Glass transition of gluten.

1: Gluten and gluten-sugar mixtures

M. T. Kalichevsky, E. M. Jaroszkiewicz and J. M. V. Blanshard
Department of Applied Biochemistry and Food Science, Nottingham University, School of Ayriculture, Sutton
Boninoton, Loughborough LE12 5RD, UK
(Received 11 December 1991; revised 2 July 1992)

The glass transition in hydrated wheat gluten has been studied usin 9 dynamic mechanical thermal analysis,
differential scannin 9 calorimetry, pulsed nuclear magnetic resonance and a three point bend test. The results
for ffluten alone are in good agreement with results obtained by other workers for 9tuten and 91utenin. In
contrast to their effect on the Tg of amylopectin, a gluten:sugar ratio of 10:1 (where the sugar is amorphous
fructose, sucrose or 9lucose) has little effect on the 9lass transition temperature, as a function of water content.
A sample containin 9 91uten and fructose in the ratio 2:1 showed plasticization due to the suyar.

Keywords: Glass transition; gluten; sugars; dynamic mechanical thermal analysis; differentialscanning calorimetry;
pulsed nuclear magneticresonance

Introduction the free volume theory of Bueche 7, and the C o u c h m a n -

Although wheat flour contains only 8-18% of protein,
the unique properties of wheat flour which enable it to
be used in a wide range of baked products, are thought
to arise chiefly from the viscoelastic properties, of
hydrated gluten proteins. Gluten is insoluble in water
but is compatible with water and plasticized by water.
It may be isolated by washing dough to remove starch
and other water and salt soluble components. When
subsequently dried, gluten contains residual starch
(10-15%) and lipid in addition to the protein.
The gluten proteins have been broadly divided into
two groups, glutenins and gliadins, on the basis of their
solubility or insolubility in 70-90% aqueous ethanol.
(For further information about gluten structure see e.g.
Ref. 1. )
A glass transition for gluten was first observed by
differential scanning calorimetry (d.s.c.) in 19842. Since
then Hoseney et al. 3 have obtained the Tg of gluten by
d.s.c, as a function of water content up to 16% water.
A glass transition has also been observed recently for
glutenin by d.s.c, and mechanical spectroscopy 4. These
d.s.c, results for glutenin are very similar to those
obtained for whole gluten 3. This suggests that either
glutenin dominates the physical properties of gluten, or
that both glutenin and gliadin have a similar glass
transition temperature.
In this study, the glass transition temperature (Tg) of
wheat gluten is studied as a function of water content
using a variety of techniques including dynamic
mechanical thermal analysis (d.m.t.a.), pulsed nuclear
magnetic resonance (n.m.r.) and d.s.c. The same
techniques are also applied to studying ternary systems
containing gluten, sugar (glucose, sucrose or fructose)
and water of various compositions. The results are
compared with theoretical curves.
In a previous study of the glass transition of
amylopectin 5 and amylopectin-sugar mixtures 6, both
Karasz s thermodynamic approach were used to model
plasticization by sugars and water. The free volume
approach underestimated the plasticizing effect of sugars
and water, but the Couchman-Karasz approach gave a
good fit to the data 6. The Couchman-Karasz equation
assumes the following form for a two component mixture:
Tg(mixture) = W~ACp,Tg, + WzACv2Tg 2 (1)
W1ACp, + W2ACp2
For a three component mixture an extra component may
be added to the equation. The subscript '1' refers to the
polymer and the subscript '2' to the diluent. W is the
weight fraction of each component, and ACp is the change
in heat capacity observed at Tg.
The Gordon Taylor (G-T) equation 9 is an empirical
equation for predicting the Tg of mixtures from the Tg
values of the components:
Tg = W, T~, + K W2 T~2 (2)
WI + KW2
In fact this equation is equivalent to the C o u c h m a n -
Karasz (C-K) equation (1) if K = ACp2 ACp, (3). The
G o r d o n - T a y l o r equation has been used by Roos and
Karel 1° to fit Tg data for starch, maltose and
maltodextrins. It is more straightforward to fit as there
is one less constant; but is not easily extended to
multicomponent systems or systems containing different
components as is the case for the C-K equation.
In contrast to the G-T equation, the C-K equation
requires values for both the Tg and the ACp of water
which have been the subject of considerable debate. The
values we have been using 6 are those obtained on vapour
condensed water by Sugisaki et al. ~1, namely 134 K and
1.94 J g - ~ K - ~ respectively. Both these values have been
subject to debate, due to the difficulty in reproducibly
obtaining amorphous solid water (ASW), which is

