Original article

Effects of the free and pre-encapsulated calcium ions on the

physical properties of whey protein edible film
Zhi Chai, Jiejing Shang, Yanfeng Jiang, Fazheng Ren & Xiaojing Leng*
CAU&ACC Joint-Laboratory, Key Laboratory of Functional Dairy Science of Beijing and Ministry of Education, College of Food Science &
Nutritional Engineering, China Agricultural University, No.17 Qinghua East Road, Haidian, Beijing 100083, China
(Received 19 February 2010; Accepted in revised form 29 April 2010)
Summary Eects of the free and the pre-encapsulated calcium ions on the physical properties of the whey protein
isolate lm were studied for improving calcium content in the edible lms. At pH 8, the lm-forming process
was hindered by serious protein aggregation and gelation caused by 0.5% (w w) free calcium ions added in
an 8% whey protein isolate solution. If the calcium ions were pre-encapsulated in the protein microparticles
(contained 17% Ca
2+
) using spray drying method, and then added in the lm-forming solution prepared
using the same protein, the calcium content could be doubled (1%, w w) without signicant eects on the
physical properties of the lm. The calcium release ability (55% at pH 1.2, 32% at pH 7.4 and more than
75% with the enzymes) of the lm was also investigated.
Keywords Calcium, edible lm, encapsulation, mechanical properties, whey protein isolate.
Introduction
Edible lms and coatings can extend shelf life and
improve the quality of food by providing barriers to
mass transfer. Their matrix is also found to be relevant as
carriers of the functional ingredients as it may be intended
for the encapsulation and controlled release of anti-
oxidants (Han & Krochta, 2007), antimicrobial agents
(Zinoviadou et al., 2009) and avours (Miller &Krochta,
1998). Many biomacromolecules including soy, gelatin
and whey proteins (WP) have been used for dierent
purposes. The latter received a particular attention and
are widely used in food products because of their high
nutritional value and their ability to form gels, emulsions
or foams (Banerjee & Chen, 1995; Fang et al., 2002;
Foegeding et al., 2002). The eects of the driving factors
including temperature, pH and concentration on the
physical properties of WP lm have been reported in
literatures (McHugh et al., 1993; Anker et al., 1998,
1999), and we thus know that the mechanical strength of
the lms reached a maximum with an 8% (w w) heat-
denatured whey proteins isolate (WPI) at pH 8.
Nevertheless, a strategic design of ingredient formu-
lation could further improve nutritional functionalities
of WP, thus increasing applications or creating new
applications in food industry. A clear understanding of
the structure and formation of protein lms is also
required to control their properties. Addition of
mineral substance such as calcium, a nutrient having
many medical and health care uses such as the
treatment of bone loss (Pak & Avioli, 1988; Hughes,
1991), can fortify nutritional values of WP lms (Fang
et al., 2002). As a traditional calcium supplementation,
calcium chloride is widely used for its high calcium
content, good solubility and low price. Its low bio-
availability can be improved by binding with WP
(Gue guen & Pointillart, 2000; Zhao et al., 2005).
However, serious protein aggregation induced by high
concentrated calcium aects the lm-forming process
(Hongsprabhas & Barbut, 1996; Parthasarathy et al.,
1999; Kulmyrzaev et al., 2000; Marangoni et al., 2000).
How to avoid such problem and achieve desired
functionalities without aecting the physicochemical
properties of the lm is an interesting challenge. The
focus of this work was to investigate the eects of the
free calcium ions and the WPI pre-encapsulated
calcium ions on the physical properties of the WPI
lm to nd a way to improve the inherit calcium
content of WPI through the evaluation of the corre-
