ARTICLE IN PRESS

Journal of Cereal Science 45 (2007) 275–284

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Relationship between the glass transition temperature and the

melt flow behavior for gluten, casein and soya
Carlos Bengoecheaa, Abdessamad Arrachidb,, Antonio Guerreroa,
Sandra E. Hillb, John R. Mitchellb
a
Departamento de Ingenierı´a Quı´mica, Universidad de Sevilla, c/ P. Garcia Gonzalez 1, 41012 Sevilla, Spain
b
Division of Food Sciences, School of Biosciences, University of Nottingham, Sutton Bonington Campus, Loughborough LE12 5RD, UK
Received 29 January 2006; received in revised form 11 June 2006; accepted 25 August 2006

Abstract

The effects of moisture content (25–45% wwb) and temperature (75–120 1C) on the viscosity of gluten, soya and rennet casein systems
was studied using a capillary rheometer. An attempt was made to relate the viscosities to the glass transition temperature measured by
differential scanning calorimetry, dynamic mechanical thermal analysis and the phase transition analyzer. The temperature where the
material flowed was also determined by the latter technique. All three-protein systems showed shear and extension thinning. Over the
shear rate range investigated (1–103 s1), gluten had a substantially lower viscosity than the other two proteins, although the difference
was less pronounced at the highest temperature studied. This low viscosity is reflected by lower values of the glass transition temperature,
the melt flow temperature and the dynamic moduli E0 and E00 in the rubbery state. The results are discussed in terms of the structure and
heat induced changes for the three proteins and their relevance to food processing considered.
r 2006 Elsevier Ltd. All rights reserved.

Keywords: Glass transition; Viscosity; Rheology; Extrusion; Differential scanning calorimetry; Dynamic mechanical thermal analysis; Phase transition
analyzer; Plasticization; Protein

1. Introduction
The rheological behavior of protein ‘‘melts’’ at relatively
low water contents (15–50% wwb) is important for
extrusion processing to produce food products such as
texturised vegetable proteins (TVP). In addition, rheologi-
cal behavior of gluten at low water contents is one factor
influencing baking performance. There have been extensive
studies on the viscosity of biopolymer melts using both
pressure capillary rheometry and on-line extrusion rhe-
ometers (Bhattacharya, 1993; Breuillet et al., 2002; Fujio
Abbreviations: C–K, Couchman–Karasz; DMTA, dynamic mechanical
thermal analysis; DSC, differential scanning calorimetry; G–T,
Gordon–Taylor; PTA, phase transition analyzer; SPI, soya protein isolate;
TVP, texturised vegetable protein
Corresponding author. PTC NESTLE YORK, Haxby Road, York
YO91 1XY, UK. Tel.: +44 1904 60 31 16; fax: +44 1904 60 48 87.
E-mail address: Abdessamad.Arrachid@rdyo.nestle.com
(A. Arrachid).
et al., 1991; Singh and Smith, 1999; Zhang et al., 1998). The
variables generally studied are temperature, shear rate and
water content (Colonna et al., 1989; Harper, 1981)
although time dependent changes such as protein associa-
tions have sometimes been taken into account (Morgan
et al., 1989; Remsen and Clark, 1978).
More recently there has been an increasing interest in the
glassy state in foods (Blanshard and Lillford, 1993). The
glass transition temperature (Tg) has been measured
extensively for both carbohydrate and protein dominated
systems, generally using differential scanning calorimetry
(DSC) or dynamic rheological methods. Of particular
interest for food applications has been the plasticizing role
of water, sugars, polyols and other low molecular weight
additives (Kalichevsky et al., 1992a, b, 1993; Morales and
Kokini, 1997; Pommet et al., 2003; Roos, 1995; Zhang
et al., 2005).
A technique that has been recently developed to provide
information both about the glass transition temperature

