Vol. 81, No.

5, 2004 567
Textural Comparisons of Gluten-Free and Wheat-Based Doughs,
Batters, and Breads
Michelle M. Moore,
1,2
Tilman J. Schober,
1,2
Peter Dockery,
3
and Elke K. Arendt
1,4

ABSTRACT Cereal Chem. 81(5):567575
Studies were conducted with two newly developed gluten-free bread
recipes. One was based on corn starch (relative amount 54), brown rice
(25), soya (12.5), and buckwheat flour (8.5), while the other contained
brown rice flour (50), skim milk powder (37.5), whole egg (30), potato
(25), and corn starch (12.5), and soya flour (12.5). The hydrocolloids used
were xanthan gum (1.25) and xanthan (0.9) plus konjac gum (1.5),
respectively. Wheat bread and gluten-free bread made from commercial
flour mix were included for comparison. Baking tests showed that wheat
and the bread made from the commercial flour mix yielded significantly
higher loaf volumes (P < 0.01). All the gluten-free breads were brittle
after two days of storage, detectable by the occurrence of fracture, and the
decrease in springiness (P < 0.01), cohesiveness (P < 0.01), and resilience
(P < 0.01) derived from texture profile analysis. However, these changes
were generally less pronounced for the dairy-based gluten-free bread,
indicating a better keeping quality. Confocal laser-scanning microscopy
showed that the dairy-based gluten-free bread crumb contained network-
like structures resembling the gluten network in wheat bread crumb. It
was concluded that the formation of a continuous protein phase is critical
for an improved keeping quality of gluten-free bread.

