LWT - Food Science and Technology 99 (2019) 476–482

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LWT - Food Science and Technology

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Waxy starch as dough component and anti-staling agent in gluten-free bread T

a b b c,∗
Mariusz Witczak , Jarosław Korus , Rafał Ziobro , Lesław Juszczak
a
Department of Engineering and Machinery for Food Industry, Faculty of Food Technology, University of Agriculture in Krakow, Balicka 122 Str., 30-149, Kraków, Poland
b
Department of Carbohydrate Technology, Faculty of Food Technology, University of Agriculture in Krakow, Balicka 122 Str., 30-149, Kraków, Poland
c
Department of Food Analysis and Evaluation of Food Quality, Faculty of Food Technology, University of Agriculture in Krakow, Balicka 122 Str., 30-149, Kraków,
Poland

ARTICLE INFO ABSTRACT

Keywords: The aim of the study was to evaluate the influence of waxy starch (WS) on structure formation and staling
Gluten-free bread retardation in starch based gluten-free bread. Corn/potato starch mix in bread formulation was partially re-
Waxy starches placed with waxy corn/waxy potato starch mix, and the resulting changes in rheological properties of the dough
Quality and quality of the bread, as well as its staling kinetics were monitored. It was concluded that the presence of
Texture
waxy starch modified rheological properties of the dough, causing an increase in values of storage and loss
Staling
moduli (G′ and G″, respectively) and zero shear viscosity, due to the increased swelling capacity of waxy starch.
Moreover 10% replacement of original starch mix with WS mix had the optimal effect on bread volume, ac-
companied with only slight changes in crumb structure. The presence of WS in amounts 10–15% had a positive
impact on texture characteristics of bread crumb, reducing its hardness and chewiness in comparison to control
and limiting increase of these parameters during storage. WS added at levels 10–15% caused a reduction in
enthalpy of retrograded amylopectin, which indicates, that it could be an effective factor reducing staling of
starch based gluten-free bread.

1. Introduction modifications applied to it have an important effect on the micro-

Structure and texture of gluten-free bakery products depend mostly
on starch used either in an isolated form, or as the major constituent of
flours in bread formulations. Basic starch sources which could be di-
rectly used for gluten-free products include corn, rice and potato
(Garzόn, Rosell, Malvar, & Revilla, 2017; Witczak, Ziobro, Juszczak, &
Korus, 2016; Zhang, Mu, & Sun, 2017). Alternative raw materials in-
volve gluten-free cereals (sorghum, millet, teff), pseudocereals (buck-
wheat, amaranth, quinoa) and other plants, such as acorn or hemp (Gao
et al., 2018; Horstmann, Lynch, & Arendt, 2017; Korus, Witczak,
Ziobro, & Juszczak, 2015a, 2017; Naqash, Gani, Gani, & Masoodi, 2017;
Witczak et al., 2016). Lack of gluten in the respective products leads to
weak dough structure and deterioration of crumb quality. Often they
also reveal lower nutritional value in comparison to wheat based ba-
kery, because of the lower content of non starch components (Melini,
Melini, Luziatelli, & Ruzzi, 2017; Segura & Rosell, 2011). Therefore
trials to seek optimum formulations for the products with proper sen-
sory parameters, nutritional quality and high consumer acceptance
persist (Melini et al., 2017; Ziobro, Juszczak, Witczak, & Korus, 2016).
Properties of the products which are based on starch are determined
by its characteristics. The type and origin of starch and possible
structure and rheological properties of the dough, water retention, and
quality of the resulting products (Gao et al., 2018; Zhang et al., 2017).
Even starch isolated from different cultivars of the same plant species
could reveal variable physico-chemical and functional properties
(Pycia, Juszczak, Gałkowska, & Witczak, 2012).
Apart from native starch, regarded as food component, gluten-free
products may contain chemically modified starch as accepted food
additives. Their presence influences water absorption, rheological
properties of the dough as well as texture and staling of the bread,
which allows the manufacture of products with strictly designed and
controllable properties. Chemically modified starches stabilize crumb
structure and reduce retrogradation tendency, thus slowing down its
staling. However the excess of cross-linked starches could lead to un-
acceptable increase of bread hardness (Witczak, Juszczak, Ziobro, &
Korus, 2012; Ziobro, Korus, Witczak, & Juszczak, 2012).
Although legally approved, the use of chemically modified starch in
food formulations is often feared by consumers. Therefore food industry
frequently replaces them with starch modified by physical and enzy-
matic methods or obtained from plants in which it has naturally mod-
ified granular or crystalline structure and/or altered amylose to amy-
lopectin ratio. Among them special attention should be paid to waxy

∗
Corresponding author.
E-mail address: rrjuszcz@cyf-kr.edu.pl (L. Juszczak).

