ARTICLE IN PRESS
Radiation Physics and Chemistry 71 (2004) 93–97
Gamma irradiation influence on physical properties
of milk proteins
K. Cieślaa,b,*, S. Salmierib, M. Lacroixb, C. Le Tienb
b
a
Institute of Nuclear Chemistry and Technology, Dorodna 16 Str., 03-195 Warsaw, Poland
Canadian Irradiation Center, research laboratories in Sciences applied to Food, INRS-Institute Armand Frappier,
University of Quebec, 531 Boulevard des Prairies, Laval, Quebec, Canada H7V 1B7
Abstract
Gamma irradiation was found to be an effective method for the improvement of both barrier and mechanical
properties of the edible films and coatings based on calcium and sodium caseinates alone or combined with some
globular proteins. Our current studies concern gamma irradiation influence on the physical properties of calcium
caseinate–whey protein isolate–glycerol (1:1:1) solutions and gels, used for films preparation.
Irradiation of solutions was carried out with Co-60 gamma rays applying 0 and 32 kGy dose. The increase in viscosity
of solutions was found after irradiation connected to induced crosslinking. Lower viscosity values were detected,
however, after heating of the solutions irradiated with a 32 kGy dose than after heating of the non-irradiated ones
regarding differences in the structure of gels and resulting in different temperature–viscosity curves that were recorded
for the irradiated and the non-irradiated samples during heating and cooling. Creation of less stiff but better ordered
gels after irradiation arises probably from reorganisation of aperiodic helical phase and b-sheets, in particular from
increase of b-strands, detected by FTIR. Films obtained from these gels are characterised by improved barrier
properties and mechanical resistance and are more rigid than those prepared from the non-irradiated gels. The route of
gel creation was investigated for the control and the irradiated samples during heating and the subsequent cooling.
r 2004 Elsevier Ltd. All rights reserved.
Keywords: Milk proteins; Gamma irradiation; Viscosity; Conformation; Gel structure
1. Introduction
In recent years the interest has increased in application of biopolymers in food industry, medicine and
environmental protection. Edible and biodegradable
packaging materials constitute one of the possible
applications. Using of edible films and coatings has
appeared to be the appropriate way for prolongation of
the shelf life of ready to eat foods and for increase its
quality with no contribution to the environmental
*Corresponding author. Institute of Nuclear Chemistry and
Technology, Dorodna 16 Str., 03-195 Warsaw, Poland. Tel.:
+48-22-8111313; fax: +48-22-8111917.
E-mail address: [email protected] (K. Cieśla).
pollution. These materials act as selective barriers for
moisture, gas and solute migration. They can also act as
carriers for food additives.
Although proteins are known for having good film
forming abilities, protein films have rather moderate
barrier properties. A necessity exists therefore for search
of new compositions and processes permitting to
obtain better products. Crosslinking induced using
gamma irradiation was found to be an effective
method for the improvement of both barrier and
mechanical properties of the edible films and coatings
based on calcium and sodium caseinates alone or
combined with some globular proteins (Brault et al.,
1997; Le Tien et al., 2000; Sabato et al., 2001; Lacroix
et al., 2002).
0969-806X/$ - see front matter r 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.radphyschem.2004.04.068
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K. Cieśla et al. / Radiation Physics and Chemistry 71 (2004) 93–97
At present the effect of gamma irradiation is
presented on the rheological properties of calcium
caseinate—whey protein isolate–glycerol (1:1:1) solutions, fresh and gelatinised at 90 C. Apart from the
studies carried out at ambient temperature, the route of
gel creation was examined during heating and cooling in
the range 20–90 C. The results were related to
modification of proteins conformation studied by FTIR.
Structural properties of proteins in solutions and gels
are related, therefore, to the functional (mechanical and
barrier) properties of the films obtained at the same
conditions using of the control and the irradiated
solutions.
Our studies concerns samples subjected to irradiation
and also to heating. It is known that both types of
treatment can induce an increase in molecular weight,
although they differ in capability to affect particular
proteins. Considering our mixed system, it seems also
worthy to remember, that the crosslinking of caseinates
was found to be more efficient by irradiation while the
crosslinking of whey proteins is more efficient by heating
(Lacroix et al., 2002).