In recent years there has been a slow and steady increase in con-
sumer interest in wheat-free foods, driven in part by an increasing
awareness of the relatively unfamiliar condition known as celiac
disease (CD) (Lovis 2003). CD, also known as gluten-sensitive
enteropathy, is characterized by inflammation of the small-intes-
tinal mucosa that results from a genetically based immunologic
intolerance to ingested gluten (Murray 1999).
In Europe, the prevalence of CD has been estimated to be 1 in
300 to 1 to 500 persons, but recent population-based screening
studies suggest that the prevalence may be as high as 1 in 100
(Mustalahti et al 2002). Recent studies show that CD is as fre-
quent in the United States as in Europe (Fasano and Catassi 2001).
The symptoms of CD can develop at any age. These symptoms
include, severe symptoms of malabsorption such as steatorrhoea,
abdominal discomfort, weight loss or gain, tiredness, anemia, and
severe diarrhea (Feighery 1999; Murray 1999; Fasano and Catassi
2001). These authors suggest that the only way that CD can be
treated is the total lifelong avoidance of gluten ingestion. The dele-
terious proteins gliadins (wheat), secalins (rye), hordeins (barley),
and possibly avenins (oats) are the predominant grains containing
toxic peptides (Murray 1999; Fasano and Catassi 2001; Kasarda
2001) and should be avoided. A review presented by Kasarda (2001)
suggests that species that are less closely related to wheat such as
sorghum, millet, teff, ragi, and Jobs tears are relatively safe grasses.
Persons with CD are unable to consume some of the most com-
mon products on the market today including breads, baked goods,
and other food products made with wheat flour (Lovis 2003). To
satisfy the demand for high-quality bread, the gluten-free breads
must have quality characteristics similar to those of wheat flour
bread (Ylimaki et al 1991). The development of such breads is dif-
ficult in view of the fact that gluten is the main structure-forming
protein in wheat flour, responsible for the elastic and extensible prop-
erties needed to produce good quality bread (Ylimaki et al 1991)
The main objective of this study was to produce gluten-free
breads that are comparable to wheat bread and also to compare
their structure with a gluten-free bread from a commercial flour
mix. Another aspect of this study was to develop healthier breads
that contain higher protein and dietary fiber levels and not only
starch. From this, two new recipes were developed: a dairy-based
gluten-free bread and a nondairy-based gluten-free bread. These
recipes were selected from a series of 200 trials for their sensory
properties as well as their nutritional improvements and food struc-
ture. The purpose of developing a nondairy-based gluten-free bread
was because 50% of the CD sufferers are intolerant to lactose
(Murray 1999). Because the villi in the small intestine of CD suf-
ferers are damaged, they cannot produce the enzyme lactase
required to break down the lactose sugar molecule (Murray 1999).
Therefore they are unable to digest lactose. The dairy-based
gluten-free bread, containing more complex cereals, was developed
with a view to improving the nutritional and keeping qualities of
bread for nonlactose intolerant CD sufferers.
Breads were studied over a five-day storage period using texture
profile analysis (TPA) to determine the staling rate associated
with the breads. Microscopic images of the dough, batters, and
bread crumbs were generated using confocal laser-scanning micro-
scopy (CLSM) to develop a fundamental understanding of the struc-
ture of gluten-free batter and gluten-free bread as a good basis for
further improvements. The advantage of CLSM is its ability to pro-
duce optical sections of a three-dimensional specimen without dam-
aging the structure (Drrenberger et al 2001). To supplement the
insight gained by CLSM, the rheological properties of the batters
and dough were compared using extrusion and penetration tests.
MATERIALS AND METHODS
Bakers flour and trytamyl gluten-free flour mix (Odlum
Group, Dublin, Ireland) were used in conjunction with instant
dried yeast (Mauripan, Burns Philip Food Ltd., UK). Salt (Salt
Union, Weston Point, Cheshire, UK), sugar (Suicra, Ireland), and
ascorbic acid (BDH, Poole, UK) were also incorporated into the
dough and the batters. The nondairy gluten-free bread and dairy
gluten-free bread were prepared using brown rice flour and
buckwheat flour (Doves Farm Foods Ltd, Berkshire, UK), corn
starch (National Starch and Chemical), soya flour, potato flour
(Wholefood Wholesalers, Dublin, Ireland), xanthan gum (Quest
International, Holland), skim milk powder (Dairygold, Mitchels-
town, Ireland), sugar syrup (Tate & Lyle Sugars, Cheshire, England),
konjac powder (The Mill, Cork, Ireland), baking powder (Super-
cook, Leeds, UK), eggs (locally supplied), and tap water.
Baking Tests
The ingredients used for wheat bread (W), commercial gluten-
free flour mix bread (C), nondairy gluten-free bread (ND), and
dairy gluten-free bread (D) are listed in Table I. The appropriate
1
Department of Food and Nutritional Sciences, University College Cork, Ireland.
2
National Food Biotechnology Centre, University College Cork, Ireland.
3
Department of Anatomy, University College Cork, Ireland.
4
Corresponding author. Phone: +353-21-4902064. Fax: +353-21-4270213. E-mail:
e.arendt@ucc.ie
Publication no. C-2004-0712-03R.
2004 American Association of Cereal Chemists, Inc.
568 CEREAL CHEMISTRY
crude protein (% db) and moisture levels are included. Moisture
and crude protein contents were determined by Standard 110/1 (ICC
1976) and Approved Method 46-30 (AACC 2000), respectively.
For all the recipes, the yeast was prefermented by suspending it in a
water and sugar solution for 20 min for revitalization. Dough W,
based on 3,000 g of flour, was mixed in a 30-qt capacity planetary
mixer (Hunt 30, Lancashire, UK) with a dough hook for 1 min at
a disk speed of 44 rpm (shaft 88 rpm) and 7 min at a disk speed
of 135 rpm (shaft 270 rpm). Water temperature was 1820C.
Dough W was rested in bulk for 30 min in a proofer/retarder
(Koma Popular, Koma, Roermond, The Netherlands) at 30C and
85% rh, scaled into 400-g portions, rounded manually, and rested
further in the proofer for 10 min at 30C and 85% rh. Dough W
was then molded in a small-scale molder (Machinefabriek Holtkamp
B.V., Almelo, Holland), placed into tins 180 mm 120 mm 60
mm (Sasa UK Ltd., Middlesex, UK), and proofed at 30C and
85% rh for 50 min.
The gluten-free breads and batters were prepared with the same
30-qt capacity planetary mixer but equipped with a batter
attachment and mixed for 30 sec at a disk speed of 44 rpm (shaft
88 rpm) and 60 sec at a disk speed of 135 rpm (shaft 270 rpm).
Batters were based on 3,000 g of gluten-free flour mix in bread C
and 2,000 g of flour in breads ND and D. The water temperature
was 1820C. The batter was then scaled to 500 g into nine
baking tins (height 7 cm, top and bottom length and breadth 16.5
cm x 10.5 cm and 13.5 cm x 7.5 cm, respectively) and proofed at
30C and 85% rh for a period of 40 min for breads ND and C and
30 min for bread D.
Baking was done at 230C top heat and 230C bottom heat for
30 min for bread W, 190C top heat and 190C bottom heat for 45
min for breads C and ND, and 180C top heat and 180C bottom
heat for 45 min for bread D in a deck oven (MIWE, Arnstein,
Germany). The oven was preinjected with steam (0.3 L of water)
and after loading was again steamed with 0.7 L of water. The loaves
were depanned and allowed to cool for 120 min on cooling racks
at room temperature. The loaves for analyses at day 2 and 5 were
then packaged in containers (polystyrol, ethylene vinyl alcohol,
polyethylene) under modified atmosphere (60% N
2
and 40%
CO
2
) and stored at 21C.
Rheology of Batters and Doughs
The batters and dough were prepared as described for the
baking experiments but without the addition of yeast and prefer-
mentation step. Dough and batter hardness characteristics were
evaluated with a texture analyzer (TA-XT2i, Stable Micro Systems,
Surrey, UK) equipped with the forward extrusion cell HPP/FE.
For batter extrusion, 200 g of sample was filled into the extrusion
vessel and the air pockets were removed with a spoon. For further
removal of randomly distributed air, the sample was precom-
pressed using the controls on the texture analyzer, while partly
closing the 10-mm diameter nozzle. Afterward, the extrusion force
was measured at a test speed of 1.0 mm/sec over a distance of 20
mm, using the 5-kg load cell of the texture analyzer. The average
force measured after reaching a plateau (818 mm) was used as
an indicator of batter firmness. Three repetitions made with the
same batter batch were averaged into one replicate value. For ex-
trusion of the wheat dough, 30 min of rest in bulk at room
temperature was allowed, the extrusion cell was filled with 200 g
of sample and measured after 5, 10, and 60 min. For the dough,
the 25-kg load cell was used. For batter penetration, 200 g of
batter was penetrated by a 20-mm cylinder probe at a test speed of
1.0 mm/sec over a distance of 10 mm and the maximum force
was recorded.
CLSM
Safranin O dye (Sigma Chemicals Co., St. Louis, MO) was
added to the respective recipes at a rate of 0.002% flour weight
basis (fwb) to stain the protein and the starch. The dye was
solubilized in the water before mixing to ensure homogenous
distribution. For microscopy of the batters, the batters were pre-
pared as described for the baking test without the yeast. Each
sample was placed onto a welled slide using a needle before the
TABLE I
Bread Recipes,
a
Moisture, and Protein Contents
Ingredients W C ND D Moisture (%) Nitrogen (% wb)
b

Wheat flour (W) 100.0
. . .

. . .

. . .
12.0 1.99
Commercial gluten-free flour mix (C)
c . . .
100.0
. . .

. . .
11.9 0.53
Brown rice flour
. . .

. . .
25.0 50.0 12.4 1.34
Potato starch
. . .

. . .

. . .
25.0 18.7 0.03
Corn starch
. . .

. . .
54.0 12.5 12.7 0.02
Buckwheat flour
. . .

. . .
8.5
. . .
13.5 1.86
Soya flour
. . .

. . .
12.5 12.5 9.1 6.48
Skim milk powder
. . .

. . .

. . .
37.5 5.9 5.69
Baking powder
. . .