0141 8130/92/050257-10
© 1992Butterworth-HeinemannLimited Int. J. Biol. Macromol., 1992, Vol. 14; October ' 257
Glass transition o f 9luten. 1: M. T. Kalichevsky et al.
kinetically unstable and may be substantially nucleated
in the cooling process. Very different calorimetric data
have been obtained from samples of ASW obtained in
various ways and the Tg is not always observed lz.
However, there appear~ to be a general consensus that
the Tg of water falls in the region between 130 145 K at
normal d.s.c, heating rates (although thermodynamically
it may be expected to occur above 170 K13). Evidence
for this comes from calorimetric studies ofhyperquenched
and vapour deposited water 11,14-17, extrapolations of Tg
values of salt solutions 13'18 and some transitions
observed (mechanically and by d.s.c.) in hydrogels 19'2°
(although the interpretation of these latter results is
debatable, an additional transition occurring at 162 K
being attributed to the Tg of freezable water19). Recent
d.s.c, values for Tg of ASW give T~..... of 136 K at a
heating rate of 30 K / m i n 14'2°, which is compatible with
our use of 134 K for comparison with Tg data obtained
from the midpoint of ACp at a heating rate of 10 K/min,
as decreasing the heating rate decreases the measured Tg.
Also a few degrees error in the Tg of water would not
significantly affect the predictions.
The ACp at Tg for ASW is even more difficult to
determine accurately. Apart from the value of Sugiski et
al. 11 (1.94 J g - 1 K - 1 ) there are few other values in the
literature. Angell and Tucker is believe this value may be
too high due to the frequently occurring relaxation
overshoot not being taken into account. They stated that
a value of approx. 1.5 J g 1K- 1 was more likely, if the
overshoot was 20% as was the case for methanol,
particularly in view of the fact that the ACp values of salt
solutions extrapolate to values between 1.05 and
1 . 3 9 J g - l K -1 for pure water TM. The recent work of
Hofer et al. 21 has shown a non-linear dependence of Tg
of vitrified dilute aqueous solutions on concentration.
However, as we are working on systems containing
5-30% water, it may be that the effective Tg and ACp of
water are not too different from extrapolations from
concentrated systems.
The recent studies of Hallbrucker et a/. 17'22 on
hyperquenched and annealed vapour deposited glassy
water give values of 0 . 0 8 9 - 0 . 1 0 6 J g - l K -1 for ACp,
which is much lower than previous values. They were
able to show that this transition is reversible, which had
not been achieved before, but it is hard to believe that
ACp for water could be so small. It has been pointed out,
from a study of molecular liquids, that increased
hydrogen bonding appears to result in increased values
of Tg and ACp. Water is clearly a highly hydrogen bonded
system, resulting in a Tg higher than methanol (103 K)
or ethanol (97 K.) 24. The Tg of methanol is also higher
than that of ethanol due to increased hydrogen bonding,
despite the fact that increasing molecular weight would
be expected to increase Tg. Unfortunately there has not
been a systematic study of ACp of alcohols. However, a
systematic study of glucose malto-oligomers 25 has
indicated that ACp increases with decreasing molecular
weight, so that the value for water might be expected to
be higher than that for the sugars (ranging from 0.48 to
0.88 J g - l K - 1 , Ref. 25). Of course, it is quite possible
that water has unique properties in this as in other ways.
If the Hallbrucker et al. value for ACp of water is of the
right order of magnitude, this would invalidate the
theoretical use of the C-K equation for systems
containing water, as it would no longer predict the
substantial plasticizing effect of water observed in many
258 Int. J. Biol. Macromol., 1992, Vol. 14, October
systems, but it may still be useful as an empirical
equation. The effect of using different ACp values for
water on predictions for mixed systems is discussed in
the results.
Experimental
Sample preparation
Commercial wheat gluten (Stadis Brand, ex RHM,
containing approx. 75% protein, 11% starch, 5% lipid
and 6% water) was employed in this study and samples
were hydrated using liquid nitrogen. The required
amount of water was sprinkled into liquid nitrogen in a
mortar and ground to a fine ice powder, using a pestle.
The appropriate amount of gluten was then added and
mixed (still in liquid nitrogen) to form a uniform mixture
of ice and protein. The liquid nitrogen was allowed to
evaporate and the sample was stored in the freezer prior
to use or pressing. The gluten was subsequently pressed
for 5 min under 20 tons psi ( = 3.1 × 108 Pa) at 60°C.
No denaturation peak is observed for gluten by d.s.c),
except possibly at temperatures in the region of 150°C 26.
Some small transitions have also been observed at
approximately 88 and 100°C 27. There is some evidence
that above about 80°C the elastic modulus of gluten (in
the absence of starch) increases substantially due to
protein network formation, at least partly due to
disulphide bonding of cysteine residues 28. The solubility
of glutenin in 1.5% SDS also decreased on heating 29
probably for the same reason. Gluten extractability has
been found to decrease on heating to temperatures above
75°C, and some reduction in loaf volume was observed
on preheating gluten to temperatures as low as 55°C 3°.
To minimize heat effects, gluten and gluten-sugar
mixtures were pressed into bars at 60°C.
For samples containing sugar, a concentrated sugar
solution was added to liquid nitrogen in place of water.
In general, the samples were pressed with a water content
of about 15% which results in a glass transition
temperature well below 60°C (the pressing temperature).
X-ray diffraction showed no sign of sugar crystallinity in
any of the samples.
Another method of preparing gluten-sugar mixtures
was also used to test whether improved mixing of the
components could be achieved. Gluten was mixed with
water or sugar solution in a pin mixer to 24-48% water
relative to gluten. The samples were subsequently pressed
and dried in an oven at 30°C or at ambient temperature,
before placing into boxes of controlled relative humidity.
Glutenin and gliadin enriched samples were kindly
supplied by Unilever Research Ltd, Colworth House.
These samples were prepared from Kadett gluten. After
extracting starch and water soluble material from the
flour, the wet gluten was suspended in distilled water.
After a resting period of 30 min the insoluble material
(glutenin) sank to the bottom and the supernatant was
decanted and centrifuged at 3000 x 9- The supernatant
contains low molecular weight glutenin and gliadin. The
glutenins were precipitated by addition of NaC1
( 1 mg/ml) and the gliadins were precipitated by addition
of more NaCl (10 mg/ml). These latter two samples were
used after freeze drying.
All samples were equilibrated at different relative
humidities (RH) using various saturated salt solutions
to obtain a variety of water contents. Water content was
determined, on samples treated identically to the