https://doi.org/10.1016/j.lwt.2018.10.009
Received 19 June 2018; Received in revised form 23 September 2018; Accepted 3 October 2018
Available online 03 October 2018
0023-6438/ © 2018 Elsevier Ltd. All rights reserved.
M. Witczak et al.
starches, which are virtually free of amylose, and thus could swell more
easily and form pastes with elevated viscosity, although more suscep-
tible to mechanical shear. Waxy starches display higher water absorp-
tion capacity and form pastes with higher viscosity in comparison to
those produced from amylose containing starch, in which the presence
of amylose delays swelling of the granules, thus reducing viscosity. The
granules of waxy starch easily absorb water, and in a swollen state are
more susceptible to mechanical damage and easily dissolve. Molar size
and branch-chain length of amylopectin affect the gelatinization be-
haviour of waxy starches and the rheological properties of their pastes
and gels. Gelatinized waxy starch forms a viscoelastic gel with a sig-
nificant share of viscous properties, that is more liquid-like at high
frequencies (Šárka & Dvořáček, 2017a, 2017b). Waxy starches could
also form clear gels with decreased hardness (Šárka & Dvořáček,
2017a), and due to these special properties could be used in a wide
range of applications within food industry (Šárka & Dvořáček, 2017b).
In baking they are used for an improvement of the quality of bread and
pastry products, as they could decrease staling (Šárka & Dvořáček,
2017a). They also stabilize emulsions and sauces by providing soft
texture of such products (Bortnowska, Balejko, Tokarczyk,
Romanowska-Osuch, & Krzemińska, 2014; Krzemińska & Bortnowska,
2017).
The possibility of using waxy flour or starch as a factor reducing
staling was first examined more than 70 years ago. Noznick, Merritt,
and Geddes (1946) observed that bread obtained from mixtures of
starch and gluten, with an addition of waxy corn starch, had glittering
crust and crumb with improved texture. Purhagen, Sjöö, and Eliasson
(2011) reported, that the addition of waxy wheat flour in an amount of
10% to white wheat bread reduces its specific volume. However ac-
cording to Hibi (2001) the presence of retrograded waxy corn starch
caused an increase of specific volume of wheat bread. Bhattacharya,
Erazo-Castrejón, Doehlert, and McMullen (2002) noticed a decrease of
crumb firmness during 5 days of storage, when wheat bread was pro-
duced with an optimum 20% share of waxy durum wheat flour. Simi-
larly Purhagen et al. (2011) observed a decrease of firmness and limited
reduction of moisture content in wheat bread with an addition of waxy
wheat flour at a level of 10%. Ghiasi, Hoseney, Zeleznak, and Rogers
(1984) reported, that the change in amylose to amylopectin ratio,
caused by the addition of waxy barley starch to high protein wheat
flour resulted in a decrease of crumb firmness, but only on the first day
after baking.
The information concerning the influence of waxy starch on struc-
ture, properties and staling of starch gluten-free bread is quite limited.
Therefore the aim of the study was to determine the influence of waxy
starch as a structure-forming factor with an ability to delay staling in
gluten-free bread (GFB). The changes caused by partial replacement of
corn/potato starch mix in bread formulation with waxy analogues were
followed in rheological properties of the dough, selected quality para-
meters of bread and staling kinetics.
2. Materials and methods
2.1. Materials
Standard amylose containing corn starch (Bezgluten, Poland), po-
tato starch (Pepees S.A., Poland), waxy corn starch (Roquette Poland),
waxy potato starch (Avebe Netherlands), guar gum (Lotus Gums &
Chemicals, India), pectin (Pektowin, Poland), freeze dried yeast Saf-
instant (S.I. Lesaffre, France), sucrose, salt, rapeseed oil (bought in local
supermarkets) and water were used as components of bread recipe.
2.2. Methods
2.2.1. Formulation for gluten-free dough
The basic recipe included 600 g of starch mix (corn starch and po-
tato starch in a ratio 4:1), 30 g freeze dried yeast, 18 g rapeseed oil, 12 g
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LWT - Food Science and Technology 99 (2019) 476–482
sucrose, 11 g salt, 10 g pectin, 10 g guar gum, and 570 g water. Part of
starch mix (5–25%, equivalent 30–150 g) was replaced with waxy
starch (corn and potato mixed at the same ratio 4:1).
2.2.2. Rheological properties of dough
Rheological properties of the dough were determined at 25 °C using
MARS II rheometer (Thermo-Haake, Germany) equipped with a system
of parallel plates (diameter 35 mm, gap size 1 mm). Dough samples
(without yeast) were placed in a measuring system of the rheometer
and left for 5 min for stress relaxation and temperature setting.
Mechanical spectra were determined in a range of linear viscoe-
lasticity at constant strain amplitude (γ = 0.05%) in a range of angular
frequency 0.1–100 rad/s. Experimental data were fitted to power law
