Journal of Food Engineering 83 (2007) 521530

www.elsevier.com/locate/jfoodeng

Whey protein isolate-based edible lms as aected

by protein concentration, glycerol ratio and pullulan addition
in lm formation
Mahamadou Elhadji Gounga, Shi-Ying Xu, Zhang Wang *
Department of Food Science and Engineering, School of Food Science and Technology, Southern Yangtze University, Wuxi 214036, PR China

Received 19 December 2006; received in revised form 4 March 2007; accepted 1 April 2007
Available online 19 April 2007

Abstract

Edible lms were prepared from whey protein isolate (WPI), and characterized in order to select a best combination of protein con-
centration and glycerol (Gly) ratio. 5%, 7% and 9% (w/v) WPI were used at three WPI:Gly ratios (3.6:1; 3:1; and 2:1). 5% WPI with a
3.6:1 WPI:Gly ratio showed the best combination with factors considered being thickness and water vapor permeability (WVP), while the
9% WPI with 3.6:1 WPI:Gly showed the best result as seen from the oxygen permeability (OP). Further studies were conducted by adding
pullulan (PUL) at dierent WPI:PUL ratios (1:0; 1:1; 2:1; 3:1; 4:1; 5:1; 6:1; 8:1; 10:1) to a selected lm in order to investigate the eect of
pullulan on thickness, OP, WVP, moisture content (MC), lm solubility (FS) and morphology using scanning electron microscopy
(SEM). WPIPUL lm had a good appearance and 1:1 WPI:PUL resulted in lms with greatest values of OP, WVP, MC, FS, and trans-
mittance. The SEM micrographs showed many pinholes and a favorable structure for the low barrier ability. However, addition of PUL
at low concentration was good enough to signicantly modify these properties, hence improving the potential characteristics of WPI-
based lms for food applications.
2007 Published by Elsevier Ltd.

Keywords: Whey protein isolate; Glycerol; Edible lms; Protein concentration; Pullulan; Thickness; Barrier properties; Microstructure

1. Introduction
Whey protein fractions (a-Lactalbumin (a-LA) and b-
Lactoglobulin (b-LG)) and whey protein isolate (WPI;
>90%) have been studied for lm formation (Fairley,
Monahan, German, & Krochta, 1996; Kaya & Kaya,
2000; McHugh, Aujard, & Krochta, 1994; McHugh &
Krochta, 1994a) and their potential as an agent to form
edible lms has in the last decade generated interest
because of their favorable functional properties and indus-
trial surplus (Chen, 1995). The lm-forming properties of
whey proteins have been applied to produce transparent,
exible, colorless, and odorless lms (Fairley et al., 1996;
*
Corresponding author. Tel.: +86 510 85884496; fax: +86 510
85863566.
E-mail addresses: bgounga@yahoo.fr (M.E. Gounga), zw@sytu.edu.cn
(Z. Wang).
McHugh & Krochta, 1994a). Edible lms from whey pro-
tein isolates have shown moderate potential as moisture
barriers (McHugh et al., 1994) and are excellent oxygen
barriers (Hong & Krochta, 2003; Mate & Krochta, 1996;
McHugh & Krochta, 1994b). Studies on WPI edible lm
and coating formulations with dierent functionalities
and applications have been carried out (Hong & Krochta,
2003; Sothornvit, Olsen, McHugh, & Krochta, 2003, 2007).
The making of protein-based lms generally needs the
incorporation of a minimal content of plasticizer to reduce
its brittleness. Film plasticizers function by weakening inter-
molecular forces between adjacent polymer chains. This
results in an increased lm extensibility and exibility with
decreased elasticity, mechanical resistance, and barrier prop-
erty of the lms. The most common plasticizers used are
polyols and mono-, di-, and oligosaccharides. Glycerol, as
a plasticizer, has been incorporated into most hydrocolloid

0260-8774/$ - see front matter 2007 Published by Elsevier Ltd.

doi:10.1016/j.jfoodeng.2007.04.008
522 M.E. Gounga et al. / Journal of Food Engineering 83 (2007) 521530