experimental samples, by drying to constant weight at
105°C and by a Karl Fischer method using the Mitsubishi
moisture meter (taking the average of four duplicates).
Both methods gave similar results (within 0.3% ) except
in the case of samples containing a large amount of sugar,
where sugar degradation (or Maillard reactions)
occurring in the oven resulted in anomalously high
moisture contents by that method. In these cases the Karl
Fischer value was used.
Instrumentation
The d.s.c., d.m.t.a., Instron Texturometer and n.m.r.
relaxation methods used have been previously described
in detail 5'6.
A Perkin-Elmer DSC-2 was used at a heating rate of
10°C/min. The transition temperature was recorded from
the midpoint of the change in heat capacity observed on
the second run (if observed) after cooling at 10 K/min
(to eliminate the effects of sample history). The
temperature and heat flow were calibrated by daily
measurement of the melting of indium (429.78 K) and
cyclohexane II (279.7 K).
The d.m.t.a. (Polymer Laboratories) was used at a
heating rate of 2°C/min, in the single cantilever bending
mode, at a frequency of 1 Hz (multifreqency scans were
also occasionally used). The lowest available strain of
× 1 was used which corresponds to a nominal peak to
peak displacement of 16/~m. Values of the elastic
modulus (E'), the loss modulus (E") and the tangent of
the phase angle (tan 6 = E"/E') were obtained as a
function of temperature.
There is no simple way to control humidity in the
d.m.t.a, furnace as humidity is temperature dependent.
Methods which have been used to seal the sample may
affect the mechanical properties, as the d.m.t.a, is very
sensitive to surface properties; oil baths have also been
used, but it is uncertain whether these significantly reduce
water loss, and in addition the oil may have a plasticizing
effect. In our experiments bars of approximately
1.5 × 6 × 25 mm were prepared. As these are not very
thin, water loss is not as great as it might be.
A sample of gluten stored at 85% RH containing
12.9% water ({weight HzO/wet weight} x 100) originally,
was found to contain 12.5% water on reaching the drop
in modulus temperature of 40°C, 12.4% at the midpoint
of the transition (peak in E") and 11.9% on reaching
62°C, above the peak in tan 6. (These values were
determined by removing samples from the d.m.t.a, during
a scan on reaching the temperatures quoted.) A difference
of 1%o in the water content would imply a 5°C error in
the tan fi peak temperature, whereas the difference of
0.4% water at the onset of the transition implies an error
of 3°C. At lower water contents, although T~ is higher,
water is lost more slowly and at higher water contents
the onset of the transition is at or below room
temperature, so that water loss will only affect the tan 6
peak temperature. It would be very time consuming to
determine the water loss for each transition in the d.m.t.a.
at each water content and the errors involved are
generally within the range of experimental error in
determining transition temperatures, as transitions for
bipolymer systems, especially as complex as gluten and
mixed systems are quite broad.
Three point bend tests were carried out at room
temperature using an Instron Texturometer at a
cross-head speed of 50 mm/s on samples previously
Glass transition of gluten. 1: M. T. Kalichevsky et al.
equilibrated at various RHs as in the experiments above.
The initial slope of the force/distance graph and the
sample dimensions were used to calculate Young's
modulus. The peak force was also recorded. A Stable
Micro Systems TA.XT2 Texture Analyser 31 was used
interchangeably with the Instron and was found to give
comparable results. Humidity was not controlled during
the experiment, but as this is a rapid test, this is not
necessary. The average of data collected from five or
more samples was used.
In the n.m.r, method, Tz relaxation measurements were
carried out using a 6 0 M H z home built n.m.r.
spectrometer constructed in this laboratory with a Varian
1.4T magnet. Samples were placed in a 7 mm OD tube.
A 90 ° pulse of 8 #s length was applied to the system; the
repetition rate was 1 s; 16 transients were co-added. The
T2 was measured straight from the free induction decay
(FID), which consisted of 1024 points. The data were
analysed by a BBC microcomputer, interfaced with the
n.m.r, spectrometer. The typical decay obtained from
samples after the application of the 90 ° pulse consisted
of two components. The first one (T2s) with a T2 of tens
of microseconds was ascribed to solid protein in the glassy
or crystalline state and the second component (T2L) of
the decay with a T2 of hundreds of microseconds was
ascribed to the protons of water molecules of reduced
mobility plus exchangeable protons of the protein.
The T2 was measured as a function of temperature
( - 8 0 ° C to 100°C). The temperature of the sample was
measured with a thermocouple, with an accuracy of
+ I°C. The sample was allowed to equilibrate at a given
temperature for 5 min, after which the measurement was
made. Below Tg the T2s relaxation is independent of
temperature, but after passing through the rigid lattice
limit temperature (TREE) it begins to increase with
temperature. This is attributed to the onset or increase
in frequency of the motion of the groups containing
hydrogen. TREEwas used as an indicator of the mobility
changes occurring in the samples investigated.
Examination of samples by s.e.m, was carried out using
a Jeol scanning electron microscope, courtesy of the
Metallurgy Department, University of Nottingham, at
15 kV. Prior to the experiment the gluten samples were
gold plated to increase electron conductivity.
Results and discussion
The effect of water on the glass transition of gluten
D.s.c. and d.m.t.a. A typical d.m.t.a, graph showing
log E' (elastic modulus), log E" and tan 6 (loss
modulus/elastic modulus [E"/E']) as a function of
temperature is shown in Figure 1. In this study the peak
in tan 6, the peak in log E" and the point at which the
slope of the log elastic modulus changes (drop in E')
were recorded for each run. The drop in modulus
temperature was determined from the intercept of the
extrapolated glassy modulus and the extrapolation of the
slope at the inflection point in the drop in E'.
These transition temperatures for gluten are plotted
against water content in Figure2; the d.s.c, glass
transition temperature is also plotted. As was previously
reported for amylopectin6'35 the d.m.t.a, tan 6 peak and
drop in elastic modulus temperatures fall above and
below the midpoint in the change in heat capacity
observed by d.s.c, respectively, thus they may be used as
an indication of the breadth of the glass transition region.
Int. J. Biol. Macromol., 1992, Vol. 14, October 259
Glass transition of gluten. 1 : M. T. Kalichevsky et al.