model (Korus, Witczak, Ziobro, & Juszczak, 2015b):
(1)
n
G( )=K
G"( ) = K " n
" (2)
where: G' - storage modulus (Pa), G″- loss modulus (Pa), ω - angular
frequency (rad/s), K′, K”, n', n” - experimental constants.
Creep and recovery tests were performed at constant strain in creep
phase σ0 = 2Pa during 150 s and 300 s of recovery phase. Experimental
data were described using Burgers' model (Witczak, Juszczak, Ziobro, &
Korus, 2017):
t
J (t ) = J0 + + J1 (1 exp t / ret
) for creep phase
(3)
0
t1
J (t ) = J1 (1 expt 1/ ret
) exp t / ret for recovery phase
0 (4)
−1
where: J is a compliance (1/Pa ), J0 – instantaneous compliance (1/
Pa−1), J1 – viscoelastic compliance (1/Pa−1), η0 – zero shear viscosity
(Pa∙s), λret – retardation time (s), and t1 – time after which stress was
removed (s).
2.2.3. Bread baking
All dough ingredients were mixed for 8 min (Laboratory Spiral
Mixer SP 12, Diosna, Germany). The obtained dough was fermented for
15 min (35 °C, 80% relative humidity), and re-mixed for 1 min, then the
portions of 250 g were weighed into greased metal pans. Final fer-
mentation continued for 20 min under the above mentioned conditions.
The loaves were baked for 30 min at 230 °C (electric oven MIWE Condo
type CO 2 0608, MIWE GmbH, Germany). The loaves were baked in two
independent batches, each containing all the analysed samples (5
loaves). After removing from pans bread was cooled down at ambient
temperature and used for further analyses or wrapped in polyethylene
bags and stored in a cabinet at 22 ± 2 °C.
2.2.4. Bread analyses
Bread volume was measured using Volscan profiler 600 (Stable
Micro Systems, England). Image analysis was performed for slices from
the internal part of each loaf (thickness – 1 cm) which were scanned by
Plustek S-12 desktop scanner. The registered images were analysed
with the help of ImageJ software v. 1.44c (Abramoff, Magelhaes, &
Ram, 2004), evaluating porosity, cell density and percentage of
pores > 5 mm.
Texture profile analysis (TPA) of bread crumb was performed using
texture analyser TA-XT2plus (Stable Micro Systems, England) at the
compression rate 5 mm/s. Sample of bread crumb with a height 2 cm
was pressed to reach 50% deformation by a P/20 aluminium cylinder
probe with a diameter 2 cm, in two cycles with a 5 s delay. The resulting
hardness (maximum force, N), cohesiveness (area under second peak
divided by area under first peak), springiness (distance during second
compression divided by distance during first compression) and chewi-
ness (hardness × cohesiveness × springiness, N) of the crumb were
used as indicators of textural changes during storage. The calculations
M. Witczak et al.
were performed using the Texture Exponent software (Stable Micro
Systems, England). The analysis were performed after 2, 24 and 48 h
after baking.
Thermal properties of gluten-free bread crumb were characterized
by means of differential scanning calorimeter DSC 204F1 Phoenix
(Netzsch, Germany). Loaves after baking were cooled for 2 h. After this
time samples were taken for DSC analyses and the rest of loaves was
stored for further analyses after 24 and 48 h. Crumb samples (approx
15 mg) in the following days of analyses were closed hermetically in
aluminium pans and heated from 25 to 100 °C at a rate of 10 K/min.
Empty aluminium pan was used as reference. Temperatures and en-
thalpy of thermal transitions were determined with the use of in-
strument's software Proteus Analysis (Netzsch, Germany). Enthalpy
were expressed as J/g of d.b.
2.2.5. Statistical analysis
In order to establish the statistical differences between means, the
data were treated by one-factor analysis of variance, and the least sig-
nificant difference (LSD) at significance level 0.05 was calculated using
Fisher post hoc test. In order to determine both the influence of waxy
starch addition level, as well as time (in the case of textural and thermal
properties), two way ANOVA was applied. The dependencies between
analysed parameters were identified using the Pearson's correlation
coefficients. The calculations were performed using statistical software
package Statistica 11.0 (StatSoft Inc., USA).
3. Results and discussion
3.1. Rheological properties of the dough
The analysis of dough rheology, especially its viscoelastic properties
allows evaluation of the influence of process or recipe modification,
including quantitative or qualitative changes in components, on dough
characteristics. Gluten-free dough subjected to small deformations re-
veals properties characteristic for weak gels (Korus et al., 2015b;
Witczak et al., 2017). Basic methods allowing determination of vis-
coelastic properties of such systems are sweep frequency and creep and
recovery tests. Fig. 1 represents the changes in storage modulus (G′) and
loss modulus (G”) recorded at increasing angular frequency of selected
gluten-free dough samples, in which 5, 15 and 25% starch mix was
replaced with waxy starches (WS). In all cases the storage modulus