. . .

. . .
2.0
. . .d

. . .d

Salt 2.0
. . .
1.8 1.8
. . .

. . .

Yeast 1.5 2.0 2.0 3.5
. . .

. . .

Sugar 1.5 2.0 1.0 1.0
. . .

. . .

Sugar syrup
. . .

. . .
2.5
. . .
19.5
. . .

Xanthan gum
. . .

. . .
1.3 0.9
. . .

. . .

Konjac powder
. . .

. . .

. . .
1.5
. . .

. . .

Ascorbic acid 0.0
. . .

. . .

. . .

. . .

. . .

Water 61.0 63.0 105.0 105.0 100.0
. . .

Egg
. . .

. . .

. . .
30.0 73.5 2.05
Sum 166.0 167.0 213.5 283.2
. . .

. . .

Total moisture in recipe 73.0 74.9 117.7 142.9
. . .

. . .

Total solid matter in recipe 93.0 92.1 95.8 140.3
. . .

. . .

Ratio of moisture to solid matter 0.78 0.81 1.23 1.00
. . .

. . .

Total nitrogen in recipe (% wb)
e
1.20 0.32 0.62 1.50
. . .

. . .

a
Relative amounts (% fwb), total of brown rice flour, corn starch, buckwheat flour, and soya flour (ND) and brown rice flour, potato starch, corn starch, and soya
flour (D) calculated as flour.
b
Two replicates, standard deviation 0.01 except for corn starch (0.02).
c
Composition (list of ingredients): wheat starch (Codex Alimentarius Quality), milk solids, modified corn starch, soya flour, glucose, salt, stabilizer, methyl-
hydroxyproply cellulose, iron, thiamine, riboflavin, niacin.
d
Moisture and protein content of minor ingredients were ignored.
e
Based on sum of ingredients.
Vol. 81, No. 5, 2004 569
application of a glass coverslip. For microscopy of the bread crumb,
the breads were baked with the dye incorporated into them as for
the baking experiments. After the bread was cooled for 2 hr, a
small sample (1 mm side length) of each was taken from the
center of the crumb, immersed in oil in a welled slice, and covered
with a glass coverslip. A confocal laser-scanning system (MRC-
1024, Biorad, UK) mounted on an upright microscope (Axioskop,
Zeiss, Germany) with 10 and 40 water immersion objectives
was used for the batter images. For bread crumb images, 10 and
63 oil immersion objectives were used. Fluorescence images
(
exc
= 488 nm,
em
= 540 nm) of a z-series of optical sections
were acquired by scanning the sample along the optical axis in 2-m
steps for the 10 objective and 1-m steps for the 40 and 63
objectives.
Bread Analysis
A series of bread analyses was performed with three loaves at
day 0, after 2 hr of cooling, before packaging. Loaf weight and
volume (rapeseed displacement method) were determined. Bake
loss and loaf specific volume (mL/g) were calculated. Crust color
was determined with a chromameter (CR-300, Minolta, Osaka,
Japan). For crumb texture analysis, bread was sliced transversely
using a slice regulator and a bread knife to obtain uniform slices
of 25-mm thickness. Two bread slices taken from the center of
each loaf were used to evaluate the crumb texture. Images of the
bread were captured using a flatbed scanner (ScanJet4c, Hewlett
Packard) and supporting software (Desk Scan II, Hewlett Packard).
The brightness levels were adjusted to 150 units and contrast to
170 units using software controls (Crowley 2000). Texture profile
analysis (TPA) was performed using a universal testing machine
(TA-XT2i, Stable Micro Systems, Surrey, UK) equipped with a
25-kg load cell and a 35-mm aluminum cylindrical probe. The
settings used were a test speed of 2.0 mm/sec with a trigger force
of 20 g to compress the middle of the bread crumb to 60% of its
original height. Water activity was determined with material taken
from the center of the crumb (Aqua Lab CX-2, Decagon Devices,
Pullman, WA). All measurements obtained with the three loaves
from one batch were averaged into one replicate value. TPA was
repeated with three loaves each at day 2 (50 hr) and day 5 (122 hr)
after baking.
Analysis of Variance
Baking tests and rheological experiments were conducted with
the different bread and batter types (W, C, ND, D) according to a
randomized block design with three blocks to achieve three repli-
cates. Rheological measurements with dough W yielded overload,
therefore only batters C, ND, and D were compared in rheology.
For the measurement of bread texture, each batch of nine bread
loaves (one replicate of each bread type) was subdivided three
times into three pieces and these were randomly assigned to the
three different storage times.
A split-plot design resulted with three blocks (replicates) and
four main plots (treatments for breads W, C, ND, D) that were
subdivided into three split plots (days 0, 2, 5). Analysis of
variance for the randomized block and split-plot design was made
as described in Mead and Curnow (1983), calculating the
respective mean squares and F-ratios. Multiple comparisons were
made with least significant differences. LSD values were
calculated with the respective t-values (two-tailed, P = 0.01) and
standard errors were multiplied. Standard errors were
r s / 2
2
(1)
for comparisons of bread/batter types within the randomized
block design,
)
`

+ ) /(
2 2
) 1 ( 2 rb
a
s
b
s b (2)
comparisons between bread types at the same storage time within
the split plot design, and
r s
b
/ 2
2
(3)
comparisons between the same bread type at different storage
times within the split plot design where s
2
is error mean square; r
is number of blocks and replicates; b is the number of split plot
levels; s
b
2
is the split-plot error mean square; s
a
2
is the main plot
error mean square. The respective degrees of freedom for the t-
values were 6 (batter rheology 4), 6, and 16, respectively. Unless
otherwise mentioned, all significance statements are based on P <
0.01.
TABLE III
Mean Squares for Textural Characteristics of Four Bread Types During Storage
a


