Characterization of some functional properties of edible lms

based on muscle proteins of Nile Tilapia

T.M. Paschoalick, F.T. Garcia, P.J.A. Sobral
*
, A.M.Q.B. Habitante
Universite de sao Paulo, FZEA-ZAZ-CP, P.O. Box 23, Pirassununga, SP 13630-900, Brazil
Received 19 July 2002; revised 18 November 2002; accepted 11 December 2002
Abstract
Recently, it was observed that the myobrillar as well as the sarcoplasmatic proteins obtained from sh are capable to form lms. The
objectives of this work was to elaborate and to characterize the water vapor permeability (WVP), the color and opacity, the mechanical
properties, and the viscoelastic properties of lms made with muscle proteins of Nile Tilapia (Oreochromis niloticus). The proteins were
obtained by nely grinding the sh muscle, followed by separation of the connective tissue and freeze-drying after liquid nitrogen freezing.
The lms were prepared from lmogenic solutions (FS) by the casting technique, as follows: 1 g of protein/100 g of FS, 1565 g of
glycerin/100 g of protein, pH 2.7 (acetic acid) and FS thermal treatment of 40, 65 and 90 8C/30 min. The WVP was determined by a
gravimetric method, and the color and opacity of the lms were determined with a colorimeter (model MiniScan XE, HunterLab). The
mechanical properties, force and elongation at puncture, were determined with the help of a texturometer (model TA.XT2i, TA Instruments),
at 25 8C. The viscoelastic properties were determined by dynamic mechanical analysis, with a DMA2980 apparatus (TA Instruments)
operating in the frequency scanning mode, at 30 8C, with the viscoelastic properties being calculated at 1 Hz. It was observed that the WVP
increased with the concentration of glycerin C
g
as expected and that an increase in temperature of FS thermal treatment also caused an
increase in the WVP of the lms. The color and the opacity of the lms decreased with C
g
; and were proportional to the thermal treatment
temperature of the FS. In general, it was observed that increasing the C
g
provoked linear reduction of puncture force and an increase on the
elongation at break, due to its plasticizer effect. It was also observed that increasing the C
g
caused depression on both the storage and loss
moduli values but increased the tan d: The presence of sarcoplasmatic proteins did not affect the quality of functional properties of lms based
on muscle proteins of Nile Tilapia.
q 2003 Elsevier Science Ltd. All rights reserved.
Keywords: Edible lms; Myobrillar protein; Water vapor permeability; Color; Mechanical properties; Viscoelastic properties; Tilapia
1. Introduction
In the middle of the nineties, Cuq, Aymard, Cuq, and
Guilbert (1995) working with Atlantic sardines, demon-
strated that the myobrillar proteins had the capacity to
form transparent and resistant lms. Since then, other works
were done with myobrillar proteins from sh (Cuq et al.,
1995; Cuq, Gontard, Cuq, & Guilbert, 1996a,b; Cuq,
Gontard, Cuq, & Guilbert, 1997a; Cuq, Gontard, &
Guilbert, 1997b; Monterrey-Quintero & Sobral, 1999,
2000; Sobral, 2000) and beef (Ocuno & Sobral, 2000;
Sobral, Ocuno, & Savastano, 1998; Souza, Sobral, &
Menegalli, 1997; Souza, Sobral, & Menegalli, 1998).
To be used in the lm elaboration process, the
myobrillar proteins have to be prepared adequately.
After slaughter and evisceration, the muscles are grounded
and washed conveniently to eliminate the sarcoplasmatic
proteins. After that, the material is minced and passed
through a screen, to separate the connective tissue (insoluble
proteins) (Cuq et al., 1995; Monterrey-Quintero & Sobral,
2000).
For making lms based on myobrillar proteins or on
other macromolecules, the utilization of plasticizers is
necessary to reduce brittleness, i.e. to improve the work-
ability of the material. The plasticizers, which are generally
polyols, reduce the intermolecular interactions between
adjacent chains of the biopolymer, resulting in an increase
of mobility of these chains and consequently, in exible
lms (Gennadios, McHugh, Weller, & Krochta, 1994;
0268-005X/03/$ - see front matter q 2003 Elsevier Science Ltd. All rights reserved.
doi:10.1016/S0268-005X(03)00031-6
Food Hydrocolloids 17 (2003) 419427
www.elsevier.com/locate/foodhyd
* Corresponding author. Fax: 55-19-35654114.
E-mail address: pjsobral@usp.br (P.J.A. Sobral).
Torres, 1994). As a consequence, at a macroscopic level, a
reduction in the mechanical resistance and an increase in the
elasticity and water vapor permeability (WVP) of the lms
may occur.
Considering that this increase in the WVP is undesirable
for the guaranty of quality of some packaged foods,
Sothornvit & Krochta (2000a,b) recommended another
approach for the reduction of intermolecular forces in the
case of whey protein isolate based lms: reduction of the
molecular mass of whey proteins by the utilization of
proteins with certain degree of hydrolysis. According to
these authors, this would make the reduction of the
utilization of plasticizers possible.