lms. It is a high boiling point plasticizer, water-soluble, was purchased from Food Ingredients Hayashibara Shoji,
polar, nonvolatile, and protein miscible. These properties Inc. (Okayama, Japan). Glycerol (Gly) and all other chem-
make glycerol a suitable plasticizer for use with a compatible icals were of analytical grade.
water-soluble polymer (Banker, 1966).
However, the limitations of protein lms in general and 2.2. Film preparation
WPI lms in particular are well dened, especially, regard-
ing their barrier properties. Several approaches have shown WPI was accurately weighed in known amounts (5%,
the use of proteins with other lm-forming polysaccharide 7% and 9% w/v) and dissolved in distilled water. The solu-
materials to strengthen barrier properties of protein-based tion was heated to 90 2 C at pH 7 for 30 min while stir-
lms. Polysaccharides are well known for having good ring continuously to denature the whey protein. The
lm-forming properties. Coatings made with these biopoly- solution was then cooled to ambient temperature and glyc-
mers or their blends are generally considered as eective erol was added at dierent ratios. It was observed that
gas barriers. In particular, the combination of protein with when Gly was added in the ratio of <3.6:1 WPI:Gly, the
polysaccharides is an eective method in improving the lms obtained were too brittle to handle. To study the
performance of the lms owing to their variability in phys- eect of Gly, three ratios of WPI to Gly were used, that
ical properties and/or their interactions. Ciesla, Salmieri, is, 3.6:1, 3:1, and 2:1. To further investigate the eect of
and Lacroix (2006) have reported that the addition of poly- PUL on WPI-based edible lms, other lm-forming solu-
saccharides has a signicant eect on the properties of pro- tions were prepared by adding PUL in dierent ratios
tein-based edible lms. In addition, the compatibility of (Table 1) while keeping the total solid content constant
blend components is an important issue when dealing with at 7% (w/v) and the WPI to Gly ratio xed at 3.6:1.
mixtures of biopolymers as this might drastically alter the The obtained solutions were degassed in an ultrasonic
performance of these materials (Diab, Biliaderis, Geraso- oscillator. Film thickness was controlled by casting
poulos, & Sfakiotakis, 2001). 2.0 mL of lm solution on a glass plate (14  11 cm). Films
Pullulan is an extremely versatile ingredient providing a were allowed to dry at ambient conditions for 24 h, peeled,
technology platform for product innovation. It is a water- and conditioned accordingly.
soluble microbial polysaccharide extracellularly produced
by the fungus-like yeast, Aureobasidium pullulans (Yuen, 2.3. Film conditioning
1974). It consists of maltotriose units interconnected via
a (16) linkages that are responsible for the irregularly Protein and polysaccharide lms, being hydrophilic, are
ordered chains and for the ensued amorphous character susceptible to moisture absorption, and due to the fact that
of this polysaccharide (Gidley, Cooke, & Ward-Smith, most of their properties are aected by relative humidity
1993). Pullulan is an excellent lm-former, producing a lm (RH) and temperature, preconditioning was necessary to
that is colorless, tasteless, odorless, transparent, exible, standardize all lms by keeping them in an environmental
highly impermeable to oil and heat-sealable with good oxy- chamber (Model LRHS-B Yue Lin Medical Instrument
gen barrier properties (Conca & Yang, 1993; Yuen, 1974). Co., Shanghai, China) at 50 3% RH and a temperature
The use of pullulan in lm-forming and coating materials of 23 2 C for 48 h before the tests (ASTM D 618,
alone (Lazaridou, Biliaderis, & Kontogiorgos, 2003) and 2000) were carried out.
its blends with dierent polysaccharides (Biliaderis, Lazari-
dou, & Arvanitoyannis, 1999) has been long reported. 2.4. Measurement of the lm thickness
More so, its eect on the properties of protein-based edible
lms has been intensively studied on soy protein isolate The lm thickness was measured using a 025 mm hand-
(Xu, Chen, & Sun, 2001) and caseinate (Kristo & Biliade- held micrometer caliper (Guanglu Digital Caliper Manu-
ris, 2006). However, no one has ever used addition of pullu- facturer Co., Ltd., China). Film thickness measurement
lan in WPI-based edible lm to study the resultant was carried out after the permeability test to avoid the
properties. The aim of this study, therefore, was to investi- eect of mechanical damage that could have occurred on
gate, on a comparative basis, the permeation properties of
WPI-based edible lms as aected by protein concentration Table 1
The WPI and PUL composition used at dierent ratios
and glycerol ratio and to study the eect of pullulan on
WPI-based edible lms. WPI:PUL ratios WPI (g) PUL (g)
1:0 7.00 0.00
2. Materials and methods 1:1 3.50 3.50
2:1 4.67 2.33
3:1 5.25 1.75
2.1. Reagents 4:1 5.60 1.40
5:1 5.83 1.17
Whey protein isolates (WPI, ca. 86.98% Kjeldahl 6:1 6.00 1.00
N  6.38) were obtained from New Zealand Milk Products 8:1 6.22 0.78
10:1 6.36 0.64
(Fonterra LTD, Auckland, New Zealand). Pullulan (PUL)
 M.E. Gounga et al. / Journal of Food Engineering 83 (2007) 521530 523