Degrees of
Mean Square Values
Freedom Hardness Resilience Cohesiveness Springiness Chewiness
Main plot
Block 2 2.268 0.002028* 0.000629 0.002787 0.160
Bread type 3 252.693*** 0.018444*** 0.046439*** 0.034573*** 58.534***
Main plot error 6 5.685 0.000195 0.000584 0.000683 0.318
Split plot
Storage time 2 506.416*** 0.238987*** 0.208472*** 0.147193*** 0.389
Bread type storage time 6 8.127 0.035180*** 0.046392*** 0.019284*** 8.162***
Split plot error 16 3.276 0.000355 0.000215 0.000651 0.294
a
*, **, *** = F-ratio significant at P < 0.05, 0.01, or 0.001, respectively.
TABLE II
Batter Rheology and Baking Characteristics of Breads
a

W
b

C
c
ND
d

D
e
Extrusion (N) >295 5.49 0.16a 6.18 0.62a 11.48 0.44b
Loaf specific volume (mL/g) 3.18 0.06a 2.83 0.09a 1.87 0.08b 2.08 0.01b
Bake loss (%)

9.87 0.12a 12.07 0.09b 11.03 0.18c 9.20 0.06d
Water activity of crumb

0.963 0.001a 0.964 0.001a 0.977 0.003b 0.969 0.002a
a
Mean value standard error of three replicates. Mean values followed by a common letter within the same row are not significantly different (P < 0.01).
b
Wheat bread.
c
Commercial gluten-free bread flour mix.
d
Nondairy gluten-free bread.
e
Dairy gluten-free bread.
570 CEREAL CHEMISTRY
RESULTS
Two gluten-free bread recipes were developed from a series of
200 preliminary experiments, one containing no dairy ingredients
(nondairy gluten-free, ND) and one containing a variety of
ingredients with cereals, especially skim milk powder and egg
(dairy gluten-free, D). These breads were compared with a wheat
bread (W) and gluten-free bread made from a commercial gluten-
free flour mix (C). Table I lists the recipes, the moisture and
protein contents of the individual ingredients, and the calculated
moisture and protein levels in the total recipes. The commercial
gluten-free flour mix (C), according to supplier information, is
based on wheat starch, milk solids, modified corn starch, soya
flour, and methyl-hydroxypropyl-cellulose. The ratio of
moisture-to-solid matter in batter C is most similar to dough W;
however, the nitrogen content is only 25% of dough W. Both
newly developed recipes are characterized by higher nitrogen
contents. Batter ND has higher nitrogen content than batter C but
distinctly lower than batter W, whereas batter D has higher
nitrogen content than batter W. Complex ingredients with high
protein content in the newly developed recipes account for higher
total nitrogen contents in ND brown rice flour (25%), buckwheat
flour (8.5%), and soya flour (12.5%), in D brown rice flour
(50%) and soya flour (12.5%) (Table I). In addition, D contains
skim milk powder and egg as protein sources. Hydrocolloids used
were xanthan gum (ND) and xanthan gum plus konjac gum (D).
D and especially ND contain comparatively high water levels, as
reflected by the moisture-to-solid matter ratio (Table I).
Batter Consistency
The consistencies of three batters (C, ND, D) were compared
with each other and also with wheat (W) dough. Two methods
were tested: 1) extrusion through a nozzle and 2) penetration with
a cylinder probe. Regarding extrusion, a significantly greater force

Fig. 1. Crumb hardness values of loaves baked from wheat (W), com-
mercial gluten-free flour mix (C), nondairy gluten-free (ND), and dairy
gluten-free (D) formulations during a five-day storage period. Mean
values standard error of three replicates. Mean values labeled with a
common lower case letter within the same bread type are not significantly
different (P < 0.01). Mean values for a given storage time labeled with a
common upper case letter are not significantly different (P < 0.01).

Fig. 2A. Crumb fracturability values of loaves baked from wheat (W), commercial gluten-free flour mix (C), nondairy gluten-free (ND), and dairy
gluten-free (D) formulations during a five-day storage period. B and C show TPA raw data curves for individual slices of ND bread.
Vol. 81, No. 5, 2004 571
was required for batter D than for C and ND, whereas no signi-
ficant difference was found between the latter two (Table II). Extru-
sion of dough W resulted in forces that exceeded the maximum
load of the instrument (295 N 30 kg). This was also the case after
prolonged relaxation times of up to 105 min. Penetration yielded
no significant differences between the batters (data not shown).
Baking Results and Texture
In baking tests, the baking parameters for loaf specific volume,
bake loss, water activity, and crust color were measured 2 hr after
baking. Textural parameters from TPA (including hardness, fractur-
ability, resilience, cohesiveness, springiness, and chewiness) were
monitored over a five-day storage period, applying a split-plot
design (main plot treatments were bread types; split-plot treat-
ments were storage for 0, 2, 5 days). Table II summarizes the baking
parameters obtained for the four bread types. Table III (split-plot)
and Figs. 16 show the results of texture measurements. Loaf
specific volume was significantly higher for breads W and C than
for ND and D. Bake loss was significantly different between all
breads; it was highest for C and lowest for D. Water activity was
significantly higher only for ND, whereas the other breads showed
no significant differences. Crust color showed no significant differ-
ences globally (data not shown). Digital images of bread crumb
and whole breads are shown Figs. 7 and 8, respectively.
With the TPA of the crumb, analysis of variance revealed for
most textural parameters that bread type (W, C, ND, D) and
storage time (0, 2, 5 days) had highly significant (P < 0.001)
effects and that there were also highly significant interactions
between them. However, hardness showed no significant inter-
actions, whereas chewiness showed no significant time effects.
Fracture occurred only for gluten-free breads (C, ND, D) at day
2 and 5. High random errors could be observed when measuring
the force at which fracture took place (Fig. 2A). With some indi-
vidual bread slices, no fracture occurred, whereas with the re-
mainder of the bread slices, fracture could be either detected
before the peak force (Fig. 2B), or the force at the fracture point
was equivalent to the peak force of the first compression cycle
(Fig. 2C). In the latter case, fracturability equaled hardness and
the peak force occurred before the probe reached its deepest
point. At day 0 for all bread types, and additionally at day 2 and 5
for bread W, no fracture occurred at all (force and variance were 0
at these points). Because equal variances for all bread types at all
storage days could not be assumed, fracturability could not be
included in the analysis of variance. Hardness of bread D was
significantly higher at all days of storage in comparison with the
other breads, except that at day 5, the difference from ND was no
longer significant (Fig. 1). Furthermore, the hardness of all breads
clearly increased over storage time, although the increase from
day 2 to day 5 was no longer significant for breads C and D.
Despite the fact that analysis of variance showed no interactions,