In addition to these studies on molecular mass reduction
of whey proteins, Japanese researchers were able to
elaborate lms with the sarcoplasmatic proteins (which
have a low molecular weight) from sh muscles (Iwata,
Ishizaki, Handa, & Tanaka, 2000; Tanaka, Iwata, &
Sanguandeekul, 2001). Iwata et al. (2000) worked with a
unique content of plasticizer, which could be considered as
elevated (50 g of glycerol/100 g of proteins), and studied the
effect of protein concentration, pH and thermal treatment of
the lmogenic solution (FS) upon certain properties of the
lms. On the other side, Tanaka et al. (2001) studied the
effect of the type and concentration of the plasticizer on
some functional properties of these lms.
Considering that the sarcoplasmatic proteins also have
the capacity to form a continuous matrix, it may be
supposed that edible lms can be produced by the mixture
of these proteins with myobrillar proteins, avoiding the
washing process of the muscles. So, the objectives of this
work was the elaboration of edible lms based on muscle
proteins of Nile Tilapia (without the proteins of stroma),
and the characterization of the WVP, color, opacity,
mechanical and viscoelastic properties of these lms as a
function of the concentration of plasticizer and the thermal
treatment of the FS.
2. Material and methods
2.1. Proteins preparation
The proteins were prepared initially by grounding the
deboned muscle (lets) of Nile Tilapia (Oreochromis
niloticus), ante rigor mortis. A paste was obtained using a
food processor for 10 min, adding ice to avoid the heating of
the material. The proteins of stroma were eliminated using a
screen (ABNT 100). The ne paste obtained was freeze-
dried after quench freezing in liquid nitrogen, in a
laboratory scale freeze-drier (Heto, model FD3). The
freeze-dried muscle proteins were grounded and tamized
in a sieve with an opening of 0.18 mm (ABNT 80),
obtaining a homogeneous powder.
This powder, that constituted the muscle protein of Nile
Tilapia (MPNT), was analyzed to determine the humidity,
lipids and proteins content, using classical methods (AOAC,
1995). The amino acid composition of MPNT was
determined after acid hydrolysis, by ionic exchange
chromatography with derivatization post-column with
ninhidrin (Monterrey-Quintero & Sobral, 2000). These
analyses were realized in duplicate.
2.2. Films elaboration
The MPNT lms were prepared by drying the FS,
conveniently applied on a support. FS were prepared under
the following conditions: protein, 1 g of MPNT/100 g of SF;
plasticizer, 1565 g glycerin/100 g protein; pH maintained
at 2.7 using acetic acid, and thermal treatment of 40, 65 or
90 8C/30 min.
Initially, the adequate amounts of glycerin and water
were added in a beaker, followed by adding MPNT, under
moderate agitation obtained with a magnetic mixer (Hanna,
HI 190 M). After that, the acetic acid was added to reduce
the pH of FS. The pH was measured every time with the
help of a digital pH meter (Tecnal, TEC-2). The FS was
thermally treated in a water bath with digital control
(^0.5 8C) of temperature (Tecnal, TE184), kept at 40, 65 or
90 8C during 30 min. Finally, the FS was conveniently
applied on Plexiglas plates (12 12 cm
2
) previously
prepared and dehydrated in an oven with air renewal and
circulation (Marconi, MA037), with PI control (^0.5 8C) of
temperature, at 30 8C and room relative humidity (55
65%), for 24 h (Monterrey-Quintero & Sobral, 2000).
Weighting (^0.0001 g) of all lms components was
accomplished using an analytical scale (Scientech, SA210).
For functional properties characterization, the lms were
conditioned at 2225 8C and 58% of relative humidity, in
desiccators with saturated solution of NaBr, for 7 days.
Then, the thickness of the lms was measured averaging
nine different positions, with a digital micrometer
(^0.001 mm) with a 6.4 mm diameter probe. All the
characterizations were accomplished in climatized room
conditions (T 2225 8C and relative humidity between
55 and 65%). Only one sample per lm was taken for test,
i.e. each lm originated only one replicate. All tests were
made in quadruplicate.
2.3. Water vapor permeability
The WVP was determined according to a method
proposed by Gontard, Guilbert, and Cuq (1993). The
edible lms were rmly xed onto the opening of cells
containing silica gel. These cells were placed in
desiccators with distilled water maintained in an oven
(Marconi, model MA415/S) with electronic control of
temperature (^0.2 8C), at 25 8C. The cells were weighted
(^0.01 g) daily, in a semi-analytic balance (Marte,
AS2000), for 8 days. The WVP was calculated with
T.M. Paschoalick et al. / Food Hydrocolloids 17 (2003) 419427 420
Eq. (1) (Gontard et al., 1993)
WVP
w
tA
x
DP
1
where x is the average thickness of the MPNT lms, A is
the permeation area (12.29 cm
2
), DP is the difference of
partial vapor pressure of the atmosphere with silica gel
and pure water (2642 Pa, at 22 8C), and the term w=t was
calculated by linear regression from the points of weight
gain and time, in the constant rate period.
2.4. Color
The color of the MPNT lms was determined with a
colorimeter (HunterLab, model Miniscan XE), working
with D
65
(day light) and a measure cell with an opening of
30 mm, being used the CIELab color parameters: L
p
; from
black (0) to white (100); a
p
; from green (2) to red (); and
b
p
; from blue (2) to yellow () (Gennadios, Weller,
Hanna, & Froning, 1996; Kunte, Gennadios, Cuppett,
Hanna, & Weller, 1997). The MPNT lms were applied in
the surface of a white standard plate, the color parameters
were measured, and transferred and calculated (Eq. (2)) in
real time for a microcomputer. The lms color was
expressed as difference of color DE
p

DE
p

DL
p

2
Da
p

2
Db
p

2
q
2
where DL
p
; Da
p
and Db
p
are the differentials between the
color parameter of the samples and the color parameter of
white standard (Table 1).