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doi:10.1016/j.jcs.2006.08.011
 ARTICLE IN PRESS
276 C. Bengoechea et al. / Journal of Cereal Science 45 (2007) 275–284
and the melt rheology of biopolymer systems, particularly
within the context of extrusion processing, is the phase
transition analyser (PTA) (Plattner et al., 2001). In the
PTA a piston applies pressure to material initially in a
closed chamber and the temperature is increased. The glass
transition temperature is associated with the point where
the material softens, giving rise to increased movement of
the piston. A flow temperature, Tf, can also be determined
by replacing the closed die at the base of the chamber by
one containing a small hole. Tf is taken as the temperature
where the material flows as evidenced by a continuous
movement of the piston at a constant temperature. For
maize these two temperatures have been shown to differ for
crops grown in different locations and have been related to
extrusion behavior (Plattner et al., 2001).
In this study, three protein systems were examined:
gluten, soya and casein. All three are used in extrusion
processes, and gluten and soya are also important in baked
products. Large amounts of information can be found in
the literature about their structure and their properties in
extrusion (Bhattacharya and Hanna, 1986; Chen et al.,
1978; Damodaran and Paraf, 1997; Jao et al., 1978;
Kitabatake and Doi, 1992). Gluten is an amorphous
mixture of polypeptides that can be divided according to
their functionality into monomeric and polymeric poly-
peptides. Monomeric polypeptides consist mainly of
storage proteins called gliadins. They can aggregate by
non-covalent interactions and contribute to the viscosity
and extensibility of gluten. The vast majority of polymeric
polypeptides are found in the glutenin fraction. Their
amino acid compositions are similar to the gliadins. They
are additionally stabilized by disulfide bonds, and tend to
contribute to the elasticity and strain hardening behavior
of gluten (MacRitchie and Lafiandra, 1997; Weegels et al.,
1996). Soya consists of mainly 7S and 11S globulins. The
7S globulin is a trimer with a molecular weight around
150–200 kD (Fukushima, 1991). The 11S globulin is
composed by six subunits and has a reported molecular
weight of 300–400 kD (Fukushima, 1991; Pearson, 1982).
Soya is used to produce TVP (Zhang et al., 2001) and is
functionally involved in gelation and emulsification (Utsu-
mi et al., 1997). Casein is the principal protein in bovine
milk and consists of four components, as1- (38%), as2-
(10%), b- (36%) and k-casein (13%), which have molecular
weights in the range of 19–26 kD and vary in hydro-
phobicity (Dalgleish, 1997). In milk, they are arranged in a
micelle. In rennet-coagulated casein, micellar aggregation
by hydrophobic bonding is thought to precede coagulation.
Rennet casein performs well in dry spinning processes
where it is extruded at temperatures of 70 1C and above
(Visser, 1988).
One objective of the work described in this paper is to
determine if more conventional methods for determining a
glass transition temperature, DSC, dynamic mechanical
thermal analysis (DMTA) and melt rheology (pressure
capillary rheometry) are consistent with the information
obtained from the PTA. A second objective was to obtain
further information about the relationship between protein
type, the glass transition temperature and the melt
rheology.
2. Materials and methods
2.1. Materials
Rennet casein was obtained from Kerry Foods Ltd
(High Protein Milk Extract, UK), gluten from RIBA S.A.
(Glutenflor Supervital, Barcelona, Spain), and soya protein
isolate (SPI) from Protein Technologies International
(SUPRO 500E, Leper, Belgium).
2.2. Methods
2.2.1. Sample preparation
2.2.1.1. Capillary rheometry. Samples were prepared by
adding the required amount of water to the powder and
mixing with a Kenwood mixer (KenWood Mixer KMC
500, KenWood) to ensure homogeneity. Gluten forms a
viscoelastic dough when mixed with water; so to prepare a
homogenous powder it was necessary to freeze the
hydrated material in liquid nitrogen, prior to milling it to
a powder using a Knifeter 1095 Sample Mill (Foss Tecator,
Hoganas, Sweden). For all three proteins water content
was measured after each experiment performed with the
capillary rheometer and was found to match the predicted
water content.
2.2.1.2. DSC and PTA. Samples were equilibrated over
P2O5 or saturated salt solutions of MgCl2, Mg(NO3)2, KI,
NaCl or KNO3 or distilled water, which produced
equilibrium relative humidities (RH) of 0%, 32.8%,
52.8%, 68.9%, 75.3%, 93.7% and 100%, respectively.
Samples were equilibrated at room temperature for at least
seven days.
2.2.1.3. DMTA. Samples were prepared by hydrating to
17% water at 100% RH overnight and then pressing to a
thickness of 0.5–1 mm in a mold under pressure
3.1  103 kPa at a temperature between 70 and 90 1C.
Samples were then cut into 20  8  1 mm strips and stored
over salt solutions of various relative humidities (as for
DSC and PTA: Section 2.2.1.2) to obtain a variety of water
contents. Samples were stored for at least a week before
measurements were made. Following equilibration, the
water content was checked for agreement with predicted
values and the measured values were used in the
interpretation of the results. Before the measurement, the
sample was coated with silicone oil (Dow Corning, USA)
to avoid water loss.
2.3. Water content
Water contents were obtained by drying to constant
weight at 105 1C.
 ARTICLE IN PRESS
C. Bengoechea et al. / Journal of Cereal Science 45 (2007) 275–284
2.4. Capillary rheometry
A Rosand RH7 twin bore capillary rheometer (Rosand
Flowmaster RH7, Bohlin Instruments, UK) was used. The
sample was driven simultaneously through a capillary die
(l ¼ 32 mm, + ¼ 0:5 mm, y ¼ 901) as well as an orifice die
(+ ¼ 0:5 mm, y ¼ 901). The samples (25%, 30%, 35%,
40% and 45% (wwb) of water content) were loaded in the
barrels, allowed to equilibrate for 10 min and extruded at
different temperatures (65, 75, 85, 95, 120 1C). The
extrusion was carried out at ram speeds of 2, 5.63, 15.9,
44.7, 126, 355 and 1000 mm/s. The pressure was recorded
as a function of the piston speed.
The data were fitted to the power law equation as used in
previous studies (Colonna et al., 1989; Harper, 1981; Singh
and Smith, 1999):
s ¼ m_gn ,
where s is the shear stress, g_ is the shear rate, n is the flow
behavior index and m is the consistency coefficient
(Brydson, 1981).
The instrument software obtains the shear viscosity ðZ ¼
s=_gc Þ from the Rabinowitsch corrected wall shear rate:
 