LOGE"
tan ~ (Pa)
,5 I I I [~1.5

LOG E" DROP IN

t " 1 " ~ ~ . ~ 4 ~ E" TEMP TAN IB
" ~ 1 t , ~ ....,,,~ \ PERK

.' • ...

•2 ............ """ """ "" . . . . . . . . . . . ' ........... ""I" "" 8

1 lift/ ,.5
i ..,,. .........

1 l I , I
TEHP (de,C}

Figure 1 A typical d.m.t.a, plot for gluten (RH = 65%), showing tan 5, log loss modulus (E") and log elastic modulus (£') as a
function of temperature

and K o k i n P correlated the peak in the loss modulus (G"

(shear modulus) in their case) with the endpoint of the
"\\
d.s.c, transition at a heating rate of 5 K/min. When our
\
E" peak temperatures are compared with d.s.c, midpoint
\ data obtained at 1 0 K / m i n in this laboratory and
\ elsewhere 3, a good correspondence is obtained (Figure 2),
120 \ particularly at higher water contents, where drying out
\ is less of a problem in the d.m.t.a. (as Tg is lower). This
\
L--;
\ is in agreement with the observations of Cocero and
Kokini 4, as increasing the d.s.c, heating rate increases
\ the measured Tg. In polymer literature Tg is usually taken
B0 as the tan 5 peak temperature• When the effects of d•s.c.
[z3_ \ z~ heating rate and d.m.t.a, frequency were studied for a
E I\D
e\ A gluten sample containing 12.9% water, equivalent
c measurement conditions may be determined• The d.m.t.a.
O
-4,- o \~ multifrequency data gave an apparent Arrhenius
"N 40 \ A activation energy of 242 kJ/mol. From this it may be
C
L o\ \ calculated that the Tg (midpoint) obtained by d.s•c, at a
heating rate of 10 K / m i n would be obtained from a
"3 d.m.t.a, tan 5 peak obtained at a frequency of
O
7.5 x 10- 3 Hz. In contrast, the heating rate which would
N
0 35 have to be used in the d.s.c, to obtain T~ at the same
16 "'~
\ temperature as the tan 5 peak at 1 Hz would be
WW% Aq \ 100 K/rain (if the extrapolation is valid).
\
\ However, it is not our intention only to obtain the
same information from these different techniques• It is
useful to know how they compare in cases where it is
Figure 2 Plot of d.m.t.a, tan 5 peak (A), log E" peak (D), only possible to use one method, but mechanical data
drop in log E' temperature (C) ) and d .s.c. midpoint temperature should also yield additional information to d.s.c, data,
( • ) for gluten as a function of water content. - - - theoretical partly because mechanical methods are more sensitive to
best fit Curve for gluten Tg by d.s.c.
the glass transition and partly because mechanical
properties are of great interest to food scientists. These
The d.s.c, results are in good agreement with those of small deformation properties should relate to perceived
Hoseney et al. 3, within experimental error. texture.
A precise comparison between mechanical and The d.s.c, data may be fitted to the Gordon-Taylor
calorimetric determinations of T~ is not simple• Cocero equation (2) to obtain a linear regression best fit. This