2. Experimental
2.1. Materials and preparation of solutions, gels and films
Calcium caseinate (CC, by New Zealand Milk
Product Inc.), whey protein isolate (WPI, by BiPro
Davisco) and chemical grade glycerol were mixed in a
weight ratio of 1:1:1 and dissolved in water. The
solutions containing 7.5% (in terms of total proteins
mass) were poured in a test tube under a flow of an inert
gas atmosphere (in order to avoid degradation) and
irradiated with 60Co gamma rays in Canadian Irradiation Centre, applying a dose of 32 kGy at a dose rate of
7 Gys 1. The solutions were dissolved till 5% (of total
proteins). A parts of these solutions were used for
viscosity studies while the other parts were heated for
45 min in an immulogical water bath at constant
temperature of 90 C. Water content of the sample
was controlled and the water loss was filled up before
further operations. The pH values of all the solutions,
irradiated and control were in the range of 6.6–6.8. Nonirradiated control solutions, gels and films were also
prepared.
Films were prepared accordingly to the procedure
described in details by Cieśla et al. (2003). The films were
stored before measurements for 48 h at 56% of humidity
(in dessicator over saturated NaBr solution). Thicknesses of the films (equal to ca. 50 mm) were measured
with a Mitutoyo Digimatic Indicator (Tokyo, Japan). In
addition to the mixed CC–WPI compositions, the films
were also made separately using CC or WPI, in order to
perform FTIR studies.
2.2. FTIR spectroscopy
Total reflectance method was applied using the
Perkin–Elmer
spectrum
one
spectrophotometer
equipped with Universal ATR sampling accessory with
diamond crystal. Spectra (100 scans) were recorded in
the region of 650–4000 nm. Measurements were done for
3–4 films of each composition and the average spectrum
was then calculated and analysed. The method based on
analysis of the second derivative of the amide I band at
1630 nm was applied in purpose to examine the proteins
conformation (Byler and Susi, 1988; Bandekar, 1992).
2.3. Studies of the rheological properties
Viscosity measurements were carried out applying
Brookfield viscometer type LVDV-II+ (Brookfield
Engineering Laboratories Inc., MA, USA) with the
close-tube system enabling to create a circulating water
bath by connecting the water jacket to the bath inlet and
outlet ports. The ULA spindle was used in measurements as well as the Wingather V2.1 programme for
data collection and calculation. Two types of experiments were performed.
The I type experiments were carried out at ambient
temperature applying shear rate equal to 112.3, 61.2,
30.6 s 1 (corresponding to the rotation speed RS of 100,
50 and 25 turns/min, respectively). This range of shear
rate was selected on the basis of the preliminary results
in regard to good reproductivity and realiabity of the
data. Moreover, almost constant viscosity values were
determined in this range and, consequently, linear
dependence of a shear stress was obtained upon a shear
rate. The average values of viscosity and of shear stress
were calculated as an average value of four measurements carried out for four separately prepared solutions
of each composition.
The II type experiments were carried out during
dynamic heating and cooling in the range of 25–90 C. In
the last case the freshly heated and still hot solution was
placed into the viscometer kept at 90 C. The solution
was equilibrated at 90 C during 3 min before cooling.
Viscosity was then recorded at several selected temperatures. All the measurements were calculated by applying
shear rate equal to 30.6 s 1 (RS equal to 25 turns/min).
Four repetitions of the experiments were done. Heating
and cooling were realised with average rates of 2.4
and 1.9 C min 1, respectively, in the total range of
temperature.
2.4. Functional properties of the films
Water vapour permeability (WVP) tests were conducted in the humidity chamber at a temperature of
30 C and relative humidity of 56% using of a modified
ASTM procedure described in detail by Sabato et al.
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(2001). Details of the procedure applied for examinations of mechanical properties are given in the paper of
Brault et al. (1997). A Stevens LFRA Texture Analyser
Model TA/100 (NY, USA) was applied. The puncture
strength (related to the film thickness in the point) and
deformation were calculated on the basis of the obtained
force-deformation curves. Viscoelastic properties were
evaluated using relaxation curves.