were higher than those of loss modulus G' > G”. This indicates the
prevalence of elastic properties over viscous and confirms earlier re-
ports concerning viscoelastic properties of starch based gluten-free
dough (Korus et al., 2015b; Witczak et al., 2017). Moreover the de-
pendence of changes in G′ and G” and their ratio represented by shift
Fig. 1. Mechanical spectra of control dough and dough with waxy starch (WS):
○ Control, □ 5% WS, △ 15% WS, ◇ 25% WS; G′ filled symbols, G″ empty
symbols.
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LWT - Food Science and Technology 99 (2019) 476–482
tangent (tan δ = G"/G′) indicate properties characteristic for weak gels.
Partial replacement of starch mix with WS modified viscoelastic prop-
erties of the dough increasing the values of G′ and G”. These observa-
tions are confirmed by parameters of power law equations used to de-
scribe sweep frequency curves (Table 1). An increase in K′,
corresponding to elastic properties increase with the level of WS starch,
until its share equalled 20%, and the significant positive change could
be noticed at the level 15%. Similarly K”, describing viscous properties,
the increase of its value could be seen up to 20% concentration of WS in
the dough. Further rise in WS quantity in the dough i.e. 25% resulted in
a decrease of both K′ and K”. No significant influence of WS con-
centration could be found in the case of other parameters (n' and n”)
used in power law model describing sweep frequency results (Table 1).
Although a significant increase of G′ and G” moduli could be observed
as an effect of growing WS share, the applied change in formulation had
no clear influence on shift tangent, which initially decreased with rising
share of WS, indicating dough strengthening, but the trend was not
statistically significant.
Example creep and recovery curves for control dough and samples
containing 5, 15 and 25% WS starch are shown on Fig. 2. The partici-
pation of WS in building dough structure resulted in a decrease of its
compliance towards applied stress, and its addition at levels 15–25%
caused significant reduction of this parameter. Parameters of Burgers
model describing creep and recovery curves were collected in Table 1.
The values of instantaneous compliance J0 and viscoelastic compliance
J1 decreased with increasing share of WS, but the changes were not
statistically significant. No statistically significant influence of WS was
observed for retardation time. On the other hand significant increase in
zero shear viscosity was observed with rising level of WS in the dough.
Despite both methods applied for rheological characterization of dough
rheology are based on different principles, the results are significantly
correlated. Strong negative linear correlation could be found for in-
stantaneous compliance J0 and parameters describing sweep frequency
test K′ and K” (r = −0.98 and −0.97, respectively) and viscoelastic
compliance J1 and K′ and K” constants (r = −0.92 and −0.91, re-
spectively). Moreover instantaneous compliance positively correlated
with the tan δ (r = 0.89). The zero shear viscosity positively correlated
with K′ and K” constants (r = 0.94 and 0.92, respectively) and nega-
tively with tan δ (r = −0.93). According to Hruśková, Bednárová, and
Novotný (2001) waxy wheat flour forms cohesive dough with viscoe-
lastic properties and the ability to retain gas, which is essential for
producing baked products with a light texture. Starch in dough systems,
prior to thermal treatment, exists in granular form, so the changes in
dough structure and rheology are strongly affected by swelling of starch
granules. When the level of other structure forming components is
constant, in this case guar gum and pectin, starch swelling is a key
factor controlling water activity in the system, and consequently
rheological characteristics of the dough. Waxy starch displays higher
swelling ability in comparison to its amylose containing counterpart
(Šárka & Dvořáček, 2017a), so its introduction into the systems should
lead to increasing water absorption and strengthening of the dough
structure by swollen starch granules. On the other hand if the share of
WS is too high it may impair hydration of non starch hydrocolloids,
such as pectin and guar gum, and reduce their role in structure for-
mation. Therefore the changes in rheological properties of the dough
are not excessive, though the observed tendency indicates its
strengthening by the share of waxy starch.
3.2. Bread characteristics
Physical parameters describing properties of control gluten-free
bread and samples baked from the dough in which part of starch mix
was replaced with WS are shown in Table 2. The presence of 5–10%
waxy starch in the dough caused an increase in bread volume in com-
parison to control, but the change was not statistically significant. On
the other hand above 20% a decrease in this parameter could be
M. Witczak et al. LWT - Food Science and Technology 99 (2019) 476–482