sponding physical properties.
Materials and methods
Materials
WPI (protein > 97%, w w) is purchased from Davisco
Foods International (Eden Prairie, MN, USA). Glycerol
(99% reagent grade), CaCl
2
, NaOH, NaBr, KBr
(analytical grade), ethylenediaminetetraacetic acid
*Correspondent: Fax: +86 10 62737761;
e-mail: xiaojing.leng@gmail.com
International Journal of Food Science and Technology 2010, 45, 15321538 1532
doi:10.1111/j.1365-2621.2010.02303.x
2010 Institute of Food Science and Technology
(EDTA), pepsin 3300 units mg
)1
(from porcine gastric
mucosa, crystalline) and pancreatin 8X (from hog
pancreas) are purchased from Sigma Chemical Co. (St.
Louis, MO, USA).
Film preparation
A 10% (w w) WPI stock solution was prepared at room
temperature and then heat denatured at 80 Cfor 30 min
in water bath. After cooling to room temperature, a
solution containing a certain quantity of CaCl
2
or
Ca
2+
WPI particles with glycerol as plasticizer was added
dropwise in the WPI solution under gentle agitation. The
nal WPI concentration was 8% and glyc-
erol WPI = 1 2 (w w). The investigated concentrations
of the free Ca
2+
were from 0% to 1%. The solution was
degassed in vacuo. The lms were cast by applying 5 g of
the solution evenly onto the polymethacrylate dishes
(8 cm diameter) and allowing to dry overnight at room
temperature. The dry lms were equilibrated at
22 3 C and 56% 8% relative humidity for at least
48 h before subsequently peeled from the casting surface
for characterisation analyses including mechanical test.
Film thickness was determined using a Digimatic Indica-
tor (Cheng-Du-Cheng-Liang Co., China) at tenrandomly
chosen locations on the lms. The average lm thickness
was 90 lm (P > 0.05).
Preparation of the encapsulated calcium
The particle-forming solutions were prepared with
Ca
2+
WPI mass ratios of 0.2, 0.4, 0.7 and 1 1 at pH
3. The solutions were spray dried using an YC-015
Spray Dryer (Ya-Cheng Pilotech Instrument & Equip-
ment Co. Ltd, Shanghai, China) with a feed rate of
10 mL min
)1
. The inlet and outlet air temperatures were
set at 180 and 90 C, respectively. The pre-encapsulated
Ca
2+
WPI (PECW) particles were collected at the
bottom of the dryers cyclone. The quantity of
Ca
2+
encapsulated in the particles was determined using
a traditional EDTA titration method (Bird et al., 1961).
The quantity of the PECW particles added in the lm
was described in the main text.
Light scattering measurement
The zeta potential and hydrodynamic size of the parti-
cles, d, was determined using a Delsa Nano Particle
Analyser (Beckman Coulter Inc, Fullerton, CA, USA)
based on the principle of dynamic light scattering. The
uctuations in time of scattered light from particles in
Brownian motion are recorded by the autocorrelation
function G(s) :
G s e
sDq
2
1
D is the diusion coecient of the particles, s the
delay time and q the scattering vector; d of the particles
is determined using Stokes-Einstein equation:
D
kT
3pg
s
d
2
k is the Boltzmanns constant, T the absolute temper-
ature and g
s
the solvent (water) viscosity.
Colour analysis
Lightness values (L values) of the lm-forming solution
vs. [Ca
2+
] were measured (CIE, 1996) using a Colour
Dierence Meter TC-P2A (XinAoYiKe Co., Ltd.,
Beijing, China) calibrated with a standard white plate
provided by manufacturer.
Mechanical properties
The puncture (PS) and tensile strength (TS) were
determined according to the method of Peleg (1979)
and ASTM D638M (ASTM, 1993) using a texture
analyser (TMS-Pro, Food Technology Corporation,
VA, USA) at room temperature.
Water vapour permeability (WVP)
WVP of the lms was measured using the modied
ASTM procedure (Gontard et al., 1992):
WVPg mm=m
2
d kPa Wx=ATDP
W is the weight gain of the cup (g), x the lm thickness
(mm), A the area of exposed lm (m
2
), T the time of gain
(h) and DP the dierence of vapour pressure across the
lm.