3n þ 1
g_ c ¼ g_ ,
4n
where g_ the wall shear rate for a Newtonian fluid is given
by
 
4Q
_g ¼ , (3)
pr3
Q is the volumetric flow rate and r the capillary radius.
The wall shear stress is given by
ðPL  P0 Þr
s¼ , (4)
2LL
where PL is the pressure drop across the long die, length
LL, and P0 the pressure drop across a zero length die
obtained from the pressure drop across the orifice die.
The Cogswell method (Cogswell, 1972) is used to obtain
an extensional viscosity ðl ¼ se =Þ, where the extensional
stress is given by
3ðn þ 1Þ
se ¼ P0
8 rheometer is that the latter extrudes at constant piston
and the extensional strain rate by
4 Z_g2
¼ . (6)
3 ðn þ 1ÞP0
2.5. DSC
Calorimetric measurements were performed using a
Perkin Elmer DSC-7 (Perkin Elmer, UK). An empty
stainless steel pan was used in the reference holder. The
sample was heated first time at 10 1C/min from 60 to
180 1C. It was then cooled to 60 1C at 50 1C/min and
277
heated a second time at 10 1C/min to 180 1C. Finally the
sample was cooled to 20 1C at 50 1C/min. The glass
transition temperature, Tg, was determined as the tem-
perature midpoint of the heat capacity change observed
during the second run.
2.6. DSC data fitting
To predict the Tg of mixtures from the Tg’s of the
components the empirical Gordon–Taylor (Gordon and
Taylor, 1952) (G–T) equation was used:
W 1 T g1 þ KW 2 T g2
Tg ¼ , (7)
W 1 þ KW 2
where W is the mass fraction of each component, Tg is the
glass transition temperature in Kelvin, and K is a system
constant inversely proportional to the plasticizing effect of
(1)
the diluent (1) on the protein (2). This model is suitable for
bicomponent systems (food component and water or other
plasticizer) and has proven to be useful to fit DSC data for
starch, maltose, maltodextrins (Roos and Karel, 1991) and
for cereal proteins (de Graaf et al., 1993; Kokini et al.,
1995; Madeka and Kokini, 1996). For multicomponent
systems, the Couchman–Karasz (Couchman and Karasz,
(2) 1978) (C–K) equation has been used:
W 1 DC p1 T g1 þ W 2 DC p2 T g2
Tg ¼ . (8)
W 1 DC p1 þ W 2 DC p2
This equation requires the value of DCp of water, which
is subject to considerable debate (Kalichevsky et al.,
1992b). G–T equation is equivalent to the C–K equation
if K ¼ DC p2 =DC p1 (4), and DCp is the change in heat
capacity observed at Tg. The values used for Tg1 and DCp1
of water are 134 K (139 1C) and 1.94 J g1 K1, respec-
tively (Kalichevsky et al., 1993; Sugisaki et al., 1968).
Values of DCp2 for the dry protein (casein, soya, gluten)
were calculated from Eq. (3), assuming a value of
1.94 J g1K1 for DCp1 for water.
2.7. PTA
The PTA is a closed-chamber capillary ‘‘rheometer’’.
One of the main differences between PTA and a capillary
(5)
speed while the PTA performs at constant pressure.
It consists of two sealed chambers, top and bottom,
separated by an interchangeable capillary die. The two
chambers house electric heaters and contain a hollow
cavity that allows a cooling fluid to be used. The pistons,
mounted together as sidebars, are held in a fixed position
during testing. Air cylinders, mounted to the bottom of the
PTA, maintain constant pressure on the sample. A linear-
displacement transducer measures the samples’ deforma-
tion, compaction, and flow relative to initial sample height
(Plattner et al., 2001). In this study the measurements of Tg
and Tf were carried out at a heating rate of 5 1C/min and an
applied pressure of 150 bar.