260 Int. J. Biol. Macromol., 1992, Vol. 14, October


was done using our data and the data of Hoseney et al. 3,
assuming that the Tg of water is 134 K. The Tg value thus
obtained for dry gluten is 435 K and K = 0.2 (if subscript
1 refers to water and 2 to gluten). This Tg value is about
65°C lower than the best fit value for waxy maize
amylopectin (which corresponds to the extrapolated
value obtained b y Orford et al. 25 for amylose or
amylopectin (500 K)).
In order to predict the Tg of three component systems
the C-K equation (1) must be used. As far as we know,
there is no published value for ACp of dry gluten. If
assumptions are made about the ACp of water, a ACp
value for gluten may be obtained using equation (3).
Using a value of 1.94 J g-XK-1 for water a ACp value
of 0.39 J g - 1K- 1 is obtained for gluten. This ACp value
is similar to the best fit value used for amylopectin
(0.425 J g- 1K- 1, using the same values for water) 6. This
indicates that gluten has similar plasticizability to starch,
but has a lower Tg at all water contents.
The magnitude of AC~ actually observed for gluten
varied greatly, but without any definite dependence on
water content. The average value was 0.22 _+ 0.05 J
g - a K -1, which would imply that ACp of water is
1.11 J g - ~ K -1, using equation (3). This value is similar
to the values of ACp of water obtained by extrapolation
of aqueous solution data 18.
These values were subsequently used to predict the
effect of sugars in the presence of water on the Tg of
gluten, using an extension of the C o u c h m a n - K a r a s z
equation. The effect of using different ACp values for
gluten and water on the predicted Tg was investigated.
Values of Tg and ACp for sugars were used as obtained
by Orford et al. 33, and these are listed in Table 1, with
the two sets of values used for gluten and water. In the
case of sugars alone, values from set A were used for the
Tg and ACp of water, as these values gave the best fit to
Tgd..... data for fructose obtained in this laboratory 34.
Other ACp values for water (1.11 or 0 . 0 8 9 J g - ~ K -~)
underestimate the plasticizing effect of water on sugars.
N.m.r. measurements. The predominant transverse
relaxation process in gluten is very rapid (10-20/~s). The
decay is Gaussian and originates from regions of gluten
with slow molecular motion. This is the part of the FID
that we are mainly interested in.
The n.m.r. TRLL measurements were carried out on
gluten powders stored at a variety of RH values. The
plasticizing effect of water on gluten is also observed by
n.m.r, measurements (Figure 3), where the theoretical
curve obtained above is plotted for comparison. The
results correspond closely to the d.s.c, results, being
Table 1 Values of Tg and ACp used in equation 1 (see text for
details)
Sample T, (K) ACp (J g 1 K - l )
Glucose 311 0.88
Sucrose 343 0.76
Fructose 280 0.83
Set A
Water 134 1.94
Gluten 435 0.39
Set B
Water 134 1.11
Gluten 435 0.22
Glass transition of gluten. 1: M. T. Kalichevsky et al.
I0C
e
Q)
4-J
o.
E
Q;
k-
t-
O
e-
20 ¸
3b
W/W% Aq
-20"
Figure 3 N.m.r. TRLL for gluten (4,), soluble glutenin (D)
and gliadin ( • ) , as a function of water content, with best fit
d.s.c, curve for comparison
possibly approximately 10°C higher. The components of
gluten (soluble glutenin and gliadin) were also examined
separately as a function of water content. There is little
difference in TRLr between them (Figure 3). When the
results are compared with those for whole gluten, there
appears to be a slight depression in TRLL,which may be
due to higher molecular weight a n d / o r aggregated
(insoluble) materials being removed during the purifi-
cation process.
The observation that the Tg values of both glutenin
and gliadin are very similar to the Tg of whole gluten
explains the good agreement between the d.s.c, results
for gluten 3 and those for glutenin 4.
The effect of sugars on the glass transition of gluten
The effect of three sugars on the glass transition of
gluten as a function of water content was studied using
n.m.r., d.s.c., and d.m.t.a. The sugars studied were
sucrose, glucose and fructose, all in the ratio of gluten
to sugar, 10:1. In addition, gluten + glucose was also
prepared in the ratio 5:1 and gluten + fructose in the
ratio 2:1.
D.s.c. and d.m.t.a. The data obtained using these
techniques are summarized in Table 2. Some preliminary
results on gluten + glycerol are also shown for
comparison. In all the samples containing sugars, a
transition is observed at low temperatures in the region
of the Tg of the sugar (at that water content), although,
in the case of gluten + fructose, this transition falls more
than 20°C above the fructose Tg at low water contents.
Generally the Tg of gluten is depressed by sugars at low
water contents (the AT values in Table 1 are obtained
from d.s.c, and log E" peak data) and sugars enhance
the Tg of gluten at higher water contents. These effects
may be caused by an unequal distribution of water
between gluten and fructose. Sugars appear to broaden
the transition, especially at low sugar contents, probably
due to sample heterogeneity.
The values used to calculate the theoretical curve in
Figures4-6 are also shown in Table2 (referring to
Int. J. Biol. Macromol., 1992, VoL 14, October 261
Glass transition of gluten. 1 ." M. T. Kalichevsky et al.

Table 2 Summary of d.s.c, and d.m.t.a, results for gluten-sugar and gluten-glycerol mixtures A and B refer to Table 1)

Change in Tg due
Values used for sugar at w/w% aq.
theoretical curve Transition breadth
Sample (A or B) (AE'--tan 6 peak T) 10% 20%

Gluten (A and B are 31 -- --

equivalent )
Gluten + sucrose (t0:1) B 43 -5°C +9°C
Gluten + glucose (10:1) -- 38 0 +8
(5:1) B 30 -4 +5
Gluten + fructose (10:1) A 41 -7 + 16
(2:1 ) A 22 -22 +2
Gluten + glycerol (10:1) -- 28 - I0 --
(3:1) -- 30 -45 -12