Two replicants of ten samples each were tested for
both film types. The SAS statistical package was applied
to analyse the results statistically. The Student-t test was
used and paired-comparison. Differences between
means were considered significant when pp0:05:
3. Results and discussion
3.1. FTIR spectroscopy
A decrease in the amount of the a-helix phase
accompanied by an increase in the total amount of the
well-ordered crosslinked b-phase was observed after
irradiation in the case of the CC films (Fig. 1a). It is
Fig. 1. Comparison of FTIR spectra recorded for the nonirradiated (continous lines) and irradiated films (dashed lines):
(a) CC (b) WPI (c) CC–WPI (1:1).
95
shown by a decrease in the intensity of the band at
1651 nm corresponding to a helix, with simultaneous
increase in the intensities of the bands at 1637 and
1630 nm corresponding to b structure. It might be also
concluded, on the basis of a split in the band at 1645 nm
to two bands at 1642 and 1647 nm, that the transformation takes place in aperiodic phase (random coil).
Changes in relative intensities of the bands at 1630,
1634 and 1637 nm corresponding to b-sheet suggest
reorganisation of this phase, while the increase
in the amount of strongly bonded and fine ordered bstrands (attributed to intermolecular b-sheets)
might be stated on the basis of increased intensity of
the band at 1618 nm. A simultaneous decrease of
the band at 1667 nm shows a decrease in the amount
of b-turns, classified as an unordered crosslinked
phase.
Rearrangement of b-sheets after irradiation, particularly an increase of the amount of b-strands might be
concluded also in the case of the films prepared from
pure WPI (Fig. 1b), respectively on the basis of the
changes in intensity of the bands at 1630, 1634 and
1637 nm and increase of the intensity of the band at
1625 nm (probably corresponding to dimers). Small
changes might be also supposed in the aperiodic a-helix
phase due to the slightly higher intensity of the band at
1640 nm. No change in the amount of a-helix phase can
be stated after irradiation. This corresponds well to the
relatively small content of a-helices, while b-sheets
constitute a typical conformation of b-lactoglobulin,
appearing to be the predominant component of whey
globulins.
For CC–WPI compositions the similar conclusions
might be drawn as for pure WPI (Fig. 1c).
The changes in conformations, in particular an
increase in b-strands, can be related to the tendency
of the protein to adopt better ordered structure
after radiation induced crosslinking. It is worth
mentioning that the essential modification of conformation similar to that caused by irradiation might
result also from aggregation induced by thermal
treatment. More essential aggregation arising after
heating performed at a rather high temperature of
90 C (Lefevre and Subirade, 2000) might also be
responsible for the smaller difference between the
content of a-helices found in the control and the
irradiated samples, as compared to the previously
discovered, after heating at 80 C (Le Tien et al., 2000),
except for the possible differences between the peculiar
protein samples.
The change in protein configuration results in
the shift of the maximum intensity of the amide I band
to the higher values (1639 to 1640, 1629 to 1631 and
1631 to 1633, in the case of the films prepared from
pure CC, pure WPI and CC–WPI composition,
respectively.
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3.2. Rheological properties of the solutions and gels
Fig. 2 presents a comparison of the dependence of
shear stress on shear rate for the fresh and gelatinised
solutions, control and irradiated. The larger values of
shear stress were recorded before heating at the same
shear rate for the irradiated solutions than for the
control ones due to the increase in viscosity caused by
proteins crosslinking (curves 2 and 1, respectively).
Viscosity of the control solutions increases while
viscosity of the irradiated ones decreases after further
heating in regard to gel creation. As a result, the
irradiated gels are less viscous than the non-irradiated
one (Fig. 2, curves 3, 4). These results corresponds well
to the conclusions arising from FTIR data. The
increasing amount of the highly ordered b strands after
irradiation leads to creation of gels have reveal an
improved ‘‘fine stranded’’ structure as the presence of bstrand structure enables to obtain more elastic and
therefore less stiff gels than the ‘‘particulate’’ ones
created on the way of association of protein particles
(Lefevre and Subirade, 2000). Due to the presence of the
better ordered structure the irradiated gels are more
elastic than gels produced from the control solutions.