278 C. Bengoechea et al. / Journal of Cereal Science 45 (2007) 275–284
2.8. Dynamic mechanical thermal analyzer
The dynamic mechanical thermal analyzer (Rheometric
Scientific, Piscataway, USA) was used with the sample
presented in the single cantilever-bending mode at a
frequency of 1 Hz and a strain of 0.03% which was in the
linear region. The heating rate used was 3 1C/min. The
results from two runs were averaged at each water content.
3. Results
3.1. Glass transition temperature
Fig. 1 displays the DSC response for the second DSC
scan for the three proteins at a single, comparable moisture
content. All three proteins showed an endothermic peak
immediately above the glass transition temperature in the
DSC first scans. This was particularly pronounced for
gluten (first run scan shown in Fig. 1). This endothermic
peak has been reported previously for a number of proteins
including gluten subunits (Castelli et al., 2000) and is
probably due to physical aging (enthalpy relaxation) an
effect well known for synthetic polymers (Drozdov, 2001;
Hay, 1993; Toufeili et al., 2002). The midpoint of the heat
capacity change from the reheat was taken as the glass
transition temperature. This change was less clear for soya
than for casein and gluten. Morales and Kokini (1997)
have reported that at, for example, a water content of 15%
the glass transition temperature of the 11S globulin is
about 40 1C higher than the 7S globulin. Since the soya
isolate used in this work is a mixture of these two proteins,
this may explain the breadth of the transition.
Fig. 2 shows the piston displacement as a function of
temperature from the PTA. As the material begins to
soften, the piston movement increases more rapidly with
temperature. The glass transition temperature was taken as
the temperature where the derivative of the displacement
temperature plot was a maximum. An interesting feature of
the displacement temperature response is the higher initial
Gluten 1st run
Heat flow - Endothermic
0 20 40 60 80 100 120 140
Temperature (C)
Fig. 1. DSC response for the three proteins during the reheat. The first
run for gluten only is represented. Moisture contents were: soya (13.7%,
wwb), casein (12.9%, wwb) and gluten (11.7%, wwb). Heating rate: 10 1C/
min.
ARTICLE IN PRESS
10
Casein
9
Soya
8
Displacement (mm)
7 Gluten
6
5
4
3
2
1
0
0 20 40 60 80 100 120 140 160 180 200
Temperature (C)
Fig. 2. Displacement temperature plots from the phase transition analyser
indicating position of glass transition temperature. Moisture contents
were: soya (13.7%, wwb), casein (12.9%, wwb) and gluten (11.7%, wwb).
Applied pressure: 150 bar. Heating rate: 5 1C/min.
bulk density and the lower degree of compression as the
temperature increases for gluten compared with the other
two proteins. This may suggest that even in the glassy state,
gluten is more deformable than the other proteins but such
an interpretation needs to be treated with caution as the
gluten samples were comminuted in a different way
(freezing followed by subsequent milling).
From DMTA data, as a function of temperature the
glass transition temperature can be taken as the tempera-
ture where a decrease occurs in E0 or the maximum in tan d
( ¼ E00 /E0 ) or E00 . In this work, the E00 maximum was taken
as the glass transition temperature since this falls in
between the other two temperatures. Fig. 3 shows the
tan d, E0 and E00 responses for all three proteins at
comparable water contents. A peak appears in the DMTA
scans around 60 1C independent of water content (Fig. 3).
Kalichevsky et al. (1992b) also reported this low tempera-
ture transition. They stated that this may be due to the
onset of short range motions, whereas the glass transition is
the onset of main chain motion. Di Gioia et al. (1999)
indicated two weak relaxations at 65 and 25 1C in the
DMTA scans attributed to secondary relaxation of
proteins. There is considerable evidence from a range of
techniques for a glassy transition in all polypeptide water
Gluten
systems at a temperature around 200 K (Ringe and Petsko,
2003) and it is possible that this mechanical relaxation is
Casein another manifestation of this. Whereas this mobility
transition may be relevant to the low temperature storage
of biological materials, it does not influence the main
Soya rheological responses of the material. Gluten shows a
higher decrease in the storage modulus 102 Pa and also a
considerably higher tan d peak than found for the other
two proteins in the glass transition region. Our results are
160 180 consistent with data of Kalichevsky et al. (1993) who
compared the tan d and E0 responses for gluten, casein,
caseinate, ovalbumin and gelatin around the glass transi-
tion. They also found a fall in E0 of approximately 102 Pa
and a tan d maximum close to 0.75 at the glass transition of
gluten at a water content of 15%. These data also
 ARTICLE IN PRESS
C. Bengoechea et al. / Journal of Cereal Science 45 (2007) 275–284 279

Fig. 3. It is appropriate to add, however, that Pouplin et al.