\\
~I00"
100
~o
L.
Q>

E
i---
p, 60 E
E
60
P,
t~

[] I--
20
20
: w!wA,
•~ "~ =. ~~o ~ WIW%Aq
5 o 15 "~ 25

-20- \ \
-20-

Figure 4 Gluten + sucrose 10:1, d.s.c, midpoint ( 0 ) , and

DMTA peaks in log E" ([]). The theoretical d.s.c, curve for
this system ( - - - ) using l.ll and 0 . 2 2 J g - l K -1 for ACp of
water and gluten respectively and the d.s.c, best fit curve for
gluten alone ( - - ) are shown for comparison
Table 1). Using the smaller ACp values (set B) results in
a greater predicted plasticizing effect of the sugar at low
water contents and a reduced effect at high water
contents. The greatest difference in the predictions occurs
at low sugar content~ a n d is less than 10°C. (Set A values
for Tg and AC v of water are used to obtain the sugar Tg
curves. )
The d.s.c, and log E" peak data for gluten containing
sucrose, glucose and fructose are plotted in Figures 4, 5
and 6 respectively. In the case of gluten:fructose 2:1 the
low temperature transition dominates the mechanical
properties, the upper transition temperatures being just
a shoulder on the main transition.
N.m.r. The n . m . r . TRL L results for gluten + sugar
(10:1) samples are plotted in Figure7. The curve
represents the TRL L values obtained for gluten alone. It
is apparent that sugars in the rato 10:1 (gluten:sugar)
do have a plasticizing effect on gluten as measured by
n.m.r. T h i s effect appears to be greater at low to
Figure 5 Gluten + glucose 10:1, d.s.c, midpoint (11), and
d.m.t.a, log E" peaks ([q); and gluten + glucose 5:1 d.s.c.
midpoint ( O ) and log E" peaks ( • ) . The theoretical d.s.c.
curves for the latter system ( - - - - - ) , for gluten alone ( - - ) and
for glucose ( ) are also plotted, as a function of water content
for comparison
intermediate water contents, especially in the case of the
sample containing fructose, which has a similar or higher
Tg than gluten alone at higher water contents ( 18% ), but
shows plasticization due to fructose at lower water
contents. Similar behaviour was observed by d.s.c.
(Figure 6). At 12 18% water the greatest plasticizing
effect of 13-30°C was shown by glucose. The average Tg
reduction due to sucrose was 5-10°C, the greatest
reduction occurring at low water contents.
General comments on gluten-sugar mixtures. The
observation of a low temperature transition and the
broadening of the transition in the presence of sugars,
suggests that these systems are phase separated. The
degree of phase separation appears to increase with
increasing water content (possibly because the greater
mobility at higher water contents allows separation to

262 Int. J. Biol. Macromol., 1992, Vol. 14, October

 Glass transition of 91uten. 1 : M. T. Kalichevsky et al.

It should also be pointed out that in the presence of

100- sugar, the water uptake of the samples is inceased at all
RH values as compared with gluten alone. When the Tg
values of samples stored at the same RH are compared
there is little difference in Tg for samples containing sugar
A
in the ratio 10:1. However, even in the sample containing
60
gluten + glucose in the ratio 5:1 the Tg at any RH is
significantly lower than for gluten alone. This effect is
enhanced in the case of gluten + fructose 2:1. Tg is not
E
a linear function of RH, largely because the water content
P, increases more at high RH values.
= 20
i.---
The effect of water and sugars on the modulus of 9luten
as determined by three point bend tests and d.m.t.a.
WIW% Aq
d \1" "\ \ d • ' ' The effect of water on the modulus of gluten has been
5
studied as a function of temperature by d.m.t.a, and as
-20 a function of water content at constant (room)
temperature by three point bend tests. As observed with
amylopectin 35, increasing the water content decreases the
rubbery modulus (high temperature limiting value of E'),
while the glassy modulus (low temperature limit E'
-60
value) changes very little with water content. This results
in an increase in the magnitude of the drop in the elastic
modulus (E') as the water content is increased as shown
Figure 6 Gluten + fructose 10:1, d.s.c, midpoint (ll), peaks in Figure 8. It has been observed 36 that E' above Tg is
in log E" ( [] ) and theoretical curve ( - - - - - ) . Gluten + fructose
highly dependent on the crosslink density of a polymer,
2:1, d.s.c, midpoint (A), peaks in log E" ( 0 ) and theoretical
curves for this system ( .... ) and for fructose ( - - - - ) . The best thus the reduction of the rubbery modulus in the presence
fit d.s.c, curve for gluten alone ( - - - ) is also plotted of a plasticizer implies reduced crosslinking. In this case
the reduction in E' may be due to polymer-polymer
hydrogen bonds being replaced by labile polymer-water
hydrogen bonds.
Although small quantities of added sugar do not
100" appear to greatly influence the Tg behaviour of gluten as
a function of water content, they do affect its mechanical
properties. When sugars are added to gluten the d.m.t.a.
results show an increase in the glassy modulus (low
eel temperature limit E' value) with increasing sugar content
~- 60 and a tendency for the rubbery modulus (high
E
temperature or high water content limit E' value) to be
decreased. Log E' plots for gluten and gluten/sugar
mixtures equilibrated at 75% RH show this effect
(Figure 9). At high sugar contents the Tg is decreased so
[] • • that the glassy modulus is only attained at low water
20
contents or low temperatures. The increase in the glassy
WlW% Aq

lo " 2; 3~
9,5

-20-

----~'~'~~ RH= 22~

Figure 7 N.m.r. T R L L for gluten:sugar 10:1 mixtures as a
function of water content. N.m.r. TRLL for gluten ( - - ) is plotted
for comparison. Gluten + sucrose ( [] ), gluten + glucose (O),
gluten + frucrose (A)

occur). The plasticizing effect of sugars is therefore

greatest at lower water contents and at higher water r+ \\
contents water redistribution between the phases may
\
also be important. The observation of a transition in the
region of the Tg of the diluent does not necessarily imply
phase separation, as diluent induced transitions are 0 50 1oo
TEMp(oc)
frequently observed in compatible systems 32. However,
the broadening observed in these systems does suggest Figure 8 Plot of d.m.t.a. Log E' data for gluten stored under
some lack of miscibility. different RH values as a function of temperature