Table 1
Parameters describing rheological properties of control gluten-free dough and dough with waxy starch addition.
Parameter Control GFB Waxy starch addition One-way ANOVA - p

5% 10% 15% 20% 25%

n′ a a a b b
K’ (Pa·s ) 1504.1 ± 89.2 1561.2 ± 98.1 1626.7 ± 73.9 1836.6 ± 106.2 1982.0 ± 143.9 1873.9 ± 99.7b < 0.001
n’ 0.194 ± 0.014 0.199 ± 0.011 0.200 ± 0.006 0.198 ± 0.006 0.199 ± 0.005 0.199 ± 0.005 0.936
r2 > 0.997 0.999 0.999 0.998 0.999 0.991 –
K” (Pa·sn”) 574.1 ± 18.0a 592.1 ± 27.4ab 617.7 ± 15.1b 686.1 ± 20.2c 743.1 ± 41.7d 694.8 ± 23.3c < 0.001
n” 0.188 ± 0.007 0.196 ± 0.005 0.191 ± 0.003 0.192 ± 0.005 0.190 ± 0.005 0.192 ± 0.002 0.320
r2 > 0.950 0.952 0.953 0.950 0.944 0.945 –
tanδ at 1 Hz 0.356 ± 0.010 0.355 ± 0.009 0.353 ± 0.012 0.348 ± 0.010 0.348 ± 0.009 0.351 ± 0.018 0.856

J0 ⋅ 103 (Pa−1) 1.00 ± 0.12 0.93 ± 0.09 0.94 ± 0.03 0.84 ± 0.03 0.78 ± 0.08 0.82 ± 0.01 0.138
J1 ⋅ 103 (Pa−1) 1.86 ± 0.26 1.64 ± 0.01 1.66 ± 0.03 1.60 ± 0.26 1.31 ± 0.05 1.39 ± 0.01 0.083
η0 ⋅ 10−3 (Pa⋅s) 53.83 ± 3.30a 52.97 ± 7.52a 57.76 ± 4.81ab 69.33 ± 3.07b 67.64 ± 5.62b 70.12 ± 3.60b 0.034
λret (s) 65.6 ± 0.3 63.0 ± 5.1 62.8 ± 9.0 73.1 ± 16.1 64.8 ± 6.0 65.8 ± 0.8 0.818
r2 > 0.987 0.982 0.986 0.984 0.981 0.984 –

Mean value of four replication ± standard deviation.

Mean values signed this same letters in particular rows are non-significant different at 0.05 level of confidence.

activity of yeast is not restrained by the lack of substrates for fermen-

Fig. 2. Creep and recovery curves of control dough and dough with waxy starch
(WS): ○ Control, □ 5% WS, △ 15% WS, ◇ 25% WS.
tation, the key factor for bread volume is an ability of the dough for gas
retention. The presence of WS initially caused an increase in volume,
which may be attributed to its better swelling and solubility in water
(Šárka & Dvořáček, 2017a). However further doses of this type of starch
caused a decrease in bread volume. Structure formation during bread
baking is related with partial starch gelatinization and interactions
between starch polymers and other structure forming substances pre-
sent in the dough. In this case an increase in WS containing mostly
amylopectin, could reduce the ability of other polymers to associate and
form stable structure responsible for gas holding properties. The share
of WS had also significant influence on cell density, which followed the
trend observed for volume, initially increasing and at larger levels of
WS (≥20%) decreasing below the value measured for control bread
(Table 2). The linear correlation coefficient between both parameters
equalled r = 0.98. On the other hand no significant influence of WS on
crumb porosity and presence of pores with diameter larger than 5 mm
could be observed.

3.3. Bread texture

observed, and the lowest volume was noticed in the case of 25% share
of WS. The reports about influence of waxy starch on bread volume
differ. Bhattacharya et al. (2002) did not found any significant influ-
ence of waxy durum wheat flour on wheat bread volume. On the other
hand Purhagen et al. (2011) observed, that 10% addition of waxy wheat
flour results in a decreased specific volume of wheat bread. Hibi (2001)
noticed an increase in specific volume of wheat bread under the in-
fluence of retrograded waxy corn starch. According to Jongsutjarittam
and Charoenrein (2014) the disadvantage of waxy flour addition in
dough may be lowering the loaf volume of bakery product. If the
Texture parameters of bakery products are decisive for their con-
sumer acceptance. Storage of bread, especially based on starch, leads to
several structural changes resulting in its deterioration. These changes
are mainly caused by migration of water from crumb to crust and
hardening of the crumb related to starch retrogradation. In order to
diminish these unfavourable phenomena several additives and com-
ponents could be applied, which control water retention and or slow
down starch recrystallization (Gao et al., 2018; Horstmann et al., 2017;
Witczak et al., 2016). Texture analysis is one of the methods commonly