The color of the freeze-dried MPNT was also determined
using the same colorimeter (Table 1), but in this case, the
powder was put in a quartz sample cup.
2.5. Opacity
The opacity of the MPNT lms was determined
according to a Hunterlab method (Sobral, 2000), with the
same equipment for color measures, also operating in the
reectance mode. The opacity Y of the samples was
calculated as the relationship among the opacity of each
sample on the black standard Y
b
and the opacity of each
sample on the white standard Y
w
: This calculation Y
Y
b
=Y
w
was made automatically by the Universal Software
3.2 (Hunterlab Associates Laboratory).
2.6. Mechanical properties
The force and the deformation at breaking point of the
lm were determined in puncture tests (Gontard et al.,
1993). The lms were xed in a 52.6 mm diameter cell and
perforated by a 3 mm diameter probe, moving at 1 mm/s.
These tests were accomplished with an instrument of
physical measures TA.XT2i (SMS, Surrey, UK). The
puncture force F and the displacement of the probe D
at break were determined with the software Texture Expert
V.1.15 (SMS) directly from the force X displacement
curves. The puncture deformation Dl
0
=l
0
can be calculated
with D considering that stress was perfectly distributed
along the lm at breaking point (Sobral, Menegalli,
Hubinger, & Roques, 2001).
2.7. Viscoelastic properties
The viscoelastic properties of the MPNT lms were
characterized by dynamic mechanical analysis, using an
equipment DMA TA2980 controlled by a TA5000 module
(TA Instruments, New Castle, DE, USA), and with the lm
grips clamps that allowed possible uniaxial traction tests.
The analysis were carried out in the frequency scanning
(0.01200 Hz) mode, with constant temperature (30 8C),
the amplitude of deformation (0.2%) and the ow of N
2
in
the measure cell (1180 ml/min).
Rectangular samples of about 19 mm 5 mm, were
submitted to oscillatory traction (senoidal stress applied)
analysis, obtaining the storage modulus E
0
; the loss
modulus E
00
and the phase angle tan d E
00
=E
0
in
function of the frequency. For the study of the plasticizing
effect of glycerin on viscoelastic properties, E
0
; E
00
and tan d
were calculated from DMA results at 1 Hz frequency
(Lazaridou & Biliaderis, 2002), with the software Universal
Analysis V1.7F (TA Instruments).
2.8. Statistical analysis
The linear regressions necessary to the calculation of
WVP R
2
. 0:98; were accomplished with Excel 2000
software (Microsoft, Seattle, WA). All linear and non-linear
regressions for the functional properties were done with the
Microcal Origin V.4.0 software (Microcal Software, North-
ampton, USA).
3. Results and discussions
The chemical analysis made in samples of freeze-dried
muscle proteins of Nile Tilapia indicated the following
average composition: 80% protein, 7% humidity and 8%
lipids. The protein content obtained was lower than that
determined by Monterrey-Quintero and Sobral (2000), but
of the same order of that obtained by Sobral (2000), who
determined concentration of proteins as 93.2 and 85%,
Table 1
Color parameters of white standard plate and of freeze-dried MPNT
L
p
a
p
b
p
White standard 94.89 20.78 1.43
Freeze-dried MPNT
1
90.02 20.92 11.34
D
2
23.72 0.02 98.21
1
Muscle protein of Nile Tilapia.
T.M. Paschoalick et al. / Food Hydrocolloids 17 (2003) 419427 421
respectively, working with myobrillar proteins of the same
species of Tilapias used in this work. However, the results of
this study were closer to the minimum amounts encountered
by Candido (1998): 84.197.7% proteins, also obtained
with samples of freeze-dried myobrillar protein of Nile
Tilapia. The content of fat obtained was also closer to that
obtained by Sobral (2000) and to the maximum amounts
obtained by Candido (1998), which were 6.75.1%,
respectively; while Monterrey-Quintero and Sobral (2000)
obtained 2.4%.
The amino acid composition of the MPNT is
presented in Table 2. It can be observed that the polar
ionic amino acids are in high concentration (aspartic
acid, glutamic acid, arginine and lysine), such as in the
myobrillar proteins obtained by Monterrey-Quintero and
Sobral (2000). The difference between the composition of
the MPNT and that of myobrillar proteins may be
explained by the presence of sarcoplasmatic proteins
(Candido, 1998).
The freeze-dried proteins obtained showed an inter-
esting uidity, i.e. without a characteristic of agglomera-
tion. However, they were not as bright as the myobrillar
proteins of Nile Tilapia, obtained by Monterrey-Quintero
and Sobral (2000), which were almost white.
In general, the lms prepared with these proteins were
well exible and easily handled, with a good visual aspect.