the lm during thickness measurement. The overall thick- in place. The cups were then placed in a desiccator equili-
ness was expressed as an average of nine readings taken brated at 80% RH and 30 C using 4.1 mol/L of (NH4)2SO4,
randomly on each lm sample. saturated solution. The cups were weighed at certain inter-
vals, and a linear regression analysis of weight gain versus
2.5. Oxygen permeability time was performed. The slope of the line in g/h provided
the water vapor transmission rate (WVTR). water vapor
Oxygen permeability (OP) was evaluated according to permeability coecient WVP was calculated according to
the ASTM Standard Method D 618, 2000. The lm was
CX
sandwiched into precut aluminum foil masks with WVP g mm=m2 d kPa 2
60.82  104 m2 eective area. Oxygen was purged into A  DP
the above cell, while nitrogen into the under cell. The oper- where C is the transmission rate of water vapor (g/d) as
ation was maintained for about 10 min until a stable oxy- shown in Table 2, X is the lm thickness (mm), A is the
gen concentration of 100 1% in the above cell and area of exposed lm (22.8906  104 m2) and DP is the va-
0 0.1% in the under cell was reached. The ow rate of por partial pressure dierence across the lm (kPa).
oxygen in the above cell was kept constant (0.5 ml/s).
The valve of nitrogen was then closed to begin detecting 2.7. Light transmission and lm transparency
the change in oxygen concentration (v/v) in the nitrogen
cell with time using an oxygen detector. A linear correla- The ultraviolet (UV) and visible light barrier properties
tion between time and oxygen concentration was obtained of the lms were measured at selected wavelengths (200
(correlation coecient was above 0.99). The slope of the 800 nm), using a UV Spectrophotometer (model UV-
line in %/h provided the permeability ratio of oxygen q 1100, Rui Li Analytical instrument Corp., Beijing, China)
(cm3/d) as shown in Table 2 and the permeability coe- following the procedure reported by Fang, Tung, Britt,
cient of oxygen was obtained according to Yada, and Dalgleish (2002). Transparency of the lms
qX was calculated as in the following equation (Han & Floros,
OP cm3 mm=m2 d kPa 1 1997):
A  DP
where X is the lm thickness (mm), A is the area of exposed A600
Transparency 3
lm (m2) and DP is the oxygen partial pressure dierence X
across the lm (kPa). where A600 is the absorbance at 600 nm and X is the lm
thickness (mm).
2.6. Water vapor permeability
2.8. Moisture content and lm solubility
Water vapor permeability (WVP) was determined using
the cup method similar to that reported by Pranoto, Salo- A lm sample (0.2 g) was weighed into aluminum cruci-
khe, and Rakshit (2005). The lm was sealed onto a glass bles and dried at 105 C in an oven with forced air circula-
permeation cup (mouth area 22.8906  104 m2) containing tion for 24 h. Moisture content was determined as
silica gel (desiccant RH 0%) with an O ring to hold the lm percentage of initial lm weight lost during drying and
was reported on wet basis.
Table 2
q, C and X as used in OP and WVP determinationa
Solubility in water was expressed as percentage of lm
dry matter solubilized after 24 h immersion in distilled
WPI (%) WPI:Gly q (cm3/d)b C (g/d)c X (mm)d
water. A portion of the lm (0.2 g dry matter) was weighed
5 3.6:1 105.67 3.29 1.95 0.20 0.0159 2.38% and immersed in 40 mL of distilled water, containing
3:1 116.57 3.14 2.16 0.27 0.0166 1.25%
2:1 125.13 6.33 2.11 0.02 0.0173 1.15%
sodium azide (0.02% w/v) to prevent microbial growth at
20 C 2 with occasional agitation. After 24 h of immer-
7 3.6:1 22.05 0.45 1.89 0.34 0.0221 0.94%
sion, the sample was vacuum ltered through a pre-
3:1 25.67 0.26 3.36 0.55 0.0226 0.92%
2:1 30.03 0.17 3.35 0.11 0.0231 0.90% weighed porous crucible. The crucible, containing any
undissolved pieces of lm, was dried at 105 C for 24 h to
9 3.6:1 3.30 0.05 1.83 0.02 0.0435 0.58%
determine the weight of dry matter not dissolved in water.
3:1 5.14 0.08 1.95 0.04 0.0445 1.13%
2:1 6.92 0.18 2.04 0.05 0.0461 0.65% The weight of the dry matter dissolved in water is equal to
a the dierence between the initial dry matter and the
Each value represents the mean value standard deviation of three
determinations. remaining dry matter (not dissolved). This was expressed
b
q = a  0.01  V  24; where a is the slope of linear correlation as a percentage of the initial dry matter.