Int. J. Biol. Macromol., 1992, Vol. 14, October 263

 Glass transition of gluten. 1." M. T. Kalichevsky et al.
q0
\ , \
\ ",
a.
o
_J
\
\ []
\
\ "--
- 50 0 50
Temperature (*C)
Figure 9 D.m.t.a. Log E' for gluten ( - - - - ) , gluten + glucose
5:1 ( ), gluten + fructose 10:1 ( ) and 2:1 ( - - - - - )
modulus may occur below the glass transition of the
sugar-rich phase, due to the reinforcement of the gluten
matrix by a sugar glass. (Due to its lower molecular
weight a sugar or sugar-gluten glass may be expected
to be more dense than a gluten glass, resulting in a higher
glassy modulus. Above Tg a sugar will be a viscous liquid
thus reducing the gluten rubbery modulus.)
Young's modulus measured by a three-point bend test
at room temperature, for gluten and gluten + fructose
in the ratios 10:1 and 2:1 is plotted as a function of water
content in Figure 10. At any particular water content
Young's modulus is lower in the presence of sugars, than
for gluten alone, except at low water contents were the
opposite appears to be the case, as was also observed by
d.m.t.a, with increasing temperature (Figure9). Both
d.m.t.a, and three point bend test results also show an
increase in the magnitude of the drop in modulus with
increasing sugar content. The arrows in Figure 10
indicate the water contents below which the samples are
brittle (this decreases with increasing sugar content).
The modulus values from the two different methods
are in reasonably good agreement, with the d.m.t.a.
values being a little lower than obtained by the three
point bend test, as observed in the case of amylopectin 5
and attributed to the higher strain rate in the three-point
bend test (0.5 m m / s ) when compared with the d.m.t.a.
(average approximately 0 . 0 4 m m / s ) and quantitative
errors in the d.m.t.a, modulus value (such as errors in
measuring the sample dimensions and end corrections
which have not been made). Generally (for many samples
studied in this laboratory) the difference in modulus
determined by d.m.t.a, and by a three point bend test is
greatest at low moisture contents, where the sample is
stiffest. This is caused by the effective sample length in
the d.m.t.a, changing with the stiffness of the sample. The
length used in the calculations is the free (unclamped)
length between the sample clamps, but when the sample
is stiff this is no longer correct. It is not straightforward
to correct for this as the magnitude of the correction
factor is sample and modulus dependent, hence the
greater reliability of moduli obtained from a three point
bend test. Of course irregularly shaped samples would
introduce errors in both methods.
264 InL J. Biol. Macromol., 1992, V0t. 14, October
10 0
ItL
\
D •0
• []
9'0'
-i
o
8"0
o
_1
7.0"
o ' ' 2b
WIW% Aq
Figure 10 Modulus measured at room temperature by a three
point bend test for gluten ( • ) and gluten + fructose in the
ratio 10:1 (IS]) and 2:1 ( , ) . (The downward pointing arrows
indicate the estimated water content at which the samples begin
to snap)
Investigation of phase separation and the effect of
changing the sample preparation method
In the case of amylopectin, sugars were observed to
have a plasticizing effect, measurable by all the techniques
used 6. The fact that the effect of sugars on gluten is
significantly different could be due to phase separation
occurring in gluten-sugar mixtures. Scanning electron
microscopy was used to investigate this and no evidence
for phase separation was found.
Gluten and gluten/sugar mixtures were also prepared
by an alternative method (described earlier), in the hope
that this would result in more homogeneous mixtures.
These samples were studied by d.m.t.a, and d.s.c.,
although it was difficult to obtain samples sufficiently
flat for use in the d.m.t.a, without using the hot pressing
method. The results for these samples proved to be similar
to those obtained by the previous (hot pressing) method,
but d.m.t.a, results were more scattered due to the
irregular sample shape.
A sample containing gluten + glucose in the ratio 5:1
gave tan 6 peak and drop in E' temperatures quite similar
to the values for gluten alone, as was previously observed
for a similar sample prepared b y the hot pressing method.
Similarly a sample containing gluten + fructose in the
ratio 2:1 showed approximately 20°C reduction in the
transition temperatures, which is similar to or a little less
than the plasticizing effect of fructose in a sample of this
composition prepared by the hot pressing method. It may