The average thickness (^standard deviation) of all the lms
utilized in the characterization of optical and viscoelastic
properties and WVP was 0.076 ^ 0.002 mm at
40 8C/30 min, 0.077 ^ 0.003 mm at 65 8C/30 min, and
0.091 ^ 0.005 mm at 90 8C/30 min. In the case of charac-
terization of the mechanical properties, the average thickness
(^standard deviation) of the lms was the following:
0.081 ^ 0.004 mm at 40 8C/30 min; 0.083 ^ 0.002 mm at
65 8C/30 min; and 0.084 ^ 0.008 mm at 90 8C/30 min.
3.1. Water vapor permeability
The results of the WVP tests of the lms elaborated with
1 g of MPNT by 100 g of SF and treated at 40, 65 and 90 8C,
are shown in Fig. 1. As expected, in general the WVP
increased with the increment of C
g
: This behavior is
common in hygroscopic edible lms and it is well-explained
in terms of molecular mobility in the specialized literature
(Cuq et al., 1997a; Gennadios et al., 1994; McHugh, Aujard,
& Krochta, 1994; Ocuno & Sobral, 2000; Sobral et al.,
2001; Sothornvit & Krochta, 2000a; Tanaka et al., 2001;
Torres, 1994).
In general, the variation of the experimental data of WVP
of the lms as a function of the C
g
; followed a parabolic
behavior being well-represented by a second order poly-
nomial equation, with satisfactory adjustments (Table 3).
On the contrary, McHugh et al. (1994) determined that the
WVP of gluten lms, plasticized by glycerin, determined at
25 8C, increased linearly R
2
0:966 with the concen-
tration of plasticizer. This same behavior has been seen by
Gontard et al. (1993) also with gluten lms plasticized with
glycerin.
The study of the WVP as a function of the effect of
thermal treatment was prejudiced by the dispersion of the
experimental data. However, it could be suggested that the
more intense SF thermal treatment (90 8C/30 min) pro-
portioned more permeable lms.
The lms produced in this work showed to be more
permeable to water vapor than those of myobrillar proteins
of Atlantic sardines elaborated by Cuq et al. (1997a),
who determined the WVP in the order of 2:7 10
24
g mm
h
21
m
22
Pa
21
in lms with 40% of glycerin, T 20 8C,
pH 3.0 and 2.6 mg of proteins/cm
2
, and those of
Table 2
Amino acid composition (g amino acids/100 g of protein) for Tilapia
proteins
Muscle proteins
1
Myobrilar proteins
2
Alanine 5.50 (0.12) 5.00
Arginine 6.15 (0.00) 2.71
Aspartic acid 9.20 (0.03) 12.08
Glutamic acid 14.69 (0.03) 12.20
Phenylalanine 3.55 (0.08) 4.07
Cystine 0.78 (0.06) 0.67
Glycine 3.97 (0.03) 4.35
Histidine 2.05 (0.03) 2.57
Isoleucine 4.19 (0.14) 5.86
Leucine 7.35 (0.04) 8.36
Lysine 8.65 (0.07) 10.30
Methionine 2.30 (0.00) 3.15
Proline 3.03 (0.06) 8.95
Serine 3.48 (0.02) 4.41
Tyrosine 2.84 (0.06) 3.43
Threonine 4.18 (0.01) 4.63
Valine 4.29 (0.10) 6.22
1
Average (standard deviation).
2
From Monterrey-Quintero and Sobral (2000).
Fig. 1. Water vapor permeability of lms based on the muscle protein of
Nile Tilapia: (W) 40 8C/30 min; (A) 65 8C/30 min; (K) 90 8C/30 min.
T.M. Paschoalick et al. / Food Hydrocolloids 17 (2003) 419427 422
myobrillar proteins of Nile Tilapia plasticized by 45% of
glycerin, pH 2.7 and treated at 40 8C/30 min, in which the
WVPcould be calculated as 4.8 10
24
g mm h
21
m
22
Pa
21
(x 0:077 mm) and 5.4 10
24
g mm h
21
m
22
Pa
21
(x 0:087 mm) (Sobral, 2000). In the present work, the
lms with glycerin concentration around 40% presented
Pva . 6 10
24
g mm h
21
m
22
Pa
21
. These lms were
more permeable to water vapor, possibly due to the
plasticizing effect of the proteins with low molecular weight
present in the freeze-dried product, contrary to the lms based
only on myobrillar proteins with fat content of the same
order (Cuq et al., 1997a; Sobral, 2000).
3.2. Color
The results of the determination of lm color, expressed
as the difference of color DE
p
in relation to the white
standard plate, are shown in Fig. 2. It can be observed that
the lms produced in this study, and which SF was treated at
30 and 65 8C/30 min, showed a DE
p
which decreased
linearly (Table 4) with the increment of C
g
; while in the case
of SF treated at 90 8C/30 min, this behavior is the opposite.
The reduction of lms color with the increase of C
g
should
probably be an effect of the dilution of the proteins because
glycerin is an uncolored compound. This means that as the
glycerin concentration increases, the lm should present
less and less color in such a manner that the difference of
color will tend to zero. On the other hand, the increase of
DE
p
; with the concentration of C
g
; at 90 8C/30 min, can be
explained by some alteration of the macromolecular
structure which may have occurred, however, more analyses
are necessary to conrm this explanation. In general, it
could be suggested that the increase of temperature caused a
slight increase of lms color, possibly due to the occurrence
of reaction among the glycerin molecules and the reactive
group of lysine.