between oxygen concentration (%) and time; V is the volume of the above
cell (70.69 cm3). 2.9. Determination of lm microstructure using SEM
c
C = a  24: where a is the slope of linear regression of weight gain
versus time.
d
Standard deviation values were too low, hence these were expressed as An Electroscan model Quanta 2000 environmental scan-
percentage of the mean value of thickness. ning microscope (Fei Company, Netherlands) was used to
524 M.E. Gounga et al. / Journal of Food Engineering 83 (2007) 521530
study representative surface structure of the lms. The
samples were coated with a ne gold layer before obtaining
the micrographs. All samples were examined using an
accelerating beam at a voltage of 10 kV.
2.10. Statistical analysis
All experiments were conducted in triplicates in a ran-
domized design and all measurements were carried out at
least twice. Statistical dierences in the lm functional
properties and thickness change between samples were ana-
lyzed by a two-way analysis of variance (ANOVA) and
Duncans multiple range test (a = 0.05 ) was carried out
using SAS Software (SASS 8.1 for window, SASS Inc.,
Cary, NC, USA). A one-way analysis of variance was also
conducted to determine the eect of pullulan addition on
the lm properties.
3. Results and discussion
Intact lms were obtained from 5%, 7% and 9% WPI
contrary to the study by McHugh et al. (1994), who
reported that, below 8% WPI, intact lms were not formed.
3.1. Eect of WPI concentration and glycerol ratio
3.1.1. Film thickness
The lm thickness increased from 0.0166 (0.0003) mm
for the 5% WPI lm to 0.0232 (0.0012) mm for the 7%
WPI lm and to 0.0447 (0.0003) mm for the 9% WPI lm
(Fig. 1). The dierence in thickness was signicant
(p < 0.05) in all comparisons of lms made from the dier-
ent concentrations of WPI. This behavior is as a result of
the protein concentration of WPI solution, when the con-
centration was high. The WPI-based lm thickness values
were lower than those reported by Vachon, Yu, Yefsa,
Alain St-Gelais, and Lacriox (2000) with thicknesses of
0.05
0.045
0.04
0.035
Thickness (mm)
0.03
0.025
0.02
0.015
0.01
0.005
0 3.6 : 1
3.6 : 1 3.0 : 1
WPI:Gly ratio
5% WPI 7% WPI
Fig. 1. Eect of WPI concentration and Gly ratio on lm thickness.
0.05 to 0.06 0.002 mm for the same material but cross-
linked by heating and irradiation. However, the values
were much higher than those reported by Simelane and
Ustunol (2005) who noted a thickness value of
0.14 0.02 mm. This was due to dierences in lm-form-
ing solution formulations and procedures used.
At the same protein concentrations, lms prepared with
a dierent ratio of plasticizer showed dierent values in
thickness. The thickness increased slightly when the glyc-
erol amount was increased in lm-forming solution. Gener-
ally WPI:Gly ratio did not signicantly aect the lm
thickness.
Thickness aects the drying kinetics of the liquid lm-
forming dispersion, which may cause dierences in lm
structure (Debeaufort & Voilley, 1995). A high lm thick-
ness produces an increase in WVP. Therefore, control of
this parameter is crucial.
3.1.2. Oxygen permeability (OP)
OP is one of the most commonly studied transport prop-
erties of edible polymer lms. The higher the OP, the lower
is the OP barrier ability of the lms.
Both WPI concentration and Gly ratio greatly aected
the OP of the lm. Fig. 2 shows that OP decreased with
increase in WPI concentration. At equal WPI:Gly ratio
(3.6:1), the mean value of OP decreased to
0.23 0.002 (cm3 mm/m2 d kPa) for 9% WPI, twelve times
lower than that of 5% WPI (2.73 0.1). The low oxygen
permeability at high WPI concentration was most proba-
bly due to the increase in density of the lm solution which
resulted in larger lm thickness. This reduced interstitial
spacing between the molecules in the cast polymer lms.
As a consequence, OP is lowered due to obstruction of
oxygen molecules passing through the more closely packed
protein network. Similar results were reported by Anker,
Stading, and Hermansson (2000) when studying the rela-
tionship between microstructure and barrier properties of
4
3.5
OP (cm 3.mm/d.m 2.kPa)
3
2.5
2
1.5
1
0.5
0
3.0 : 1 2.0 : 1
2.0 : 1
WPI:Gly ratio
5% WPI 7% WPI 9% WPI
9% WPI
Fig. 2. Eect of protein concentration and glycerol ratio on the OP of
WPI edible lms.
 M.E. Gounga et al. / Journal of Food Engineering 83 (2007) 521530 525