1.E+10 (1999) reported that the tan d maximum varied with water
Soya
content and also reported an absolute value for E0 which
Casein was 102 Pa lower than shown in Fig. 3 for the sample
1.E+09
Gluten containing 11.7% water.
Fig. 4 compares the Tg values for the three proteins from
E' (Pa)

1.E+08 the different techniques. DMTA gave the highest Tg and

DSC the lowest. Kalichevsky et al. (1992a) also indicated a
1.E+07 higher value for the glass transition temperature obtained
from DMTA at 1 Hz than the glass transition temperature
from DSC, though the differences were smaller than in our
1.E+06
-100 -50 0 50 100 150 200 study. Of particular interest are the values given by the
Temperature (C)

a
1.E+09 200
CASEIN DMTA
Soya 180
DSC
Casein 160

Temperature (C)
PTA
Gluten 140
1.E+08
120
E" (Pa)

100
80
1.E+07 60
40
20
1.E+06 0
-100 -50 0 50 100 150 200 0 5 10 15 20 25 30
Temperature (C) Moisture content (%, wwb)

b
0.9 200
DMTA
Socv
180 SOYA
0.8 DSC
0.7
Casein 160
Temperature (C)

PTA
0.6
Gluten 140
Tan delta

0.5 120
0.4 100
0.3 80
0.2 60
40
0.1
20
0
-100 -50 0 50 100 150 200 0
0 5 10 15 20 25 30
Temperature (C)
Moisture content (%, wwb)
Fig. 3. E0 and E00 and tan d responses from DMTA for the three proteins.
Moisture contents were soya (13.7%, wwb), casein (12.9%, wwb) and
c
200
gluten (11.7%, wwb). Frequency: 1 Hz. Heating rate: 3 1C/min. GLUTEN DMTA
180
DSC
160
Temperature (C)

PTA
140
suggested a much lower glass transition temperature for 120
gluten than obtained for casein and caseinate, as also found 100
in our work. Of the proteins in this investigation, only 80
gelatin gave a comparable E0 decrease to gluten and 60
showed a large tan d peak (0.9). The mechanically 40
measured glass transition temperature for gelatin as judged 20
from the temperature of the tan d peak was higher than 0
0 5 10 15 20 25 30
gluten but lower than the casein systems. This relatively
Moisture content (%, wwb)
large fall in modulus of 102 Pa has also been reported for
dry gluten by Pouplin et al. (1999), who found a value of Fig. 4. Comparison of the effect of the water content on Tg values
the tan d maximum of 0.75 compared with 0.8 shown in measured using DSC, DMTA and PTA for casein, soya and gluten.
 ARTICLE IN PRESS
280 C. Bengoechea et al. / Journal of Cereal Science 45 (2007) 275–284

PTA. This measures a temperature where the modulus of 250

Tg CASEIN
the powder decreases and might be expected to relate most
Tf
closely to the temperature where the storage modulus E0 200

Temperature (C)
falls. The lower temperature for the PTA determined Tg
compared with the DMTA value taken from the E00 150
maximum is consistent with this. It should be appreciated
that the glass transition is a kinetic phenomenon which will 100
depend not only on the criteria used to but also on the time
scale of measurement. 50
All three techniques show that gluten has the lowest glass
0
transition temperature at all moisture contents, and casein 0 5 10 15 20 25 30
the highest. Table 1 shows the results from the fitting of the Moisture content (%, wwb)
G–T to the DSC values for Tg. The glass transition
predicted for dry gluten using this approach is in good 250
SOYA Tg
agreement, within experimental error, with the values Tf
200
reported in the literature, (433 K (Hoseney et al., 1986);