Comparing the behaviors and the values of DE
p
presented in Fig. 2 with the results (not shown) of the
differences of chrome DL
p
; Da
p
; Db
p
; it could be suggested
that the behavior of color difference was mainly due to the
variation of chrome b
p
: On another side, the initial values of
DE
p
were well related with the color DE
p
11:04 of
freeze-dried MPNT. The important difference noted in the
parameter b
p
(Table 1) indicated that the lms color was
tending to yellow.
Using equations determined by Sobral (2000), for lms
elaborated with 1 g of myobrillar proteins/100 g of SF,
45% of glycerin, pH 2.7 and SF thermal treatment of
40 8C/30 min, it can be calculated DE
p
values around 7 and
8 for lms with 0.077 and 0.087 mm of thickness,
respectively. These values were comparable to those
determined in lms of this work elaborated with 45% of
glycerin and treated at 40 8C/30 min (Fig. 2). The increase
Table 3
Parameters of the second order polynomial equation Y A BX CX
2

calculated by non-linear regression

Properties Thermal
treatment
(8C/30 min)
A B C R
2
WVP 40 3.719 0.097 23.647 10
24
0.969
65 1.132 0.192 21.460 10
23
0.722
90 20.227 0.371 23.700 10
23
0.885
E
0
40 935.98 224.130 0.187 0.963
65 974.62 227.103 0.216 0.984
90 1128.92 236.636 0.321 0.961
E
00
40 100.23 21.672 0.009 0.947
65 118.02 22.731 0.020 0.991
90 150.67 24.333 0.036 0.960
Fig. 2. Color difference of lms based on the muscle protein of Nile Tilapia:
(W) 40 8C/30 min; (A) 65 8C/30 min; (K) 90 8C/30 min.
Table 4
Parameters of the linear equation Y A BX calculated by linear
regression
Properties Thermal treatment
(8C/30 min)
A B R
2
DE
p
40 10.403 20.055 0.834
65 14.425 20.110 0.929
90 9.201 0.082 0.571
Opacity 40 6.046 20.036 0.754
65 8.721 20.043 0.335
90 7.575 20.096 0.921
Puncture force 40 7.45 20.078 0.978
65 9.54 20.115 0.951
90 5.81 20.059 0.972
Puncture deformation 40 2.99 0.054 0.674
65 1.98 0.098 0.908
90 2.16 0.068 0.738
tan d 40 0.110 1.47 10
23
0.961
65 0.112 1.40 10
23
0.991
90 0.117 1.85 10
23
0.966
T.M. Paschoalick et al. / Food Hydrocolloids 17 (2003) 419427 423
of protein concentration in SF or treatment temperature
provoked an increase of lm color in relation to those
elaborated with myobrillar proteins of Nile Tilapia
(Sobral, 2000). In addition, the lm color elaborated
in this work was higher than that based on egg albumins
DE
p
1:72:3; x 0:099 mm (Gennadios et al., 1996)
and pigskin gelatin DE
p
, 3; x , 0:090 mm (Sobral,
1999). However, it was comparable to the color of soybean
protein lms DE
p
8:511:6; x 0:0540:065 mm
(Kunte et al., 1997).
3.3. Opacity
With relation to the opacity, it could be observed that it
also decreased with the increase of C
g
(Fig. 3), possibly due
to the diluting effect of glycerin, which is a transparent
compound (low opacity). The opacity behavior in function
of the C
g
could be represented by the linear equation with
satisfactory regression coefcients (Table 4), except in the
lms treated at 65 8C/30 min due to data dispersion,
problem reported in some works about this property (Sobral,
1999, 2000).
The opacity of lms produced in this work was greater
than the opacity of pigskin gelatin lms Y , 0:5; x , 0:1
mm; which were extremely transparent, but were compar-
able to the opacity of lms based on myobrillar proteins of
Nile Tilapia Y , 3:5; x , 0:090 mm; especially in the
case of lms elaborated with more than 40% of glycerin.
3.4. Mechanical properties
The mechanical properties determined by perforation
tests, also were inuenced by C
g
; as expected. In Fig. 4, it
could be observed that the increase of C
g
provoked a linear
reduction (Table 4) of the puncture force, in the domain of
C
g
studied. This behavior was according to Cuq et al.
(1997a) and Monterrey-Quintero and Sobral (1999), who
also observed a linear reduction of puncture force of similar
lms, between 0 and 40 g of glycerol/100 g of myobrillar
protein of Atlantic Sardine, and 30 and 70 g of glycerol/
100 g of myobrillar proteins of Nile Tilapia, respectively.
On the other hand, Sobral et al. (1998) observed that the
puncture force in perforation tests with lms based on
myobrillar protein from beef and acidied by acetic or
lactic acid, was reduced exponentially with the C
g
between
25 and 100% of glycerin. This same exponential behavior
was observed by Ghorpade, Gennadios, Hanna, and Weller
(1995) in soybean protein lms and by Sothornvit and
Krochta (2001) in lms of b-lactoglobulin, both working
with traction tests.