Table 2
Physical parameters of control gluten-free bread and bread with waxy starch addition.
Bread volume (cm3) Porosity Cell density (cm−2) Percentage of pores > 5 mm

bc b
Control GFB 597.5 ± 12.58 0.421 ± 0.010 2.929 ± 0.201 0.142 ± 0.014
5% WS 607.5 ± 12.58bc 0.416 ± 0.014 3.040 ± 0.268b 0.132 ± 0.013
10% WS 615.0 ± 10.00c 0.424 ± 0.010 3.036 ± 0.311b 0.142 ± 0.017
15% WS 605.0 ± 12.91bc 0.422 ± 0.011 3.030 ± 0.294b 0.136 ± 0.016
20% WS 590.0 ± 11.55b 0.429 ± 0.012 2.867 ± 0.301ab 0.144 ± 0.014
25% WS 562.5 ± 9.57a 0.420 ± 0.015 2.693 ± 0.262a 0.147 ± 0.018

One-way ANOVA - p < 0.001 0.098 0.002 0.069

Mean value of four replication ± standard deviation.

Mean values signed this same letters in particular columns are non-significant different at 0.05 level of confidence.

479
M. Witczak et al. LWT - Food Science and Technology 99 (2019) 476–482

Table 3
Texture parameters of control gluten-free bread and bread with waxy starch addition.
Sample Day Hardness (N) Springiness (-) Cohesiveness (-) Chewiness (N)

b g e
Control GFB 1 1.47 ± 0.13 1.00 ± 0.00 0.89 ± 0.01 1.31 ± 0.11bc
2 10.61 ± 1.39hi 0.96 ± 0.01bcde 0.58 ± 0.03d 5.86 ± 0.70i
3 8.64 ± 0.17fgh 0.97 ± 0.02def 0.48 ± 0.06abc 4.01 ± 0.57gh
5% WS 1 1.45 ± 0.14b 1.00 ± 0.00g 0.86 ± 0.03e 1.25 ± 0.15b
2 8.44 ± 1.68fg 0.97 ± 0.01ef 0.55 ± 0.05cd 4.45 ± 0.58h
3 9.85 ± 1.71ghi 0.97 ± 0.01def 0.45 ± 0.08a 4.28 ± 1.04gh
10% WS 1 0.96 ± 0.21a 1.00 ± 0.00g 0.86 ± 0.01e 0.83 ± 0.18a
2 2.88 ± 0.64c 0.97 ± 0.02ef 0.58 ± 0.07d 1.61 ± 0.29c
3 5.35 ± 0.46d 0.96 ± 0.01cde 0.49 ± 0.06abc 2.51 ± 0.11de
15% WS 1 1.36 ± 0.21b 1.00 ± 0.00g 0.87 ± 0.01e 1.19 ± 0.19b
2 5.79 ± 0.50d 0.96 ± 0.01bcde 0.53 ± 0.06bcd 2.96 ± 0.47ef
3 5.29 ± 0.31d 0.94 ± 0.02b 0.42 ± 0.06a 2.11 ± 0.36d
20% WS 1 0.87 ± 0.10a 1.00 ± 0.00g 0.88 ± 0.02e 0.77 ± 0.09a
2 6.43 ± 0.37de 0.96 ± 0.01bcde 0.55 ± 0.07cd 3.40 ± 0.55fg
3 5.34 ± 0.58d 0.95 ± 0.00bcd 0.46 ± 0.05ab 2.29 ± 0.10d
25% WS 3 11.81 ± 1.90i 0.93 ± 0.02a 0.47 ± 0.06ab 5.08 ± 1.00hi
2 7.43 ± 0.97ef 0.95 ± 0.02bc 0.60 ± 0.05d 4.23 ± 0.58gh

Two-way ANOVA-p
Factor A (level) < 0.001 < 0.001 0.291 < 0.001
Factor B (time) < 0.001 < 0.001 < 0.001 < 0.001
Factor A× Factor B < 0.001 0.260 0.863 < 0.001

Mean value of two replication ± standard deviation.

Mean values signed this same letters in particular columns are non-significant different at 0.05 level of confidence.