Temperature (C)
435 K (Kalichevsky et al., 1992b); 412–448 K (for different
150
subunits of gluten) (Noel et al., 1995; Sartor and Johari,
1996); 423 K (Cherian and Chinachoti, 1996); 460 K 100
(Pouplin et al., 1999); 448 K (Micard et al., 2001); 401 K
(Toufeili et al., 2002). Similar agreement was found with 50
the Tg values for dry casein by Kalichevsky et al. (1992b)
(Tg ¼ 417 K) and Mizuno et al. (1999) (Tg ¼ 408 K)) and 0
for soya (387 (7S)–433 (11S) (Morales and Kokini, 1997); 0 5 10 15 20 25 30
423–473 K (by DMA) (Ogale et al., 2000)). Moisture content (%, wwb)
The very high Tg in the absence of plasticizers compared 250
with most synthetic polymers (Aklonis and MacKnight, Tg GLUTEN
1983; Ferry, 1970) could be explained by the presence of a Tf
200
Temperature (C)

significant incidence of polymer-polymer interactions, by a

high density of hydrogen bonding, or by ionic or 150
hydrophobic interactions (Pouplin et al., 1999).
100

3.2. Flow temperature (Tf)

50

Fig. 5 compares Tf and Tg both measured at a pressure 0

of 150 bar for the three proteins at different moisture 0 5 10 15 20 25 30
contents. It can be seen that gluten which has the lowest Tg, Moisture content (%, wwb)
also starts to flow at much lower temperatures than the
other two proteins. For example, at a water content of 15% Fig. 5. Effect of the moisture content on Tg and the melt flow temperature
Tf for casein, soya and gluten. Tg and Tf were obtained at a pressure of
Tf for gluten is 75 1C compared with 140 1C for soya 150 bar and a heating rate of 5 1C/min.
and 120 1C for casein. Another important feature is the
constant difference in temperature between Tg and Tf for soya can be related to the longer transition observed in the
the three proteins irrespective of the water content. This DSC plot as well as in the DMTA (E0 ).
difference is 37 1C for gluten, 39 1C for casein and
75 1C for soya. This result suggests a strong link between 3.3. Capillary viscosity
the glass to rubber transition and the ability of the proteins
to flow. The higher difference between Tg and Tf found for Fig. 6 shows the results for the shear and extensional rate
dependence of the viscosity from the capillary rheometer at
Table 1 the highest and the lowest water contents (45% (wwb) and
Gordon-Taylor and Couchman-Karasz fitting parameters obtained for 25% (wwb)) and at temperatures of 120 and 75 1C. All the
gluten, soya and casein proteins show shear and extensional thinning behaviors for
Protein Tg (K) K DCp [k ¼ DC p2 =DC p1 ] (J g1 K1) all the conditions studied. The extensional viscosities are
much higher than the shear viscosities with a Trouton ratio
Gluten 434.9 4.9370.61 0.394 of 10–70 at a common deformation rate of vicinity of
Soya 445.0 4.4270.79 0.439
1.101 s1. In all cases, the shear viscosity at the lower shear
Casein 479.4 4.9770.73 0.391
rates is much lower for gluten than it is for the other two
 ARTICLE IN PRESS
C. Bengoechea et al. / Journal of Cereal Science 45 (2007) 275–284 281

1.E+09 1.E+10
Extensional and Shear viscosity

Extensional or shear viscosity

1.E+08 1.E+09
1.E+07 1.E+08
1.E+07
1.E+06
1.E+06
1.E+05
(Pa.s)

(Pa.s)
1.E+05
1.E+04
1.E+04
1.E+03
Casein 1.E+03 Casein
1.E+02 1.E+02
Soya Soya
1.E+01 1.E+01
Gluten Gluten
1.E+00 1.E+00
1.E-03 1.E-02 1.E-01 1.E+00 1.E+01 1.E+02 1.E+03 1.E+04 1.E-03 1.E-02 1.E-01 1.E+00 1.E+01 1.E+02 1.E+03 1.E+04
Extensional or shear rate (1/s) Extensional or shear rate (1/s)

1.E+09
1.E+10

Extensional or shear viscosity

Extensional or shear viscosity

1.E+09 1.E+08
1.E+08 1.E+07
1.E+07 1.E+06
1.E+06 1.E+05

(Pa.s)
(Pa.s)

1.E+05 1.E+04
1.E+04
1.E+03
1.E+03 Casein Casein
1.E+02
1.E+02 Soya Soya
1.E+01
1.E+01 Gluten Gluten
1.E+00 1.E+00
1.E-03 1.E-02 1.E-01 1.E+00 1.E+01 1.E+02 1.E+03 1.E+04 1.E-03 1.E-02 1.E-01 1.E+00 1.E+01 1.E+02 1.E+03 1.E+04

Extensional or shear rate (1/s) Extensional or shear rate (1/s)

Fig. 6. Extensional and shear viscosity of the three proteins at different temperatures and moisture contents. Extensional viscosities are represented by the
highest values. Conditions were: (a) 75 1C–25% (wwb); (b) 75 1C–45% (wwb); (c) 120 1C–25% (wwb); (d) 120 1C–45% (wwb).