It can be observed in the work of Monterrey-Quintero
and Sobral (1999), that the lms of 1.25% of myobrillar
protein of Nile Tilapia in SF and with 30 and 50% glycerin,
presented a puncture force of about 6.7 and 4.3 N,
respectively. This was equivalent to that of the lms
produced in this project under similar conditions. However,
all these lms were less resistant than the lms of
myobrillar protein of beef, with 30% of glycerin and
acidied by acetic acid, which presented a puncture force
around 8.7 N. Possibly, these disagreements may be
explained by differences in the amino acids compositions
between these two myobrillar proteins that caused
different macromolecular interactions.
It can be observed in Fig. 5 that the puncture deformation
of the lms increased linearly (Table 3) with C
g
: This
behavior agrees with the results observed by Sobral et al.
(1998), working with lms of myobrillar protein of bovine
meat and acidied by acetic acid. However, Gontard et al.
(1993) observed an increase of 620% in the puncture
deformation of lms based on gluten, caused by the increase
of 1633 g of glycerin/100 g of dry material, following a
segment of parabola, while Cuq et al. (1997a), working with
lms of myobrillar protein of Atlantic Sardine, observed a
sigmoid behavior, for values of C
g
lower than 40%.
Fig. 3. Opacity of lms based on the muscle protein of Nile Tilapia: (W)
40 8C/30 min; (A) 65 8C/30 min; (K) 90 8C/30 min.
Fig. 4. Puncture force of lms based on the muscle protein of Nile Tilapia:
(W) 40 8C/30 min; (A) 65 8C/30 min; (K) 90 8C/30 min.
T.M. Paschoalick et al. / Food Hydrocolloids 17 (2003) 419427 424
The lms of MPNT produced in this work presented values
of puncture deformation slightly lower to that of the
respective lms of myobrillar proteins of Nile Tilapia
(Monterrey-Quintero and Sobral, 1999). However, they were
equivalent to lms of myobrillar protein of Atlantic Sardine
at similar conditions of formulation (Cuq et al., 1997a).
3.5. Viscoelastic properties
The viscoelastic properties of the lms of MPNT varied
subtly as a function of the oscillation frequency of the strain
applied by the dynamic-mechanical analyzer (except
between 100 and 200 Hz due to resonance problem).
Examples of responses obtained during analyses at 30 8C
of the lms elaborated with 15% of glycerin and thermal
treatment of 40 8C/30 min, and 65% of glycerin and thermal
treatment of 90 8C/30 min could be observed in Fig. 6: E
0
was always greater than E
00
in the entire frequency domain,
which is a characteristic of physical gels; and tan d
decreased smoothly with the increase of frequency, while
E
0
increased after 0.1 Hz, a typically post glass transition
material behavior (Ferry, 1980). Effectively, based on the
glass transition of the lms of myobrillar protein of Nile
Tilapia (Sobral, Monterrey-Quintero, & Habitante, 2002), it
could be supposed that under the conditions of these
analyses, the lms of this work were in the rubbery state. A
similar behavior of E
0
; between 0.1 and 100 Hz, could be
observed in the work of Lazaridou and Biliaderis (2002).
The values of E
0
and E
00
calculated at 1 Hz, are presented
in Figs. 7 and 8, respectively, as a function of C
g
: These
properties decreased with the increase of C
g
due to the
plasticizing effect of glycerin. In general, the values of E
0
and E
00
dropped around 80 and 70%, respectively, following
a parabolic segment in both the cases. Thus, these behaviors
could be represented by a second order polynomial equation
with very good regression coefcients (.0.94) observed in
Table 3.
It can be observed in Figs. 7 and 8, that the increasing of
temperature of thermal treatment caused more important
reduction in E
0
and E
00
as a function of C
g
: In molecular
terms, this would occur due to possible reduction of
molecular weight of proteins, which was not probable.
Normally, heating of SF could provoke the formation of
aggregates by disulphide bonds involving residues of amino
acids with sulfur (Perez-Gago & Krochta, 2001; Vachon
et al., 2000), which might implicate on an increment of
apparent molecular weight of the protein in such a way that,
for a same concentration of glycerin, this lm would be less
plasticized. This way, the observed behavior, contrarily to
the one described, was difcult to explain.
The results of the last viscoelastic property, the phase
angle, well called tan d and calculated as the relation
between E
00
and E
0
; are shown in Fig. 9. It could be observed
that, contrarily to E
0
and E
00
; tan d increased with the C
g
in
Fig. 5. Puncture deformation of lms based on the muscle protein of Nile
Tilapia: (W) 40 8C/30 min; (A) 65 8C/30 min; (K) 90 8C/30 min.
Fig. 6. Examples of results of dynamic mechanical analysis of lms based on the muscle protein of Nile Tilapia: () C
g
15%; 40 8C/30 min; (- - -)
C
g
65%; 90 8C/30 min.
T.M. Paschoalick et al. / Food Hydrocolloids 17 (2003) 419427 425
all the thermal treatments. This could be explained,
according to Ferry (1980), by the fact that both the solvent
(glycerin) and the solute (proteins) contributed to E
00
; while
only the solute contributed to E
0
: This way, the greater
inuence (reduction) of glycerin on E
0
; caused the increment
in tan d:
Moreover, considering that the reductions of E
0
and E
00
followed the same behavior, evidently with different
intensities, the consequent increase in tan d was linear. It
could be observed in Table 4, that the adjustments of the
linear equation to the experimental points, in general, were
very good with only one value of R
2
lower than 0.96.