be that preparing samples at a higher moisture content
actually allows a greater degree of phase separation to
occur (the d.m.t.a, results have also suggested a greater
degree of phase separation in wetter samples).
These results indicate that the Tg measured by d.m.t.a.
for these samples is not highly dependent on the sample
preparation method. The lack of or reduced plasticizing
effect of small amounts of sugar on gluten when
compared with amylopectin/sugar mixtures 6 appears to
be inherent in the system and not due to a lack of mixing
during sample preparation. In fact the results indicate
that the second sample preparation method may result
in greater phase separation and reduced plasticization
due to sugars, when compared with samples prepared by
the hot pressing method.
Our studies have concentrated on gluten sugar
mixtures due to their practical importance in foods. It
has been suggested (by a reviewer) that glycerol might
be more miscible with gluten than sugars, due to its lower
molecular weight and size. Preliminary studies on
gluten + glycerol prepared by the pressing method in the
ratios 10:1, 5:1 and 3:1 (gluten:glycerol) indicate that
this is the case. In a sample containing gluten + glycerol
in the ratio 10:1 in the log E" peak temperature is
depressed by 9-29°C (the greatest amount at low water
contents). For the sample in the ratio 5:1 this is increased
to 11-50°C and for 3:1 to 17-68°C. These samples
contain between 4 19% water. The Tg of glycerol is low
compared with the sugars (approximately 180K37),
which results in a greater plasticizing effect (predicted
using 37 ACp~-0.88Jg-2K -1 and observed). The
minimum predicted plasticizing effect of glycerol is
obtained when 1.94 and 0.39 J g- 1K- 1 are used for ACp
of water and gluten respectively. The Tg observed
approaches this predicted value for the 10:1 samples, but
is much higher than predicted for the higher glycerol
contents, which may be due to the limits of compatibility.
A low temperature transition may be observed in the
region - 5 0 to -60°C, which is much higher than the
Tg of glycerol (about -93°C), but may be due to a
glycerol-rich phase.
In summary, therefore, gluten is more compatible with
and more plasticized by glycerol than by sugars, due to
the lower molecular weight and Tg of glycerol.
Compatibility of gluten with carbohydrates appears to
be greatest at lower water contents, the water distribution
between gluten and sugars may also affect the value of
T~ observed.
Plasticization and molecular interactions
Although gluten is not water soluble, it is clearly water
plasticizeable. It has a low Tg (approx. 435 K) compared
with amylopectin (500K25), which may be due to a
reduced degree of association in the case of gluten.
Nakamura et al. 38 have shown that the Tg of
polyhydroxystyrene derivatives depends on the number
of hydroxyl groups (i.e. hydrophilicity), with an
increased number resulting in a higher Tg as a result of
increased hydrogen bonding. In the case of gluten there
may be fewer entanglements and a lower degree of
crosslinking than in the case of starch, due to the lower
molecular weight, greater hydrophobicity and the
multicomponent nature of gluten.
Both amylopectin and gluten are substantially
plasticized by water, but sugars behave differently in these
two systems. There are various factors which may cause
Glass transition of gluten. 1: M. T. Kalichevsky et al.
the reduced plasticizing (Tg reducing) effect of sugars on
gluten when compared with amylopectin6. In the case of
amylopectin, sugars are very similar to or identical to
starch monomers and as such are readily incorporated
into the system at low sugar contents, with a resulting
reduction in Tg due to the relatively high mobility of the
sugar molecules. In the case of gluten it may be more
difficult to attain uniform mixing due to incompatibility
of the sugar, resulting in a reduced effect on Tg (even
when the method of sample preparation is changed). The
presence of incomplete compatibility is shown by the
broadening of the transition region as observed by
d.m.t.a, in the presence of sugars and by the appearance
of a transition in the region of the sugar Tg. Glycerol
appears to be more miscible with gluten due to its lower
molecular weight.
The n.m.r, results appear to measure Tg close to the
onset of the glass transition region, in a similar way to
the drop in E' temperature. The n.m.r, results show more
of a plasticizing effect due to the sugars than shown by
d.s.c., in the same way as the onset of the transition (as
shown by AE') occurs at lower temperatures as the
transition is broadened by the presence of sugars.
Another possible reason for the reduced plasticizing
effect or even Tg increasing effect of sugars is that
the sugar molecules may be preferentially hydrated,
decreasing the water content (and therefore increasing
Tg) of the gluten matrix. It has also been observed 39 that
sugars have a stabilizing effect on proteins, increasing
hydrophobic interactions. These would also tend to
increase the Tg of the system. Thus several effects are in
operation making it very difficult to predict the Tg of
these three component systems, although these experi-
mental results may give some clues for qualitative
predictions.
Summary
The glass transition of gluten and gluten-sugar mixtures
has been studied as a function of water content using a
variety of techniques. There is good agreement between
the different techniques and the d.s.c. Tg obtained for
gluten is in agreement with the results of Hoseney et al. 3.
At a higher sugar content (gluten:fructose 2:1) two
transitions were clearly observed by d.m.t.a., suggesting
the occurrence of phase separation, but with some mixing
of the components, as the Tg of the gluten-rich phase was
depressed by 20-40°C. This plasticization due to the
sugar was not as great as predicted by the use of the
Couchman-Karasz equation. Smaller quantities of
added sugar had little plasticizing effect on the gluten,
except at low water contents, although similar quantities
of glycerol had a substantial plasticizing effect. At higher
water contents (above about 18% ), fructose, sucrose and
glucose showed some increase in Tg by d.s.c. This may
be caused by some degree of water redistribution in
favour of the sugar, or by increasd hydrophobic bonding
in the protein. Glycerol has a significant plasticizing effect
on gluten, even when added in the ratio 10:1 (gluten:
glycerol), due to its low Tg and low molecular weight
and size.
When applying these results to foods it should also be
noted that the presence of sugar increases the water
uptake of the system especially at higher RH values. This
means that even if the Tg of the system is unchanged by
the presence of sugar at any water content, when a sample
Int. J. Biol. Macromol., 1992, Vol. 14, October 265
G l a s s t r a n s i t i o n o f 91uten. 1: M . T. K a t i c h e v s k y et al.

is stored at a particular RH the presence of sugar may 15 Johari, G. P., Hallbrucker, A. and Mayer, E. Nature 1987, 330,
552
still result in a decrease of Tg at that RH (due to an 16 Handa, Y. P. and Klug, D. D. J. Phys. Chem. 1988, 92, 3323
increase in the water content). 17 Hallbrucker, A., Mayer, E. and Jobari, G. P. J. Phys. Chem.
1989, 93, 4986
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Acknowledgements 19 Wilson, T. W. and Turner, D. T. Macromolecules 1988, 21, 1184
This paper has arisen from research pursued under the auspices 20 Hofer, K., Mayer, E. and Johari, G. P. J. Phys. Chem. 1990,
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of the A C T I F (Amorphous Crystalline Transitions in F o o d s ) 21 Hofer, K., Hallbrucker, A., Mayer, E. and Johari, G. P. J. Phys.
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and enthusiastic interest of the consortium of industrial 22 Hallbrucker, A., Mayer, E. and Johari, G. P. Philosophical Ma9.
companies and the Ministry of Agriculture, Fisheries and Food. B 1989, 60, 179
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266 Int. J. Biol. M a c r o m o l . , 1992, Vol. 14, O c t o b e r