proteins. The difference is greatest for the conditions Table 2

closest to the glass transition temperature (75 1C and 25% Model fitting parameters for the capillary shear data for gluten, soya and
casein
moisture content) with gluten having a shear viscosity
between one and two orders of magnitude lower than soya Protein K0 n DE (kJ/mol) a R2
and casein. This is consistent with the Tf values measured
with the PTA which at 20% water is about 50 1C lower Gluten 2340 0.45 5.70 4.96 0.78
Soya 7.86 0.34 30.3 5.83 0.91
than the other two proteins. The glass transition tempera-
Casein 0.399 0.27 29.8 8.04 0.95
ture is also lower for gluten than the other two proteins as
previously discussed. Soya always gives the highest values
of extensional viscosities compared with the two other that the constants obtained will be dependent on the
proteins. The difference is highest for the low temperature. time–temperature history and thus should be regarded only
Some general appreciation of the effect of deformation as an empirical representation of the data set. As
rate, moisture content and temperature on viscosity can be mentioned in the introduction, models have been proposed
obtained by fitting an equation of the form (Colonna et al., to take crosslinking into account (Morgan et al., 1989;
1989): Remsen and Clark, 1978), but to include this requires a
much more complex and extensive experimental procedure.
Z ¼ k0 expDE=RT expaMC g_ n1 , (9)
Tables 2 and 3 show the fitted values for the constants in
where Z is the viscosity, k0, a constant, DE, the activation Eq. (9) for the shear and extensional viscosities, respec-
energy, MC, the moisture content of the sample, R, the tively. The values of n show that the materials appear to
universal gas constant, T, the absolute temperature, g_ in show more extension thinning than shear thinning as it can
this case is the shear or extensional rate, n, the flow be clearly seen from Fig. 6. The values also show that, in
behavior index, and a, a constant, to the data obtained at general, the gluten shear viscosity is less dependent on
all five temperatures and moisture contents. Since dena- temperature and moisture content than the other proteins,
turation and time dependent crosslinking reactions will whereas the temperature dependence of the extensional
occur at the higher temperatures it should be appreciated viscosity is much higher for soya.
 ARTICLE IN PRESS
282 C. Bengoechea et al. / Journal of Cereal Science 45 (2007) 275–284
Table 3
Model fitting parameters for the capillary extensional data for casein,
gluten and soya
Protein K0 n DE (kJ/mol) a R2
Gluten 22000 0.17 3.02 6.78
Soya 0.050 0.16 42.5 7.38
Casein 3790 0.21 14.8 8.69
4. Discussion
Results showed the difficulty to relate viscosity (shear
and extensional) behavior to glass transition temperature.
The extensional viscosity is related to the pressure drop at
the entrance to the capillary of the capillary rheometer and
will be influenced by features such as the resistance to
molecular elongation and the extent of molecular entangle-
ment between these molecules. Extensional viscosity does
not appear to relate to the glass transition temperature in
the way that shear viscosity does. It might be suggested
that a higher then expected Trouton ratio for gluten could
be related to the strain hardening properties of gluten in
extension (Breuillet et al., 2002), whereas in comparison
with casein, soya possess more ordered globular structures
resisting orientation.
Of particular interest is the very low shear viscosity
found for the gluten melt compared with the other two
proteins. The zero shear viscosity is related to the stress
relaxation modulus G(t) by the expression:
Z 1
Z¼ tGðtÞd ln t, (10)
0 and gelatin, which also show a large fall (102 Pa) in the
where the frequency dependence of G0 mirrors the time
dependence of the stress relaxation modulus G(t) (Ferry,
1970) (high frequencies will be equivalent to short times
and the extensional modulus EðtÞ ¼ 3GðtÞ). If it is assumed
that time temperature superposition holds, then a low
viscosity relates both to a low Tg, a low value of G* or G0 in
the rubbery state, a narrow transition and a narrow
rubbery region in terms of time or temperature. For a
completely amorphous high molecular weight polymer
with increasing temperature, there will be a glass to rubber
transition which is evidenced by a fall in the elastic
modulus of about 103 Pa to a value governed by the density
of non-specific and ill defined chain entanglements
(Aklonis and MacKnight, 1983; Ferry, 1970). In addition
to the fall in modulus, the glass transition is evidenced by a
peak in tan d. The height of the tan d peak relates
quantitatively to the volume fraction of the relaxing phase
and for a completely amorphous polymer the height of the
peak is 1.0, and the breadth of this transition is typically
10–20 1C (Wetton et al., 1986). The extensive studies of
water plasticised biopolymers have invariably shown
smaller and broader transitions than would be expected
for an ideal high molecular weight synthetic polymer. For
example, the fall in modulus is often only an order of