The values of the viscoelastic properties (E
0
; E
00
and
tan d) determined in this work, were of the same order of
magnitude of the values observed in the papers of Cuq et al.
(1997b), who worked with lms of myobrillar protein of
Atlantic Sardine, and Cherian, Gennadios, Weller, and
Chinachoti (1995) and Gontard and Ring (1996), who
worked with gluten lms containing various plasticizers. In
general, it is very difcult to compare these types of results,
because most of the authors worked with temperature
scanning, and above all, because they veried the plasticizer
effect of the sample humidity, and not necessarily of the
added plasticizer agent, as in the present work.
4. Conclusion
The utilization of muscle proteins of Nile Tilapia that is,
including the sarcoplasmatic proteins and excluding the
proteins from stroma, for the elaboration of the edible lms
with glycerin is an alternative for the utilization only of the
myobrillar proteins, once that reduces the washing stage of
the ground muscle. By this way, the industrial process can
be considered starting from sh collecting, slaughter,
cleaning, evisceration and lleting, where the llet will be
directly taken to the elaboration line of lms, starting by
grinding.
The presence of sarcoplasmatic proteins caused little
alteration of the functional properties of lms, in relation to
the lms elaborated only with myobrillar proteins. But, the
WVP, the color, the opacity, and the mechanical and
viscoelastic properties of the lms elaborated in this work
were of the same order of magnitude of those based on the
myobrillar protein of Nile Tilapia. Moreover, it should be
emphasized that the differences of the functional properties
would not constitute necessarily a disadvantage, because
there could be a demand for packages with these
characteristics.
Acknowledgements
To FAPESP, for the nancial support (00/14091-8,
02/03203-5) and IC fellowship of TMP (00/14466-1); to
CAPES for the MS fellowship of FTG and to CNPq for the
research fellowship of PJAS (522953/95-6).
Fig. 7. Storage modulus, at 1 Hz, of lms based on the muscle protein of
Nile Tilapia: (W) 40 8C/30 min; (A) 65 8C/30 min; (K) 90 8C/30 min.
Fig. 8. Loss modulus, at 1 Hz, of lms based on the muscle protein of Nile
Tilapia: (W) 40 8C/30 min; (A) 65 8C/30 min; (K) 90 8C/30 min.
Fig. 9. Phase angle tan d; at 1 Hz, of lms based on the muscle protein of
Nile Tilapia: (W) 40 8C/30 min; (A) 65 8C/30 min; (K) 90 8C/30 min.
T.M. Paschoalick et al. / Food Hydrocolloids 17 (2003) 419427 426
References
AOAC (1995). Ofcial Methods of analysis of AOAC International (16th
ed). Washington: Association of Ofcial Analytical Chemists.
Candido, L. M (1998). Obtencao de concentrados e hidrolisados proteicos
de Tilapia do nilo (Oreochromis niloticus): composicao, propriedades
nutritivas e funcionais (207 pp.). Dr These. Campinas: Faculdade de
Engenharia de Alimentos da UNICAMP.
Cherian, G., Gennadios, A., Weller, C., & Chinachoti, P. (1995).
Thermomechanical behavior of wheat gluten lms: effect of sucrose,
glycerin and sorbitol. Cereal Chemistry, 72, 16.
Cuq, B., Aymard, C., Cuq, J. L., & Guilbert, S. (1995). Edible packaging
lms based on sh myobrillar proteins: formulation and functional
properties. Journal of Food Science, 60(6), 13691374.
Cuq, B., Gontard, N., Cuq, J. L., & Guilbert, S. (1996a). Functional
properties of myobrilar protein-based biopackaging as affected by lm
thickness. Journal of Food Science, 61(3), 580584.
Cuq, B., Gontard, N., Cuq, J. L., & Guilbert, S. (1996b). Stability of
myobrillar protein-based biopackagings during storage. Lebensmittel-
Wissenschaft-und-Technologie, 29(4), 344348.
Cuq, B., Gontard, N., Cuq, J. L., & Guilbert, S. (1997a). Selected functional
properties of sh myobrillar protein-based lms as affected by
hydrophilic plasticizers. Journal of Agricultural and Food Chemistry,
45, 622626.
Cuq, B., Gontard, N., & Guilbert, S. (1997b). Thermal properties of sh
myobrillar protein-based lms as affected by moisture content.
Polymer, 38, 23992405.
Ferry, J. D. (1980). Viscoelastic properties of polymers. New York: Wiley.
Gennadios, A., Mchugh, T. H., Weller, C. L., & Krochta, J. M. (1994).
Edible coatings and lms based on proteins. In J. M. Krochta, E. A.
Baldwin, & M. Nisperos-Carriedo (Eds.), Edible Coatings and Films to
Improve Food Quality (pp. 210278). Lancaster: Technomic Pub. Co.,
Inc.
Gennadios, A., Weller, C. L., Hanna, M. A., & Froning, G. W. (1996).
Mechanical and barrier properties of egg albumen lms. Journal of
Food Science, 61(3), 585589.
Ghorpade, V. M., Gennadios, A., Hanna, M. A., & Weller, C. L. (1995).
Soy protein isolate/poly(ethylene oxide) lms. Cereal Chemistry, 72(6),
559563.
Gontard, N., Guilbert, S., & Cuq, J.-L. (1993). Water and glycerol as
plasticizer affect mechanical and water vapor barrier properties of an
edible wheat gluten lm. Journal of Food Science, 58(1), 206211.