whey protein lms. Miller and Krochta (1997) reviewed the
oxygen barrier properties of edible lms and concluded
that the OP decreases at low free volume.
The eect of plasticizer ratio on OP was also examined
(Fig. 2). At the same concentration of WPI (5%, 7% or
9%), a slight, yet signicant increase in OP was observed
for lms formed when the Gly amount was increased in
the lm solution. Gly may compete with water for the
active sites on the polymer, thus promoting water cluster-
ing and increased free volume in the polymers at low mois-
ture levels. The eect of Gly concentration on diusivity
was greater than that on solubility and resulted in increased
permeability values (Lieberman & Gilbert, 1973).
All in all, OP values were shown to be low. This could be
related to the more polar nature and more linear structure
of WPI hence leading to a higher cohesive energy density
and a lower free volume. The results were in accordance
with those reported by McHugh and Krochta (1994a)
who concluded that edible lms made from WPI/Gly had
low OP. Olivas and Barbosa-Canovas (2005) conrmed
that lower OP can be achieved by the use of edible lms
and coatings than when conventional plastic lms were
used.
3.1.3. Water vapor permeability (WVP)
It was observed that the lm WVP values increased with
increase in WPI concentration (Fig. 3). Mean values of
WVP for the 9% WPI lms were much higher than 7% lms
which were also higher than 5% lms. Besides, it was also
observed that there was a signicant dierence between
WVP values of lms made with 5% and 7% on one side
and 7% and 9% on another side (p < 0.05). The results
clearly showed how varying the concentration of WPI
could inuence the water barrier property of the lms as
studied by McHugh et al. (1994). This might be due to an
increased number of air bubbles when the concentration
became relatively high. The formation of bubbles could
probably be explained by the larger pores formed at high
14
12
WVP (g.mm/m 2.d.kPa)
concentrations. When the pore size increases, the liquid
phase (water and plasticizer) increases too. Due to the
hydrophilicity of water molecules, their easiest way through
the lm matrix is by the hydrophilic water and plasticizer
phase. This phase increases as larger pores are formed,
hence the WVP will increase. Similar results were reported
by McHugh et al. (1994) who noted that when the concen-
tration of WPI was increased from 8% to 12% (w/w), the
WVP increased too. Anker et al. (2000) concluded from
their investigation that when the concentration of WPI is
increased, more aggregated structures are formed, with a
denser protein matrix and larger pores, and this greatly
aects the water barrier property of WPI lms.
An increase of plasticizer concentration normally causes
an increase in the WVP of hygroscopic lms due to a reor-
ganization of the proteinic network and consequent
increase of free volume (Cuq, Gontard, Aymard, & Guil-
bert, 1997). Hence, it was observed that at equal concentra-
tion of WPI, the lms with a WPI:Gly ratio of 2:1 were
more permeable to water vapor than those of 3:1 ratio
and in addition were less water vapor resistant compared
to the 3.6:1 ratio lms (Fig. 3). This could be explained
by the fact that Gly reduces internal hydrogen bonding
and increases intermolecular spacing, thereby decreasing
the permeability of WPI lms. McHugh et al. (1994)
reported the same eect when examining the WVP proper-
ties of plasticized WPI. The hydrophilic nature of plasti-
cizer (nature and amount) signicantly aects the
moisture barrier ability of protein lms.
The increase of WVP value could also be explained by
increase in lm thickness as shown by other studies.
According to Cuq, Gontard, Cuq, and Guilbert (1996),
the eect of thickness is a signicant variable when barrier
properties are taken into account. A recent study by Bra-
vin, Peressini, and Sensidoni (2006) showed that experi-
mental WVP data and thickness of polysaccharide and
polysaccharide-oil lms tted well with rst-order regres-
sion WVP for starch-MC lm that showed a signicantly
higher dependence on thickness than lm containing oil.
Banker, Gore, and Swarbrick (1966) and Barrer (1951)
attributed the thickness eect to lm swelling due to
waterlm interaction. Hydrophilic lms exhibit increased
WVP values as lm thicknesses are increased due to the