Decrease in viscosity takes place during heating both
the irradiated and the control solutions (Fig. 3a) due to
increase in molecular mobility at higher temperature.
Possible dissociation of dimers into monomers pointed
Fig. 2. Comparison of dependence of shear stress on shear rate,
obtained in the case of: (1) control solution, (2) solution
irradiated with a 32 kGy dose, (3) heated at 85 C for 45 min, (4)
irradiated with 32 kGy and heated at 85 C for 45 min. The
values of shear stress determined at shear rate of 122.3, 61.2 and
30.6 s 1, respectively, were equal to 2.38, 1.18, 0.61 (curve 1);
2.84, 1.43, 0.73 (curve 2); 2.85, 1.42, 0.74 (curve 3), 2.64, 1.30,
0.66 (curve 4).
Fig. 3. Viscosity determined at selected temperature for the
control and the irradiated CC–WPI solutions during: (a)
dynamic heating with an average rate of 2.4 C min 1; (b)
cooling with an average rate of 1.9 C min 1.
out as the first step taking place during fine-stranded gel
creation (Lefevre and Subirade, 2000) can reveal the
similar effect. More essential and faster preliminary
decrease in viscosity observed for the irradiated samples
than for the control ones can be related to the more
intensive dissociation occurring after irradiation. As a
result, viscosity values determined for both samples
became closed already at 40 C, although those of
irradiated samples were still slightly higher at all
temperature ranges than those of the controls.
Increase in viscosity was faster during cooling for the
control than for the irradiated solutions (Fig. 3b).
Viscosity of the irradiated solution was larger at the
temperatures higher than 50 C than the viscosity of the
control solution but smaller in the range of lower
temperature. These results corresponds well to the
smaller viscosity values determined at ambient temperature for the irradiated and afterwards heated solutions
as compared to these which were only heated (Fig. 1).
3.3. Mechanical and barrier properties of the films
Stronger films with the improved barrier properties
were prepared from the irradiated solutions than from
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97
Table 1
Functional properties of the films
Dose
(kGy)
Tensile strength
(N mm 1)
Deformation
(mm)
Viscoelasticity coefficient
WVP
( 10 g mm/m2 d mmHg)
I
II
III
IV
V
0
32
53.972.6
77.473.2
4.4670.29
4.0770.35
0.52470.01
0.56170.01
168.6710.1
114.979.6
the non-irradiated ones, as shown by the larger puncture
strength and smaller water vapour permeability
(Table 1). Simultaneously, smaller deformation values
and larger viscoelasticities indicate higher rigidity of the
irradiated films as compared to the non-irradiated ones.
Statistical analysis has proved that all the functional
properties measured for all the irradiated films differs
significantly from those determined for the control
films.
4. Conclusion
Increase in viscosity of the irradiated proteins solutions as compared to the controls was found due to
radiation-induced crosslinking. It might be stated, on
the basis of the lower values of viscosity found after
heating for the irradiated solutions as compared to the
controls that gels obtained from the irradiated solutions
reveal better developed ‘‘fine-stranded’’ structure than
gels prepared from the control solutions. Creation of the
better ordered gels after irradiation corresponds well to
rearrangement of the crosslinked b-phase (accompanied
by reorganisation of aperiodic phase) found by FTIR. In
particular, higher content of the strongly bonded bstrands was detected in the irradiated and heated
samples, as compared to those which were only heated.
Using of gamma irradiation therefore causes better
improvement in well organised b conformation than
thermal treatment alone. Presence of the better ordered
protein conformations in gels obtained from irradiated
solutions leads to production of the more ‘‘crystalline’’
films. These films are characterised by improved
barrier properties and mechanical resistance and
higher rigidity than those prepared from the nonirradiated solutions.
Acknowledgements
The financial support of International Atomic Energy
Agency (fellowship of K. Cieśla, C6/POL/01003P)
enabling to perform the experiments, is kindly acknowledged.
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