enzymatic treatments and thermal effects on
edible soy protein films. Journal of Food Science 59, 1332–1337.
Were, L., Hettiarachchy, N.S., Coleman, M., 1999. Properties of cysteine-added soy
protein–wheat gluten films. Journal of Food Science 64 (3), 514–518.