Differential scanning calorimeter (DSC)
The fusion temperature of the lms was analysed using a
dierential scanning calorimeter (DSC Q10, TA, USA)
calibrated with pure indium (melting point 156.4 C).
About 10 mg of the lm samples was weighed and
sealed in an aluminium pan with an encapsulating press.
The sample was heated from )125 to 200 C at a rate of
20 C min
)1
. An empty sample pan was used as a
reference.
Controlled release studies
The controlled release study can provide the informa-
tion about the lm capacity to release the free calcium
by the PECW particles in simulated gastric and intes-
tinal conditions. Calcium release was determined by
incubating an amount of lms in an investigated
Effect of encapsulated Ca
2+
on whey protein lm Z. Chai et al. 1533
2010 Institute of Food Science and Technology International Journal of Food Science and Technology 2010, 45, 15321538
medium with continuous agitation at 37 C for 6 h
(close to the food retention time in human digestive
organs). The investigated medium included: (i) HCl
water solution at pH 1.2; (ii) HCl water solution at pH
1.2 with 0.1% (w v) pepsin; (iii) phosphate-buered
water solution (PBS, pH 7.4); (iv) phosphate-buered
water solution (PBS, pH 7.4) with 1% (w v) pancreatin.
The quantity of Ca
2+
released was determined using
EDTA titration (Bird et al., 1961).
Light transmission
The lm light transmission was evaluated from 800 to
200 nm using a UV Spectrophotometer (Model UV-
1100, Rui-Li Analytical instrument Corp., Beijing,
China) following the procedure reported by Tang et al.
(2005). The transparency was determined as:
T A
600
=x
where A
600
is the absorbance at 600 nm and x the lm
thickness (Perez-Mateos et al., 2009).
Statistical analysis
Analysis of variance (SPSS, version 11.5, SPSS Inc.,
Chicago, IL, USA) for a completely random design to
determine the least signicant dierence at 0.05 was used
to analyse the statistics of the results. Three replicates
were tested at least for each measurement.
Results and discussion
Film-forming solution properties
Figure 1 showed the variation of the WPI aggregate size
vs. the free [Ca
2+
] at pH 8. The insert image was for the
corresponding autocorrelation functions, G(s). Table 1
showed the zeta potential variation of the WPI aggregate
vs. [Ca
2+
] at pH 8. Without addition of the free calcium,
the aggregates size was 809 nm (Fig. 1), and the zeta
potential was at )23.8 mV. When [Ca
2+
] increased from
0% to 0.5% (domain I), the size decreased from 809 to
509 nm (P < 0.05), and the zeta potential varied from
)23.8 to )11.6 mV. The decrease in the potential
indicated that the negative surface charges of the protein
were neutralised by the free Ca
2+
, which led to the
decline of the intermolecular electrostatic repulsion (Ju &
Kilara, 1998; Balnois et al., 2000; Marangoni et al.,
2000), and made the shrinkage of the aggregates, and the
microstructure became dense. When [Ca
2+
] continued to
increase from 0.5% to 0.7% (domain II), the potential
decreased to )6.6 mV, but the size increased to 670 nm.
This point suggested that the further charge neutralisa-
tion with excess Ca
2+
led the smaller dense aggregates to
joint together to form larger aggregates. Table 1 showed
the colour analysis as well, where the internal L-values
increased rapidly (solution became opaque) when [Ca
2+
]
>0.5%, and we could directly observe the solution
rapidly getting into gel state.
Physical properties
Figure 2 showed the variations of PS and TS of the lms
vs. [Ca
2+
]. PS and TS without addition of the free
Ca
2+
were 28.1 N mm
)1
and 33.7 N mm
)2
, respectively.
When [Ca
2+
] increased from 0% to 0.1%, PS and TS
increased signicantly (P < 0.05) to 54.6 N mm
)1
and
53.8 N mm
)2
, respectively; but no more increase
0.0 0.2 0.4 0.6 0.8
400
600
800
1000
1200
10
0
10
1
10
2
10
3
10
4
10
5
10
6
10
7
0.0
0.1
0.2
0.3
0.4
0.5
Domain II Domain I
d