applied to monitor the extent of bread staling. The results of analyses
performed on 3 consecutive days of storage were given in Table 3. It
could be observed that both the level of WS addition and the time of
storage, as well as the interactions between both factors significantly
affected bread hardness. On the day of baking control bread had the
hardest crumb, but the smallest addition of WS (5%) did not cause a
significant change in this parameter. Larger amounts of WS resulted in a
significant decrease in crumb hardness. On the second day of storage
the smallest hardness could be noticed in the case of bread with 10%
WS, while higher levels of WS addition caused an increase in this
parameter, though not as large as in the case of control. On the third
day of baking the softest crumb was observed in bread with 10–20%
addition of WS, while significant increase in hardness could be found
for 25% level. The results are in general agreement with other reports.
Bhattacharya et al. (2002) observed lower firmness during 5 days of
storage of wheat bread with optimum 20% share of waxy durum wheat
flour. Also Purhagen et al. (2011) found a decrease in firmness and
water loss in wheat bread containing 10% waxy wheat flour.
Jongsutjarittam and Charoenrein (2014) found that the addition of
waxy wheat flour maintains the moistness and softness of bread after
storage at room or refrigerated temperature. On the other hand Ghiasi
et al. (1984) reported that the change in amylose to amylopectin ratio,
caused by addition of waxy barley starch to high-protein wheat flour,
resulted in a decrease in crumb hardness but only on the first day after
baking. Springiness was the second analysed texture parameter
(Table 3). The applied two way analysis of variance proved a significant
influence of both the level of WS and the time of bread storage on
springiness, but no significant influence of interactions between both
these factors could be found. In all cases crumb springiness decreased
over time, which was negatively correlated with the observed increase
in hardness. The changes in springiness measured for samples with WS
at levels 5–10% were similar to control, but above 15% a decrease of
this parameter below the value of control sample could be observed.
The smallest springiness was measured for bread with the highest share
of WS on the third day of storage, which also displayed the highest
hardness (Table 3). A significant linear correlation between bread vo-
lume and its springiness on consecutive days of storage could be found
(r = 0.89; 0.93; 0.81, respectively). In the case of cohesiveness, time of
storage was the only significant factor, causing its decrease. However
the largest cohesiveness, comparable with control sample could be
detected in the case of 10% addition of WS. The last analysed texture
parameter was chewiness. Both the level of waxy starch and the time of
storage, and the interactions between these factors significantly af-
fected this parameter. In all cases chewiness increased during storage.
The lowest values on the first day were measured for samples with 10,
20 and 25% share of WS. On the following days, the chewiness in-
creased, and the smallest chewiness on the second and third day of
storage was observed for bread with 10–15% share of WS. Textural
characteristics of bread during storage indicates, that the level of about
10% WS positively influences stability and texture of analysed bread,
but larger amounts of its addition (> 15%) could deteriorate it. These
observations seem to confirm earlier findings. Jongsutjarittam and
Charoenrein (2013) demonstrated that waxy rice flour can be used as a
substitute for wheat flour at a level of 10% in freeze-thawed cake, but
higher amounts of substitution (> 15%) diluted the gluten, and the
cake had a gummy layer and collapsed upon cooling.
3.4. Thermal properties
Thermograms registered during heating of bread crumb reveal the
presence of a peak characteristic for decomposition of recrystallized
amylopectin, and its area could be regarded as enthalpy of this process.
The results indicate that the level of WS, time of storage and interac-
tions between both these factors had significant influence on transition
enthalpy. Directly after baking all bread samples in which part of starch
mix was replaced with WS were characterized by lower enthalpy than
control (Fig. 3), which correlated to the values of crumb hardness
(Table 3). Nevertheless the level of WS addition was not significant.
Similarly on the second day of storage, bread with WS revealed smaller
enthalpy in comparison to control sample, with an exception of the
sample with the highest content of WS. More obvious differences could
be found on the third day after baking. The largest enthalpy were
measured for bread with 5% share of WS, and the smallest with 10%.
These observations indicate, that the presence of WS starch in gluten-