10 increased water vapor partial pressure conditions to which

8 the lm is exposed during the analysis. McHugh, Avena-
Bustillos, and Krochta (1993) attributed the thickness
6 eects to relative humidity. They observed that as the lm
4 thickness increased, there was reduced resistance to water
vapor transfer across it. As a consequence, a stagnant air
2 layer formed, characterized by a high water vapor partial
0 pressure on the inner lm surface.
3.6 : 1 3.0 : 1 2.0 : 1
WPI:Gly ratio 3.2. Eect of addition of pullulan on barrier properties of
5% WPI 7% WPI 9% WPI
WPI lms

Fig. 3. WVP of WPI-based edible lms as aected by both protein Films made from 5% WPI with a 3.6:1 ratio of WPI to
concentration and Gly ratio. Gly were initially selected for further study with addition of
526 M.E. Gounga et al. / Journal of Food Engineering 83 (2007) 521530

Pullulan (PUL) to investigate the eect of the amorphous 3.2.2. Water vapor permeability
polysaccharide on barrier and structural properties of The results (Fig. 4) showed that the WVP was highest
WPI-based edible lms. However, when the concentration when the ratio of PUL/WPI was 1. Water vapor barrier
of total solid content (WPI and PUL) was kept at the 5% ability was then the lowest. This situation can be explained
level in dierent WPI:PUL ratios, the lms could not be by the fact that pullulan contains much hydroxyl groups
peeled after xing the WPI:Gly ratio at 3.6:1. This was pre- (OH) which greatly increase the hydrophilic property of
sumably due to the lack of intermolecular interaction upon the lm material, limiting its ability to form lms with good
lm dehydration. Hence, the 7% WPI lm with WPI to Gly moisture barrier property, as compared to the synthetic
in a ratio of 3.6:1 was selected as it also presented one of polymer lms, such as low-density polyethylene (LDPE)
the best OP and WVP in the study. or polyvinylidene chloride (PVDC) (Perez-Gago, Nadaud,
Film thickness did not change signicantly because the & Krochta, 1999). On critical analysis, macromolecular
total solid content (WPI + PUL) was kept at 7% while swelling at the stagnant air gaplm interface would deter-
the ratio of WPI to Gly was kept at 3.6:1 even after addi- mine higher water diusion due to the increase in mobility
tion of pullulan to lm-forming solution (results not of polymeric chains brought about by the dominating pres-
shown). ence of pullulan in the mixture. Fig. 4 shows the inuence
of the actual water vapor permeability by addition of pullu-
3.2.1. Oxygen permeability lan in this work.
Dierent ratios of WPI to PUL ranging from 1:0 to Structural modications (Fig. 5) in the lm relation to
10:1 (Table 1) were employed to investigate any eect changes in thickness explain the results (Cuq et al., 1996).
of pullulan addition on OP. The quantitative ratios were The WVP value decreases drastically from 42.38 2.8 to
expressed as PUL/WPI (Fig. 4). OP values tended to 30.14 3.5 g mm/m2 d kPa when the ratios were 1 and
increase with addition of pullulan. The OP then decreased 0.5, respectively. At this level, the presence of pullulan mol-
with decrease of pullulan content and the result showed ecule in the mixture dominates the hydrophobic property
signicant dierences at a = 5% (Fig. 4). It has been of whey protein due to the presence of its own hydrophobic
reported that pullulan has a very low oxygen penetration amino acids. In general, the WVP decreased slightly with
(Conca & Yang, 1993; Yuen, 1974). However, when the decrease in pullulan amount in the lm composition
ratio of WPI to PUL was 1 the OP value reached its (Fig. 4).
maximum 1.043 0.03 cm3 mm/m2 d kPa. This situation All in all, it was evident that the addition of pullulan in
could be explained by the fact that PUL, mixed with WPI lm solution improved the WVP of WPIPUL lms
WPI at the same ratio, dominated WPI by its hydrophi- shown by their low values as compared to the control. A
licity, since it has been recognized as a plasticizer. At low similar eect was reported by Ciesla et al. (2006) using
concentrations, PUL contributed to a cooperative func- potato starch and sodium alginate on WPI.
tionality with WPI. In this lm system, the OP may be
improved by taking advantage of each constituent (WPI 3.2.3. Light transmission and lm transparency
and PUL). The former may give rise to a very tight The transmittance (T) in visible range (350800) of con-
structure by inter- and intramolecular folding; while the trol lm (PUL = 0) ranged from 68.9 6.5% to
latter may impart structural cohesion and serve as a 88.0 3.7% (Table 3). Very low values of transmission
structural matrix (Wu, Weller, Hamouz, Cuppett, &
Schnepf, 2002).

1.2 50
a OP WVP
Water Vapor Permeability

45
1
Oxygen Permeability

b 40
(cm3.mm/m2.d.kPa)

(g.mm/m2.d.kPa)

b
a 35
0.8 bc bc
c c 30
cd
0.6 d 25
b 20
0.4 15
c g
h i 10
0.2 d e f 5
0 0
0 1 0.5 0.33 0.25 0.2 0.17 0.125 0.1
PUL/WPI ratios