(
n
m
)
Ca
2+
(%)
[Ca
2+
] = 0
[Ca
2+
] = 0.1%
[Ca
2+
] = 0.3%
[Ca
2+
] = 0.5%
[Ca
2+
] = 0.7%
G
(

Figure 1 Light scattering measurements of whey proteins isolate (WPI)

aggregate size vs. [Ca
2+
] at pH 8. Insert: autocorrelation functions.
The bars are the standard deviation, and the dash line shows the trend
of variation. The insert image exhibited the aggregation state with
[Ca
2+
].
Table 1 Variation of the zeta potential and the lightness L-values of whey proteins isolate (WPI) aggregate vs. the free [Ca
2+
] in the lm-forming
solution at pH 8
Ca
2+
(%) 0 0.1 0.3 0.5 0.7 1.0
Zeta potential (mV)

)(23.8 0.4) )(22.5 0.5) )(17.6 1.1) )(11.6 0.3) )(6.6 1.3) *
L-value

0 0.01 0.4 0.02 0.3 0.04 0.6 0.01 2.6 0.09 21.3 1.00
*Gelation not suitable for the test of zeta potential.

Mean standard deviation for four replicates.

Effect of encapsulated Ca
2+
on whey protein lm Z. Chai et al. 1534
International Journal of Food Science and Technology 2010, 45, 15321538 2010 Institute of Food Science and Technology
(P > 0.05) for both could be observed when [Ca
2+
]
>0.1%. This observation indicated that the addition of
0.1% Ca
2+
strengthened the crosslink network of the
protein lm; however, the excess Ca
2+
induced complex
protein aggregation and made the lm microstructure
heterogenous and therefore aected the promotion of
PS and TS (Termonia, 1990).
Figure 3 showed the variations of the water vapour
permeability (WVP) vs. [Ca
2+
], where WVP was kept at
about 20 g mm m
)2
d kPa and no signicant variations
could be observed with the free [Ca
2+
] (P > 0.05).
Similar data were also reported by Fang et al. (2002).
The concentrated Ca
2+
reinforced the crosslink network
of the lm, reduced the intervals between the protein
molecules, and was expected to decrease WVP; however,
the hygroscopic salt also enhanced the hydrophilicity
and hygroscopicity of the system and favoured the water
molecules to permeate into the matrix. Therefore, the
nal WVP values were the results of the balance of the
above opposite eects.
PECW particle properties
Figure 1 indicated that the quantity of the free calciumin
the lm-forming process could not exceed a certain
critical value. The key to improve the inherit calcium
content of WPI is how to control the activity of the
calciumions in the system, i.e. if the activity of the calcium
ions is pre-limited into the tiny particles rather than the
entire solution, the large-scale aggregation in the solution
could be avoided. Table 2 showed the state of the lm-
forming solutions vs. the quantity of the added PECW
particles. The PECWparticles usedinthis workcontained
17%Ca
2+
(If more than17%, the PECWparticles became
moist and dicult to collect from the dryers cyclone).
The maximum quantity of the PECW particles added in
the lm-forming solution was 0.5%(w w), which allowed
having 1%(w w) Ca
2+
in the lm. Obviously, if the drying
conditions in the particle preparation process could be
improved, the calciumcontent in the lmcould be further
enhanced. Considering the point of using the encapsula-
tion method in this work was to increase the calcium
content in the lm, only the lmcontaining the maximum
quantity of the calcium through PECW method was thus
used to do the comparison.
DSC analysis
Figure 4 showed the thermograms of three dierent
WPI lms: without Ca
2+
, with 0.4% free Ca
2+
or with
PECW particles. The fusion temperature (T
f
) of the
lm without Ca
2+
was at 130 C (denoted using the
0.0 0.2 0.4 0.6 0.8 1.0
0
10
20
30
40
Film containing Ca
2+
-WPI particles
Ca
2+
(%)
W
V
P

(
g

m
m

2
4
h

1

m

2

k
p
a

1
)
Figure 3 Variation of the water vapour permeability (WVP) of the
whey proteins isolate (WPI) lms vs. [Ca
2+
]. The PECW particle
incorporated WPI lm is denoted with the arrow. Mean standard
deviation for three replicates.
20
40
60
80
100
0.0 0.2 0.4 1.0
20
30
40
50
60
70
T
S