Fig. 3. Crumb cohesiveness values of loaves baked from wheat (W),
commercial gluten-free flour mix (C), nondairy gluten-free (ND), and
dairy gluten-free (D) formulations during a five-day storage period. Mean
values standard error of three replicates. Mean values labeled with a
common lower case letter within the same bread type are not significantly
different (P < 0.01). Mean values for a given storage time labeled with a
common upper case letter are not significantly different (P < 0.01).

Fig. 4. Crumb springiness values of loaves baked from wheat dough (W),
commercial gluten-free flour mix (C), nondairy gluten-free (ND), and
dairy gluten-free (D) formulations during a five-day storage period. Mean
values standard error of three replicates. Mean values labeled with a
common lower case letter within the same bread type are not significantly
different (P < 0.01). Mean values for a given storage time labeled with a
common upper case letter are not significantly different (P < 0.01).

Fig. 5. Crumb resilience values of loaves baked from wheat (W), com-
mercial gluten-free flour mix (C), nondairy gluten-free (ND), and dairy
gluten-free (D) formulations during a five-day storage period. Mean values
standard error of three replicates. Mean values labeled with a common
lower case letter within the same bread type are not significantly different
(P < 0.01). Mean values for a given storage time labeled with a common
upper case letter are not significantly different (P < 0.01).

Fig. 6. Crumb chewiness values of loaves baked from wheat (W), com-
mercial gluten-free flour mix (C), nondairy gluten-free (ND), and dairy
gluten-free (D) formulations during a five-day storage period. Mean values
standard error of three replicates. Mean values for a given storage time
labeled with a common upper case letter are not significantly different (P
< 0.01).
572 CEREAL CHEMISTRY
a certain trend can be seen in Fig. 1 that the increase in firmness
over storage time is less pronounced for bread D than for the other
bread types. Cohesiveness, springiness, and resilience showed com-
parable trends (Figs. 35). These parameters showed little depen-
dence on storage time for bread W, whereas for the gluten-free
breads C and ND, and less so for D, they decreased over storage
time. This decrease was significant between all days of storage
for cohesiveness and springiness, and significant between day 0
and day 2 only for resilience. The fact that the changes over
storage time depended on bread type for all three parameters (W
behaved differently from C, ND, and D, and D showed weaker
effects than C and ND) is in agreement with the interactions
found by analysis of variance (Table III). With regard to differ-
ences between bread types, it is remarkable that at day 0, W did
not show the highest values for all three parameters, whereas at
day 2 and day 5, it was significantly higher than all other treat-
ments. Furthermore, at day 2 and day 5, D was significantly higher
than C and ND in springiness and cohesiveness. Chewiness (Fig. 6)
was characterized by the clearest interactions; it increased over
storage time for W, decreased for D, and varied little for C and
ND. Averaged over the different bread types, however, storage
time showed no significant effect on chewiness, the F-test was not
significant (Table III). Chewiness was generally high for W and D
and low for C and ND, although this also depended on the storage
day (Fig. 6).
CLSM
The microstructures of batters, dough W, and breads were studied
by CLSM (Figs. 9 and 10). To identify the individual components
in the batters (potato starch, corn starch, brown rice flour,
buckwheat flour, soya flour, and skim milk powder), suspensions
of these components were prepared and observed under the
microscope (data not shown).
The microstructure of dough W at 10 was characterized by an
oriented gluten network. At 10, individual strands were visible,
between which, more amorphous gluten areas were located. Gluten
appeared very bright, the fluorescent dye Safranin O was strongly
adsorbed. At 40, globular starch granules (dark) with 1020 m
diameters were visible embedded in gluten. These adsorbed the
dye obviously to a lesser degree than protein (gluten) and thus
appear dark. Batter C at 10 showed mainly starch granules (wheat
starch, according to information from the supplier, see Table I)
visible as light grey globules with diameters of 20 m. No oriented
network was visible at 10 but isolated white areas were found
(protein in wheat starch and soya flour). At 40, besides the
greyish wheat starch granules (diameter 1020 m as in dough
W), these white protein areas were found again along with some
fine structures.
Batter ND was similar to the batter C, however the white protein
areas were more frequent (10), reflecting the higher (soya) protein
content of the recipe. Corn starch (54% of the flour basis, Table I)
appeared as slightly smaller granules compared with the wheat
starch (40). Batter D appeared most heterogeneous at 10, with
numerous white areas (soya protein) and large (50 m) dark
starch granules (potato starch). At 40, a continuous white area
(milk and egg protein) was visible, in which starch granules of
different sizes were embedded.