Gontard, N., & Ring, S. (1996). Edible wheat gluten lm: inuence of water
content on glass transition temperature. Journal of Agricultural and
Food Chemistry, 44, 34743478.
Iwata, K., Ishizaki, S., Handa, A., & Tanaka, M. (2000). Preparation and
characterization of edible lms from sh water-soluble proteins.
Fisheries Science, 66, 372378.
Kunte, L. A., Gennadios, A., Cuppett, S. L., Hanna, M. A., & Weller, C. L.
(1997). Cast lms from soy protein isolates and fractions. Cereal
Chemistry, 74(2), 115118.
Lazaridou, A., & Biliaderis, C. G. (2002). Thermophysical properties of
chitosan, chitosan-starch and chitosan-pullulan lms near the glass
transition. Carbohydrates Polymers, 48, 179190.
Mchugh, T. H., Aujard, J. F., & Krochta, J. M. (1994). Plasticized whey
protein edible lms: water vapor permeability properties. Journal of
Food Science, 59, 416419. see also pp. 423.
Monterrey-Quintero, E. S., & Sobral, P. J. A. (1999). Caracterizacao de
propriedades mecanicas e oticas de biolmes a` base de prote nas
miobrilares de tilapia do nilo usando uma metodologia de superf cie-
resposta. Ciencia e Tecnologia de Alimentos, 19(2), 294301.
Monterrey-quintero, E. S., & Sobral, P. J. A. (2000). Preparo e
caracterizacao de prote nas miobrilares de tilapia do nilo (Oreochro-
mis niloticus) para elaboracao de biolmes. Pesquisa Agropecuaria
Brasileira, 35(1), 179189.
Ocuno, D., & Sobral, P. J. A. (2000). Permeabilidade ao vapor de agua de
biolmes a` base de prote nas miobrilares de carne. Brazilian Journal
of Food Technology, 3, 1116.
Perez-Gago, M. B., & Krochta, J. M. (2001). Denaturation time and
temperature effects on solubility, tensile properties, and oxygen
permeability of whey protein edible lms. Journal of Food Science,
66(5), 705710.
Sobral, P. J. A. (1999). Propriedades funcionais de biolmes de gelatina em
funcao da espessura. Ciencia & Engenharia, 8(1), 6067.
Sobral, P. J. A. (2000). Inuencia da espessura sobre certas propriedades de
biolmes a` base de prote nas miobrilares. Pesquisa Agropecuaria
Brasileira, 35(6), 12511259.
Sobral, P. J. A., Menegalli, F. C., Hubinger, M. D., & Roques, M. A. (2001).
Mechanical, water vapor barrier and thermal properties of gelatin based
edible lms. Food Hydrocolloids, 15(4/6), 423432.
Sobral, P. J. A., Monterrey-quintero, E. S., & Habitante, A. M. Q. B. (2002).
Glass transition of Nile tilapia myobrillar protein lms plasticized by
glycerin and water. Journal of Thermal Analysis and Calorimetry,
67(2), 499504.
Sobral, P. J. A., Ocuno, D., & Savastano, H., Jr. (1998). Preparo de
prote nas miobrilares de carne e elaboracao de biolmes com dois
tipos de acidos: propriedades mecanicas. Brazilian Journal of Food
Technology, 1(1/2), 4452.
Sothornvit, R., & Krochta, J. M. (2000a). Water vapor permeability and
solubility of lms from hydrolyzed whey protein. Journal of Food
Science, 65(4), 700703.
Sothornvit, R., & Krochta, J. M. (2000b). Oxygen permeability and
mechanical properties of lms from hydrolyzed whey protein. Journal
of Agricultural and Food Chemistry, 48, 39133916.
Sothornvit, R., & Krochta, J. M. (2001). Plasticizer effect on mechanical
properties of b-lactoglobulin lms. Journal of Food Engineering, 50,
149155.
Souza, S. M. A., Sobral, P. J. A., & Menegalli, F. C (1997).
Desenvolvimento de lmes comest veis a` base de prote nas miobri-
lares extra das de carne bovina. Proceedings of Workshop sobre
Bipol meros, Pirassununga (SP) (pp. 102106).
Souza, S. M. A., Sobral, P. J. A., & Menegalli, F. C (1998). Glass transition
of a meat myobrilar protein based edible lm. Proceedings of
Workshop on Biopolymer Science, Montpellier, France (pp. 183188).
Tanaka, M., Iwata, K., Sanguandeekul, R., et al. (2001). Inuence of
plasticizers on the properties of edible lms prepared from sh water-
soluble proteins. Fisheries Sciences, 67(2), 346351.
Torres, J. A. (1994). Edible lms and coatings from proteins. In N. S.
Hettiarachchy, & G. R. Ziegler (Eds.), Protein Functionality in Food
Systems (pp. 467507). New York: Marcel Dekker.
Vachon, C., Yu, H.-L., Yefsah, R., Alain, R., St-Gelais, D., & Lacroix, M.
(2000). Mechanical and structural properties of milk protein edible
lms cross-linked by heating and g-irradiation. Journal of Agricultural
and Food Chemistry, 48, 32023209.
T.M. Paschoalick et al. / Food Hydrocolloids 17 (2003) 419427 427