Fig. 4. Eect of addition of pullulan on oxygen and water barrier

properties. Each data point represents average of three determinations.
Any two points on the same curve followed by the same letter are not
signicantly dierent. The PUL/WPI ratios X values: 0 = 0/1; 1 = 1/1;
0.5 = 1/2; 0.33 = 1/3; 0.25 = 1/4; 0.2 = 1/5; 0.17 = 1/6; 0.125 = 1/8; Fig. 5a. Scanning electron micrographs of WPIPUL lms: (a) surface of
0.1 = 1/10. WPI:PUL 1:0.
 M.E. Gounga et al. / Journal of Food Engineering 83 (2007) 521530
Fig. 5b. Scanning electron micrographs of WPIPUL lms: (b-1)
WPI:PUL 1:1; (b-2) detail of the surface.
Fig. 5c. Scanning electron micrographs of WPIPUL lms: (c) WPI:PUL
10:1.
527
were noted at 200 nm (0.6%) and 280 nm (1.6%) in UV
light. The results showed that the control lm had excellent
barrier properties in the 200280 nm UV light region. On
the other hand, synthetic polymer lms did not prevent
the passage of UV light above 280 nm except for the poly-
ester (PE) lm. The results suggest that WPI lms can
potentially retard lipid oxidation induced by UV light in
food systems. This is in agreement with other studies done
on whey protein isolate (Fang et al., 2002), sh skin gela-
tin-based lms (Jongjareonrak, Benjakul, Visessanguan,
Prodpran, & Tanaka, 2006), and surimi lms (Shiku, Ham-
aguchi, Benjakul, Visessanguan, & Tanaka, 2004). How-
ever, the T values in this study were lower than those
reported for the WPI lms (Fang et al., 2002). The dier-
ence could have arisen from the concentration of WPI used
and other lm solution components, such as Ca2+.
When pullulan was added to the lm solution at a
weight ratio of 1, the transmittance was lowest for all
ranges (0071.4%). The T value of WPI:PUL lms
increased when pullulan content was decreased, but it
remained below the transmittance of WPI alone. Pullulan
had good resistance to UV and visible light, hence playing
an important role in UV barrier properties of WPI lms
and color loss. The 1:1 ratio of WPI:PUL lm eectively
blocked most of the light in the UV and visible light but
had the worst OP and WVP barrier abilities (Fig. 4).
The control lm had a transparency of 3.41% indicating
that the lm was more transparent than that made from
marlin myobrillar protein (Shiku, Hamaguchi, & Tanaka,
2003) and some other synthetic polymer lms. The trans-
parency had a higher value at a WPI:PUL ratio of 1:1
and decreased gradually with decrease in the amount of
pullulan. The results indicated that WPIPUL lms were
clear enough to be selected and used as see-through coating
and/or packaging material and prevent color deterioration
and lipid oxidation.
3.3. Moisture content and lm solubility
Five proportions of WPI/PUL were chosen to study the
eect of pullulan addition on the moisture content (MC)
and the solubility of WPI-based edible lms (Table 4).
The control lm had the lowest MC (16.48 1.5%). The
MC increased with increased content of PUL and reached
its maximum when the proportion of WPI/PUL was 1. The
results showed that MC of WPI-based lms diered signif-
icantly (p < 0.05) with change of PUL content in the mix-
ture. The reason why pullulan is more readily
disintegrated by absorbing water and having poorer barrier
properties could be its hygroscopic property and its high
hydrophilicity.
The results showed that the control lm was not dis-
persed after 24 h immersion in water and had no visual loss
in integrity. This was related to the relatively low solubility
value (50.58 4.8%) when no PUL was added and reached
the highest value (97.98 2.2%) at WPI/PUL = 1. The rel-
atively low solubility of WPI lms in water has been
528 M.E. Gounga et al. / Journal of Food Engineering 83 (2007) 521530

Table 3
Light transmission (%) and transparency (A600/mm) of WPI-based edible lms as aected by PUL
Films WPI:PUL Wavelength (nm)a Transparencyb
200 280 300 350 400 500 600 700 800
1:0 0.6(0.1) 1.6 68.9(6.5) 81.4(3.6) 84.0(4.6) 85.0(4.3) 86.9(4.0) 87.8(3.8) 88.0(3.7) 3.41
1:1 0.0 0.0 34.0(2.4) 37.1(0.9) 45.0(0.8) 59.8(3.3) 66.7(2.3) 69.9(2.2) 71.4(2.5) 7.42
2:1 0.0 0.2 50.7(1.9) 67.9(1.3) 74.1(1.2) 77.5(1.1) 79.2(1.4) 79.7(1.2) 80.3(0.8) 4.37
3:1 0 0.2 39.2(2.8) 57.6(3.5) 64.5(3.1) 68.8(2.1) 71.3(1.5) 72.9(1.0) 74.6(1.3) 7.05
4:1 0 0.8 38.5(1.1) 63.3(1.8) 69.9(2.2) 73.4(2.0) 75.8(1.6) 76.8(1.4) 77.7(1.3) 5.93
5:1 0 1.0 35.8(1.9) 56.8(1.5) 63.6(1.5) 68.3(1.5) 71.6(1.5) 73.0(1.0) 74.5(0.6) 6.47
6:1 0 1.0 51.8(1.2) 58.9(1.6) 63.9(0.8) 68.6(0.9) 71.6(0.8) 73.5(0.8) 74.8(0.8) 6.03
8:1 0 1.3 51.4(4.7) 60.5(3.36) 66.3(3.7) 72.0(3.7) 75.9(3.3) 78.2(3.3) 79.6(2.5) 5.55
10:1 0.2(0.1) 1.4 60.7(3.8) 72.2(3.3) 76.5(3.3) 77.8(2.0) 81.0(1.1) 82.9(0.9) 84.1(1.0) 4.07
Synthetic lmsc
LDPE 13.1 67.5 79.9 83.4 85.6 86.9 87.8 83.6 3.05
OPP 4.6 80.0 86.2 87.9 88.8 89.1 89.3 89.6 1.67
PE 0.3 0.3 68.3 73.6 82.1 83.5 84.2 84.9 1.51
PVDC 0.3 79.1 83.8 86.6 87.5 90.0 87.9 84.9 4.58
a
Each value represents the mean value and (standard deviation) of three determinations.
b
Transparency was calculated according to the formula: A600/X.A600 is absorbance at 600 nm, and X is thickness of the corresponding lm.
c
From Shiku et al. (2003) LDPE: low-density polyethylene; OPP: oriented polypropylene; PE: polyester; PVDC: polyvinylidene chloride.