Fig. 8. Digital images of wheat bread (W), commercial gluten-free bread (C), nondairy gluten-free bread (ND), and dairy gluten-free bread (D).
Fig. 7. Digital images of wheat bread crumb (W), commercial gluten-free
bread crumb (C), nondairy gluten-free bread crumb (ND), and dairy
gluten-free bread crumb (D).
Vol. 81, No. 5, 2004 573
The bread crumbs were studied at 10 and 63, however, at
10, no characteristic differences were found between the bread
types. Pore walls 0.11 mm thick that surrounded the voids were
visible (data not shown). At 63, pore walls in bread crumb W
were characterized by a sponge-like structure of continuous asso-
ciated strands (denatured gluten). Globular dark bodies embedded
in these white strands might either be voids or deformed (gelatin-
ized) starch granules. In crumb C, only diffuse, greyish areas
could be found (probably gelatinized starch), overall lacking any
network-like structure. Crumb ND appeared similar, however, in
the grey areas, globular structures were still visible, and also white,
well-stained areas. Overall, crumb ND resembled batter ND. It
appears plausible that due to gelatinization, the corn starch granules
increase in size and partly loose integrity, but are still visible in
crumb ND. No continuous white protein network was visible.
Crumb D showed greatest similarity to crumb W. Well-stained
protein structures were visible with film-like areas and strands.
Dark bodies (diameter 20 m) embedded in W were either voids
or gelatinized starch.
DISCUSSION
The present study included three different recipes, all of which
resulted in acceptable gluten-free breads. However, the bread struc-
ture was achieved in different ways. The commercial (C) flour
mix contained mainly starch with little protein originating from the
soya and dairy ingredients present. The newly developed nondairy
bread (ND) was rich in whole meal cereals and soya flour and
thus higher in protein, although still much lower than a typical
wheat (W) formulation. The dairy bread (D), however, had the
highest protein levels due to the presence of soya flour, skim milk
powder, egg, and whole meal cereals (brown rice flour).
Most of the differences between the breads can be explained by
the characteristic properties of starch, protein, or fiber. Gelatin-
ized starch tends to regain a microcrystalline form at room tem-
perature (retrogradation). Many researchers have found that the
firming of wheat bread crumb is influenced by many factors,
including protein content, moisture, temperature, loaf specific
volume, and flour content (Ponte et al 1962; Axford et al 1968;
Maleki et al 1980). Changes in crumb properties associated with
staling include an increase in crumbliness of the crumb, starch
crystallinity, opacity, firmness, loss of flavor, and a decrease in
soluble starch and hydration capacity of the crumb (Herz 1965;
DAppolonia and Morad 1981). Retrogradation in wheat bread
solely depended on temperature and moisture. Zeleznak and Hos-
eney (1986), Davidou et al (1996), and Rogers et al (1998) reported
that bread moisture content influenced the firming rate and starch
retrogradation during storage of bread. Rogers et al (1998) reported
that the firming rate of wheat bread was retarded in bread that
contained a higher moisture content than that of wheat bread of
lower moisture. The high water activity of the ND bread reported
here did not, however, affect a retarded rate of firming.
Bran particles from brown rice flour or buckwheat contain high
concentrations of fiber and swell extensively. Bran supplemen-
tation usually weakens the structure and baking quality of wheat
dough and decreases bread volume and elasticity of the crumb
(Pomeranz et al 1977; Gan et al 1992). For wheat, Gomez et al
(2003) concluded that water absorption was increased with the
addition of dietary fiber, which in turn may explain the resulting
low volume. Therefore, in analogy, a higher water level was neces-
sary in ND batter to obtain a consistency comparable to that of
batter C due to the swelling of such particles. The significantly
higher firmness of batter D in comparison with that of batter C,
despite a higher water content reflects its high egg, soya, and milk
protein content, the high percentage of brown rice flour, and the
addition of konjac flour plus xanthan gum. Dairy ingredients are
used in bread for nutritional benefits including calcium content
and protein efficiency ratio and functional benefits including
flavor and texture enhancements and storage improvement
(Kenny et al 2001). The significantly higher firmness of batter D
in comparison with that of C, despite a higher water content,
reflects its high protein content. Konjac gum is derived from the
tuber of Amorphophallus konjac (Nishinari and Takahashi 2003).
Konjac gum contains a high molecular weight glucomannon
consisting of mannose and glucose in a molar ratio of 3:2 with
beta-1,4-linkages (Tye 1991). Xanthan gum is a high molecular
Fig. 9. Confocal laser-scanning micrographs of wheat dough (W), com-
mercial gluten-free batter (C), nondairy gluten-free batter (ND), and dairy
gluten-free batter (D) stained with Safranin O dye. Magnification bar 10
(100 m, left column); 40 (50 m, right column).