magnitude and the height of the tan d peak is less than 0.5
(Kalichevsky et al., 1993). The relationship between
polysaccharide structure and a calorimetrically determined
Tg has been extensively discussed by Bizot et al. (1997) who
consider the increase in Tg with various types of chain
immobilization. For a 1-4 linked polysaccharide chains, it
0.95 is not possible to observe a glass transition calorimetrically
0.94
possibly because of specific inter residue hydrogen bonds
0.88
(Gidley et al., 1993). Other interactions which contribute to
immobilize the polymer e.g. crystallization, the presence of
ordered secondary structure (e.g., b-sheets or a-helices in
proteins), covalent crosslinks e.g., disulfide bonds, and
other interchain non-covalent interactions sometimes
called hyperentanglements (Morris et al., 1981) would be
expected to increase Tg, and if they are preserved at
temperatures above Tg, they would reduce the size and
increase the width of the glass transition. All these effects
will increase the viscosity.
The low shear viscosity of gluten relative to the two
other proteins studied along with the higher value of tan d
and low moduli in the rubbery region, suggests that the
immobilizing interactions are lower in this protein system
than in most other biopolymers i.e., it is closer to an ideal
synthetic polymer. The reason for this is not clear. The
greater hydrophobicity as shown by the lack of water
solubility may be a factor. Another possibility could be
differences in the content of proline+hydroxyproline
residues in the proteins. These residues are not compatible
with most ordered secondary structures (a-helix, b-sheet)
and, therefore, will be another factor contributing to an
expanded disordered structure rather than one that is
compact and ordered (Mohammed et al., 2000). Gluten
storage modulus at the glass transition (Kalichevsky et al.,
1993), both have a higher praline/hydroxyproline content
than soya and casein (Mohammed et al., 2000).
It should be appreciated that the range of factors
contributing to the immobilization of the polypeptide
chain makes a detailed interpretation difficult. For
example, Mizuno et al. (2000) demonstrated an increase
in Tg on crosslinking casein with microbial transglutami-
nase as would be expected, but a counter-intuitive decrease
for soya. The latter was attributed to a reduction in b sheet
secondary structure associated with the crosslinking reac-
tion. An important complicating factor is that heating will
result in additional crosslinking. This is indicated by
exotherms observed in DSC studies of gluten at low water
contents and discussed in detail by Sartor and Johari
(1996). This behavior of gluten protein could be explained
by disulfide/thiol exchange and thiol oxidation that take
place upon heating, leading to an increase of the covalent
coupling by disulfide bonds of gluten proteins (Morel et al.,
2000; Schofield et al., 1983; Weegels and Hamer, 1998).
Crosslinks involving tyrosine have also been suggested
(Tilley et al., 2001).
Protein crosslinking, which in general would be expected
to increase with temperatue and water content, will
increase the viscosity of the protein melt.
 ARTICLE IN PRESS
C. Bengoechea et al. / Journal of Cereal Science 45 (2007) 275–284
The relative contributions of the glass transition and
crosslinking reactions can be understood qualitatively from
Eq. (10). If the time dependence of the relaxation modulus
is considered, crosslinking will increase both the width and
height of the rubbery plateau and an increase in the glass
transition temperature will increase the width of the glassy
zone. Both these effects will increase viscosity, which is an
integral of the stress relaxation modulus over time. The
observation that under all conditions gluten has a lower
shear viscosity than the other two proteins, particularly at
low shear rates where Eq. (10) is likely to be most relevant,
suggests that what dominates its low viscosity is the low Tg.
The low values for DE and a for gluten compared with the
other two proteins is probably due to crosslinking reactions
becoming more dominant at higher temperatures and
water contents resulting in a lower decrease in viscosity
with increasing temperature and water content. Cross-
linking could explain the increase in the modulus (E0 ) seen
for casein and gluten at high temperatures (Fig. 3) though
water loss might also be a factor.
5. Conclusions
The PTA gives values for the glass transition tempera-
ture and also the temperature of flow which are consistent
with the information obtained from DSC, DMTA and
capillary rheometry. Gluten gives a much lower shear
viscosity than soya and casein which is partly related to a
lower Tg and the involvement of more of the protein in the
mobilization occurring above Tg. This is shown not only by
transition techniques (DSC, DMTA, capillary rheometry)
but also by the measurements of Tg and Tf using the PTA.
This may be expected to have implications not only for
extrusion processing but for the performance of gluten in
baked products.
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