Table 4 PUL lm at a 1:1 ratio. Therefore, it was elucidated that

Moisture content (%) and lm solubility of WPI-based edible lms as
aected by PULa
Films WPI:PUL Moisture content (%)b Film solubility (%)b
1:0 16.48 1.5d 50.58 4.8c
1:1 26.13 1.0a 97.98 2.2a
2:1 22.63 1.4b 60.57 2.6b
5:1 20.58 1.1bc 62.26 1.1b
10:1 17.93 2.4cd 59.11 2.6b
a
Mean values standard deviation.
b tact with water during processing and storage. The oppo-
Any two means in the same column followed by the same letter are not
signicantly dierent (p > 0.05).
reported (Fairley et al., 1996; McHugh & Krochta, 1994b).
Low solubility could be explained by the high interaction
density and presence of intermolecular disulde bonds as
a result of the heat treatment (McHugh et al., 1993;
McHugh & Krochta, 1994b). Perez-Gago et al. (1999) con-
rmed that native whey protein produces water-soluble
lms, but the heat-denatured solution of WPI produces
lms whose protein is insoluble.
The study showed that PUL greatly aected the MC and
FS of WPI-based edible lms. The larger solubility of WPI
PUL lms was certainly due to the hygroscopicity of pullu-
lan and its water-soluble nature that cannot be modied by
heat treatment contrary to WPI. Pullulan solubility can only
be modied by esterication and ethoxylation (Yuen, 1974).
The highest solubility value (97.98 2.2%) showed that
lms prepared with pullulan dissolve in water more signi-
cantly. Yuen (1974) further reported that lms and contain-
ers prepared with pullulan dissolve in water three to four
times faster than those prepared with polyvinylalcohol
(PVA) and left no trace. The phenomenon could be
explained by the fact that although intermolecular disulde
bonds can be formed during the drying of protein solution,
the presence of SS bonds was not observed in the WPI to
hydrogen bonds and hydrophobic interactions, combined
with ionic bonds, played an important role in the formula-
tion of WPIPUL lms. The result conrmed that the
lm-forming mechanism of the control was quite dierent
from the 1:1 WPI:PUL lm. The hydrophobic interactions
were predominant for the former, while the hydrogen bonds
were predominant for the latter.
Low water solubility is important when lms are in con-
site is desirable when the intent is to design a package
with premeasured dry food amounts to be dissolved in
water or in hot food (Guilbert & Biquet, 1989).
3.4. Microstructure of lm
The control lm and that from two other ratios of WPI:-
PUL (1:1; and 10:1) were chosen due to their remarkable
results regarding their barrier properties. It was evident
from the micrographs (Fig. 5) that all the lm surfaces dif-
fered in their microstructure with pinholes observed. How-
ever, the size and amounts of holes that elevate moisture to
pass through the lms varied from lm to lm with dier-
ent amounts of pullulan, plasticizer ratio being the same
for all lms. Both surface and cross section of 1:1 WPI:-
PUL had numerous small pinholes (Fig. 5b). A detail of
the surface (b-1) is presented in g. b-2. This could be
responsible for the higher WVP values of the lms
(Fig. 4), compared with the control lm which had a less
number of holes. The control lm images revealed a
sponge-like structure where WPI aggregates appear to be
linked by ne stands to form a continuous network. This
network was reected in the relative low solubility reported
about the lm (Table 4). The results are in agreement with
the properties of WPI-based lms formed at pH 7 (Anker
et al., 2000; Fang et al., 2002).
 M.E. Gounga et al. / Journal of Food Engineering 83 (2007) 521530
4. Conclusion
Whey protein isolate edible lms have good physical
properties for food systems. Film thickness, oxygen and
moisture permeability were inuenced by protein and glyc-
erol at dierent levels of concentrations. In this way, the
lm consistence and its barrier properties can be controlled
using the concentration of the basic material and plasticizer
used in the dispersion. Further investigation resulted in
reduction of OP in WPI-based lms, through the addition
of PUL. More so, at low PUL concentrations, WPIPUL
lms showed low solubility and high transparency when
compared with synthetic polymer lms. This was a result
worth noting, considering the possibility of applying the
lms to food processes, such as in packing material.
As far as the structural and physical features of lms
obtained in the presence and/or absence of pullulan are
concerned, microstructure analysis showed good compati-
bility of the plasticizer (Gly) with the WPI. On the other
hand, absence of empty spaces in the WPIPUL lm indi-
cated good compatibility of pullulan with the WPI. There-
fore, the application of the lms studied is related to the
objective of producing a coating material which can
improve barrier properties of specic foods or drugs. To
further dene the kind of material suitable for coating,
studies on the mechanical and thermomechanical proper-
ties need investigation.
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