Fig. 10. Confocal laser-scanning micrographs of wheat bread crumb (W),
commercial gluten-free bread crumb (C), nondairy gluten-free bread crumb
(ND), and dairy gluten-free bread crumb (D) stained with Safranin O dye.
Magnification bar 10 (100 m, left column); 63 (50 m, right column).
574 CEREAL CHEMISTRY
weight natural exopolysaccharide produced by the bacterium
Xanthomonas campestris (Su et al 2003). Xanthan gum forms a
single- or double-stranded helix (Sato 1984; Rodd 2000) and is
classified as a cold-set gel. Hydrocolloids according to Toufeile et
al (1994), are added as gluten substitutes in the formulation of
gluten-free breads because these gums could act as polymeric
substances that mimic the viscoelastic properties of gluten in
wheat bread dough. Research reported by Tye (1991) showed that
konjac flour and xanthan gum interact to form a gel with unique
viscoelastic properties. This author also reported that konjac flour
interacts with most starches, thus increasing the viscosity of the
systems.
Nevertheless, all batters, including batter D, were softer than
dough W, which could not be extruded under the conditions
applied. Wheat gluten is unique in its ability to form a large,
aggregated, viscoelastic network (Paolo et al 2001). However, the
lack of gluten does not preclude the achievement of a high volume,
as demonstrated by bread C, which was mainly dominated by
starch. The main drawbacks of this approach are of nutritional
nature because important components such as fiber and naturally
occurring vitamins are lacking. Furthermore, such starch-based
breads are characterized by a relatively flat starchy aroma. The
presence of whole meal cereals adds aroma and dietary fiber, how-
ever at the expense of volume (Krishnan et al 1987; Chen et al
1988; Pomeranz et al 1997; Sievert et al 1990). In bread ND,
CLSM illustrated that the structure was visibly dominated by
starch, which will result in the formation of a weak gel structure.
As explained above, bran particles or fiber reduce the volume
even in whole meal wheat breads, where the structure is stabilized
by gluten. When no gluten is present, this negative effect on volume
can be assumed to be even worse. In agreement with these find-
ings, breads D and ND were lower in volume in comparison with
C, although D was slightly higher than ND.
The texture and staling showed several common traits charac-
teristic of all gluten-free breads. All were brittle at day 2 and also
at day 5, unlike bread W. This was reflected by different texture
parameters. Fracture occurred during the first bite; that is, the
bread structure was no longer cohesive as it cracked or crumbled
under the probe. Consequently, the measured cohesiveness derived
from the two-bite curve (ratio of the positive force area during
second compression to that during first compression) (Bourne
1978) was also much lower at day 2 and day 5. Springiness was
also lower at day 2 and day 5; that is, the bread recovered less in
height during the first and second bite (Bourne 1978) as it lost
elasticity during storage. The reduction in resilience also
characterizes loss of elasticity. Whereas in the determination of
the springiness, the bread crumb can recover for 5 sec between
the two compression cycles, in the determination of resilience, the
ratio of the area under curve of the second half of the first cycle
(upward stroke) to the first half (downward stroke) is measured.
For high resilience, a relatively quick, instantaneous recovery is
required, whereas delayed recovery also may contribute to springi-
ness. In agreement with this consideration, resilience was more
quickly lost over storage time; it strongly dropped from day 0 to
day 2 and only slightly and nonsignificantly from day 2 to day 5.
In contrast, springiness, dropped more gradually over storage
time; that is, a delayed recovery could be maintained even longer
in stale bread. These various changes characterizing brittleness
are in agreement with what was reported by Toufeili et al (1994)
for gluten-free flat bread. These authors found that after overnight
storage, all gluten-free formulations cracked extensively on appli-
cation of stress, had a markedly reduced ability to resist shearing
forces, and were notably less cohesive when compared with
regular bread aged for a similar time period.
In the present study, bread W showed none of these changes,
except a slight drop in resilience after five days of storage in
accordance with what was found by other authors (Nussinovitch
et al 1992). Only the increase in firmness was the same as for the
gluten-free breads. Although bread W was not most elastic and
cohesive at day 0, it did preserve these properties to day 5 and
thus exceeded the gluten-free breads at day 2 and day 5. In wheat
bread, it may be considered that a continuous elastic structure of
denatured gluten strands surrounding the starch may mask some
of the changes originating from starch retrogradation. It has also
been reported that gluten may serve as a reservoir to buffer any
changes in the hydration capacity of starch and retard crumb
firmness development (Gray and BeMiller 2003) A similar situ-
ation may be anticipated for bread D. It was the only gluten-free
bread where CLSM showed continuous film-like protein structures
similar to gluten. Because milk and soy proteins in the other
recipes did not show such films, it is most likely that egg protein
is responsible for this. Regarding egg proteins, interfacial film
formation is essential for foam and emulsion development. In
foaming systems, the formation of strong cohesive viscoelastic
films are essential for stable foaming (Kato et al 1990).
Despite the common staling patterns of all gluten-free breads
described so far, several effects were weaker for bread D relative
to breads C and ND; hardness was generally higher (reflecting the
stronger structure caused by egg protein as seen in CLSM), but
the increase over time was less (reflecting the lower starch
content). Accordingly, resilience, cohesiveness, and springiness
were better maintained. Hence, bread D remained elastic for a
longer period of time and overall became less brittle during
storage. Even though a firm structure might be regarded as a
disadvantage of bread (bread D), this property coincides with the
elastic and cohesiveness characteristics recorded for wheat bread.
The chewiness was the most indicative characteristic of bread D.
It is calculated by multiplying hardness, cohesiveness, and springi-
ness (Bourne 1978), and reflects the time required to masticate a
solid food to ready for swallowing (Cauvain 1987). Chewiness
was much higher for bread D than for C and ND. The low values
of C and ND at all days reflected the fact that a strong increase in
firmness was compensated by a strong drop in cohesiveness and
also in springiness; that is, less chewing is required because the