(
N

m
m

2
)
P
S

(
N

m
m

1
)
Film containing Ca
2+
-WPI particles
Ca
2+
(%)
Figure 2 Mechanical properties (PS and TS) of the whey proteins
isolate (WPI) lms vs. [Ca
2+
]. Solid symbol: PS; open symbol: TS. The
pre-encapsulated Ca
2+
WPI (PECW) particleincorporated WPI lm
is denoted with the arrow. Mean standard deviation for three
replicates.
Table 2 State of the lm-forming solution and the total Ca
2+
content in the lm vs. the quantity of the pre-encapsulated Ca
2+
WPI (PECW)
particles added in the lm-forming solution
PECW particle quantity (%, w w) 0 0.3 0.5 0.7 1.0
Film-forming solution state Solution Solution Solution Gel Gel
Ca
2+
(%, w w) 0 0.6 1.0 1.3 2.0
Effect of encapsulated Ca
2+
on whey protein lm Z. Chai et al. 1535
2010 Institute of Food Science and Technology International Journal of Food Science and Technology 2010, 45, 15321538
maximum value of the peak). The broad shape of the
fusion peak was mainly because of the incorporated
water in protein matrix. When the free Ca
2+
or the
PECW particles were added, T
f
of the lms increased to
142 and 138 C, respectively. T
f
was related to the heat
stability of the protein microstructure. The stronger the
cross linkage of the network structure was, the higher T
f
the lm had. The increase in T
f
of the sample with free
Ca
2+
indicated that the cross linkage of the network
structure was enhanced. In contrast, the increase in the
T
f
of the PECW particleincorporated WPI lm was
weaker. Figure 5 schematically illustrated the eects
of the free Ca
2+
and PECW particles on the microstruc-
ture of the WPI lms. High concentration of free
Ca
2+
could induce serious protein aggregation and
made the matrix dense. If Ca
2+
was pre-encapsulated,
the cations mobility was restricted in particle matrix
(more details were shown in Figure 6), and the extent of
aggregation in the whole systemcould be largely reduced.
Calcium-controlled release studies
Figure 6 compared the Ca
2+
-release capacity of the
PECW particles alone (a) and the PECW particle
incorporated WPI lm (b). For the PECW particles
(Fig. 6a), more than 65% Ca
2+
could be released from
the particle matrix at pH 1.2 but less than 50% at pH 7.4
in 6 h. The higher release capacity at acid environment
was attributed to the electrostatic repulsions between
Ca
2+
and the positive charges of the protein. For the lm
(Fig. 6b), the Ca
2+
release was reduced (55% at pH 1.2
Figure 5 Schematic image of the Ca
2+
eect on the microstructure of whey proteins isolate (WPI) lms.
0 1 2 3 4 5 6
0
20
40
60
80
100
Time (h)
pH = 1.2
pH = 1.2+pepsin
pH = 7.4
pH = 7.4+pancreatin
R
e
l
e
a
s
e

o
f

C
a

(



)
R
e
l
e
a
s
e

o
f

C
a

(



)
(a)
(b)
0 1 2 3 4 5 6
0
20
40
60
80
100
Time (h)
pH = 1.2
pH = 1.2+pepsin
pH = 7.4
pH = 7.4+pancreatin
Figure 6 Comparison of the Ca
2+
-release capacity of the PECW
particles alone (a) and the PECW particleincorporated whey proteins
isolate (WPI) lm (b).
Figure 4 Dierential scanning calorimeter (DSC) thermograms of the
three dierent whey proteins isolate (WPI) lms.
Effect of encapsulated Ca
2+
on whey protein lm Z. Chai et al. 1536
International Journal of Food Science and Technology 2010, 45, 15321538 2010 Institute of Food Science and Technology
and 32% at pH 7.4 in 6 h), because the protein
molecules in the lm matrix hindered the movement of
the Ca
2+
released from the particle matrix. In the use of
the digestive enzymes, the Ca
2+
could be largely released
(more than 75% with pepsin and more than 95% with
pancreatin in 6 h), and no signicant dierence between
the particle system and the particle-incorporated lm
system was observed. The higher release capacity with
the pancreatin was caused by its stronger ability to
hydrolyse the protein molecules.
Light transmission and transparency
Figure 7 compared the light transmittance and trans-
parency (insert image) of the lms without the free
Ca
2+
(a) and with the PECW particles (b), and no
signicant dierence was found between both systems
(P > 0.05). The two light transmittance curves were
almost superimposed and both showed about 80%
transmittance between 400800 nm while less than 6%
transmittance between 200300 nm. The UV absorption
was chiey because of the aromatic side chains as
tryptophan, phenylalanine, and tyrosine in protein
(Goldfarb et al., 1951). PS, TS and WVP of the PECW
particleincorporated WPI lm were inserted in Figs 2
and 3, respectively. In these gures, the dierences
between the lms without Ca
2+
and with the PECW
particles were not signicant (P > 0.05).
Conclusions
Taking into account the divalent cations as calcium
inducing serious protein aggregation and thus prevent-
ing the formation of lm, the quantity of calcium
added to WPI lms could not exceed a critical
concentration. Using a pre-encapsulation method, the
critical concentration of calcium could be exceeded
without signicant variation of the physical properties
including PS, TS, WVP and transparency of the lm
under the present experimental conditions. This meth-
od could potentially be used to incorporate the other
minerals or organic substances that could cause protein
aggregations.
Acknowledgments
This work was supported by China High-Tech (863)
project (2007AA10Z311), and National Science and
Technology Support Programme (2006BAD04A06).
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; (b)
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