bread breaks in the mouth like a biscuit. In contrast, bread D had
a higher chewiness value that was more comparable to bread W
after day 2 and day 5. The overall better keeping quality of the
high protein bread D was its outstanding advantage.
CONCLUSIONS
Starch-based gluten-free breads may achieve a high volume,
but at the expense of quick staling. Increased water levels in
combination with whole meal cereals could not notably delay the
staling. However, the addition of the right protein sources in
sufficient amounts improved the keeping quality. The formation
of a continuous protein phase and film-like structures appears to
be critical because they can partially mask changes caused by
starch retrogradation. Due to its high resolution and its ability to
scan through thick samples, confocal laser-scanning microscopy
proved very useful in detecting these desirable protein structure
elements in bread crumb.
ACKNOWLEDGMENTS
This project was funded by the Irish government under the National
Development Plan, 2000-2006. We would like to thank Charmaine Clarke
and Tom Hannon for valuable contributions made to this article.
LITERATURE CITED
American Association of Cereal Chemists. 2000. Approved Methods of
the AACC, 10th Ed. Method 46-30. The Association: St. Paul, MN.
Arendt, E. K., OBrien, C. M., Schober, T. J., Gallagher, E., and Gormley,
T. R. 2002. Development of gluten-free cereal products. Farm Food 21-
27.
Axford, D. W. E., Colwell, K. H., Confor, S. J., and Elton, G. A. H. 1968.
Vol. 81, No. 5, 2004 575
Effect of loaf volume on the rate and extent of staling in bread. J. Sci.
Food Agric. 19:95-101.
Bourne, C. M. 1978. Texture profile analysis. Food Technol.32:62-66.
Cauvain, P. S. 1987. Characterizing baked products using texture profile
analysis. FMBRA Bull. 2:21-28.
Chen, H., Rubenthaler, G. L., Leung, H. K., and Baranowski, J. D. 1988.
Chemical, physical, and baking properties of apple fiber compared
with wheat and oat bran. Cereal Chem. 65:244-247.
DAppolonia, L. B., and Morad, M. M. 1981. Bread staling. Cereal
Chem. 58:186-190.
Drrenberger, B. M., Handschin, S., Conde-Petit, B., and Escher, F. 2001.
Visualization of food structure by confocal laser scanning microscopy
(CLSM). Lebensm. Wiss. Technol. 34:11-17.
Fasano, A., and Catassi, C. 2001. Current approaches to diagnosis and
treatment of celiac disease: An evolving spectrum. Gastroenterology
120:636-651.
Gan, Z., Galliard, T., Ellis, P. R., Angold, R. E., and Vaughan, J. G. 1992.
Effect of the outer bran layers on the loaf volume of wheat bread. J.
Cereal Sci. 15:151-163.
Gomez, M., Ronda, F., Blanco, A. C., Caballero, A. P., and Apesteguia, A.
2003. Effect of dietary fibre on dough rheology and bread quality. Eur.
Food Res. Technol. 216:51-56.
Herz, K. O. 1965. Staling of breadA review. Food Technol. 19:1828-
1841.
ICC. 1976. Standard Methods of the International Association for Cereal
Science and Technology. Method 110/1. The Association: Vienna.
Kasarda, D. D. 2001 Grains in relation to celiac disease. Cereal Foods
World 46:209-210.
Kato, A., Ibrahim, H., Watanabe, H., Honma, K., and Kobayashi, K. 1990.
Enthalpy of denaturation and surface functional properties of heated
egg white proteins in the dry state. J. Food Sci. 55:1280-1282.
Kenny, S., Wehrle, K., Auty, M., and Arendt, E. K. 2001. Influence of
sodium caseinate and whey protein on baking properties and rheology
of frozen dough. Cereal Chem. 78:458-463.
Krishnan, P. G., Chang, K. C., and Brown, G. 1987. Effect of commercial
oat bran on the characteristics and composition of bread. Cereal Chem.
64:55-58.
Loewe, R. 1993. Role of ingredients in batter systems. Cereal Foods
World 38:673-677.
Lovis, L. J. 2003. Alternatives to wheat flour in baked goods. Cereal
Foods World 48:61-63.
Maleki, M., Hoseney, R. C., and Mattern, J. P. 1980. Effects of loaf
volume, moisture content, and protein quality on the softness and staling
rate of bread. Cereal Chem. 57:138-140.
Martin, M. L., and Hoseney, R. C. 1991. A mechanism of bread firming.
II. Role of starch hydrolyzing enzymes. Cereal Chem. 68:503-507.
Mead, R., and Curnow, R. N. 1983. Statistical Methods in Agriculture and
Experimental Biology. Pages 47-54, 96-103. Chapman and Hall:
London.
Murray, J. A. 1999. The widening spectrum of celiac disease. Am. J. Clin.
Nutr. 69:354-365.
Mustalahti, K., Lohiniemi, S., Collin, P., Vuolteenaho, N., Laippala, P.,
and Markku, M. 2002. Gluten-free diet and quality of life in patients
with screen-detected celiac disease. Eff. Clin. Pract. 5:105-113.
Nishinari, K., and Takahashi, R. 2003. Interaction in polysaccharide
solutions and gels. Curr. Opin. Colloid Interface Sci. 8:396-400.
Nussinovitch, A., Steffens, M. S., and Chinachoti, P. 1992. Elastic prop-
erties of bread crumb. Cereal Chem. 69:678-681.
Paolo, M., Cavella, S., and Piazza, L. 2001. An interpretation of the
rheological behavior of wheat flour dough based on fundamental tests.
Pages 75-90 in: Bread Staling. P. Chinachoti and Y. Vodorotz, eds.
CRC Press: Boca Raton, FL.
Pomeranz, Y., Shogren, M. D., Finney, K. F., and Bechtel, D. B. 1977.
Fiber in breadmakingEffects on functional properties. Cereal Chem.
54:25-41.
Ponte, J. G., Titcomb, S. T., and Cotton, R. H. 1962. Flour as a factor in
bread firming. Cereal Chem. 39:437-444.
Rodd, A. B., Dunstan, D. E., and Boger, D. V. 2000. Characterisation of
xanthan gum solutions using dynamic light scattering and rheology.
Carbohydr. Polym. 42:159-174.
Rogers, D. E., Zeleznak, K. J., Lai, C. S., and Hoseney, R. C. 1998. Effect
of native lipids, shortening, and bread moisture on bread firming.
Cereal Chem. 65:398-401.
Sato, T., Norisuye, T., and Fujita, H. 1984 Double-stranded helix of
xanthan in dilute solution: Evidence from light scattering. Polym. J.
16:341-350.
Sievert, D., Pomeranz, Y., and Abdelrahman, A. 1990. Functional prop-
erties of soy polysaccharides and wheat bran in soft wheat products.
Cereal Chem. 67:10-13.
Su, L., Ji, K. W., Lan, Z. W., and Dong, Q. X. 2003. Chemical modi-
fication of xanthan gum to increase dissolution rate. Carbohydr. Polym.
53:497-499.
Toufeili, I., Dagher, S., Shadarevian, S., Noureddine, A., Sarakbi, M., and
Farran, T. M. 1994. Formulation of gluten-free pocket-type flat breads:
Optimization of methylcellulose, gum arabic, and egg albumen levels
by response surface methodology. Cereal Chem. 71:594-601.
Tye, J. R. 1991. Konjac flour: Properties and applications. Food Technol.
3:86-92.
Ylimaki, G., Hawrysh, Z. J., Hardin, R. T., and Thomson, A. B. R. 1991.
Response surface methodology in the development of rice flour yeast
breads: Sensory evaluation. J. Food Sci. 5:751-759.
Zeleznak, K., and Hoseney, R. C. 1986. The role of water in the retro-
gradation of wheat starch gels and bread crumb. Cereal Chem. 63:407-
411.
[Received November 14, 2003. Accepted March 23, 2004.]