480
M. Witczak et al.
Fig. 3. Effect of storage time on melting enthalpy of retrograded amylopectin in
gluten-free bread with waxy starch (WS): day 1, day 2, day 3.
free formulation causes a reduction of amylopectin recrystallization,
and thus bread staling. Moreover, the results of thermal analysis sug-
gest, that the optimum WS content is 10%. The results are in agreement
with earlier reports. Bhattacharya et al. (2002) observed a decrease in
enthalpy of decomposition of retrograded amylopectin with rising le-
vels of waxy durum wheat flour in wheat bread in comparison sample.
The authors stated, that 20% addition of flour was optimum, but this
discrepancy could only be apparent, because of the presence of non-
starch constituents, which might have additional effects on staling.
Although amylopectin retrogradation is not the only factor affecting
bread ageing, including the increase of its hardness, high linear corre-
lation between bread hardness and enthalpy of retrograded amylo-
pectin decomposition could be found (r = 0.81).
4. Conclusion
The results show that waxy starch could be used in order to modify
texture and extend shelf life of gluten-free bread based on a formulation
consisting potato and corn starch. Bread with 10% share of WS ex-
hibited the largest volume and only slight changes in crumb structure in
comparison to control formulation. The application of WS significantly
affected bread texture, namely its hardness, springiness and chewiness.
On the day of baking bread containing WS revealed lower hardness in
comparison to control, and its limited increase during storage could be
attributed to the introduction of WS, especially at levels 10–15%. Bread
with 10% share of WS revealed high springiness and the lowest che-
winess, which increased over storage to the least extent. The partici-
pation of WS in bread formulation caused a reduction in enthalpy of
retrograded amylopectin decomposition, and the smallest value of this
parameter on the third day of storage was measured for bread with 10%
WS. The results suggest that the optimum level of WS, in this case 10%,
could effectively improve quality of gluten-free bread. On the other
hand the excessive (25%) level of waxy starch in bread formulation
significantly reduces its volume and intensifies staling.
The mechanism of waxy starch on staling retardation in gluten free
bread seems to be similar to wheat bread. The key role is played by
short-chain branched molecules of amylopectin, which are less likely to
associate and form crystalline structures than linear chains. However
the results show that the excess of waxy starch could negatively impact
texture and physical properties of bread crumb. Some differences be-
tween gluten-free and wheat based bread could be expected on mole-
cular level. Wheat bread structure strongly depends on the presences of
gluten, and gluten-starch interactions are thought to be the major factor
in its staling. In gluten free bread, especially deprived of protein, the
481
LWT - Food Science and Technology 99 (2019) 476–482
primary role in staling should be attributed to the interactions between
swollen starch granules and dissolved starch polymers, probably with
some side effects caused by the presence of non starch hydrocolloids, in
this case pectin and guar gum.
Acknowledgement
This research was financed by the Ministry of Science and Higher
Education of the Republic of Poland.
References
Abramoff, M. D., Magelhaes, P. J., & Ram, S. J. (2004). Image processing with ImageJ.
Biophotonics International, 11, 36–42.
Bhattacharya, M., Erazo-Castrejón, S. V., Doehlert, D. C., & McMullen, M. S. (2002).
Staling of bread as affected by waxy wheat flour blends. Cereal Chemistry, 79(2),
178–182.
Bortnowska, G., Balejko, J., Tokarczyk, G., Romanowska-Osuch, A., & Krzemińska, N.
(2014). Effects of pregelatinized waxy maize starch on the physicochemical proper-
ties and stability of model low-fat oil-in-water food emulsions. Food Hydrocolloids, 36,
229–237.
Gao, Y., Janes, M. E., Chaiya, B., Brennan, M. A., Brennan, C. S., & Prinyawiwatkul, W.
(2018). Gluten-free bakery and pasta products: Prevalence and quality improvement.
International Journal of Food Science and Technology, 53, 19–32.
Garzόn, R., Rosell, C. M., Malvar, R. A., & Revilla, P. (2017). Diversity among maize
populations from Spain and the United States for dough rheology and gluten-free
breadmaking performance. International Journal of Food Science and Technology, 52,
1000–1008.
Ghiasi, K., Hoseney, R. C., Zeleznak, K., & Rogers, D. E. (1984). Effect of waxy barley
starch and reheationg on firmness of bread crumb. Cereal Chemistry, 61(4), 281–285.
Hibi, Y. (2001). Effect of retrograded waxy corn starch on bread staling. Starch Staerke,
53, 227–234.
Horstmann, S. W., Lynch, K. M., & Arendt, E. K. (2017). Starch characteristics linked to
gluten-free products. Foods, 6, 29.
Hruśková, M., Bednárová, M., & Novotný, F. (2001). Wheat flour dough rheological
characteristics predicted by NIR Systems 6500. Czech Journal of Food Sciences, 19,
213–218.
Jongsutjarittam, N., & Charoenrein, S. (2013). Influence of waxy rice flour substitution
for wheat flour on characteristics of batter and freeze-thawed cake. Carbohydrate
Polymers, 97, 306–314.
Jongsutjarittam, O., & Charoenrein, S. (2014). The effect of moisture content on physi-
cochemical properties of extruded waxy and non-waxy rice flour. Carbohydrate
Polymers, 114, 133–140.
Korus, J., Witczak, M., Ziobro, R., & Juszczak, L. (2015a). The influence of acorn flour on
rheological properties of gluten-free dough and physical characteristics of the bread.
European Food Research and Technology, 240(6), 1135–1143.
Korus, J., Witczak, T., Ziobro, R., & Juszczak, L. (2015b). Linseed (Linum usitatissimum L.)
mucilage as a novel structure forming agent in gluten-free bread. LWT-Food Science
and Technology, 62, 257–264.
Korus, J., Witczak, M., Ziobro, R., & Juszczak, L. (2017). Hemp (Cannabis sativa subsp.
sativa) flour and protein preparation as natural nutrients and structure forming agents
in starch based gluten-free bread. LWT-Food Science and Technology, 84, 143–150.
Krzemińska, N., & Bortnowska, G. (2017). Effects of native waxy maize starch on phy-
sicochemical properties of model sauces made using poultry meat. Food. Science.
Technology. Quality, 24(2), 40–50 111.
Melini, F., Melini, V., Luziatelli, F., & Ruzzi, M. (2017). Current and forward-looking
approaches to technological and nutritional improvements of gluten-free bread with
legume flours: A critical review. Comprehensive Reviews in Food Science and Food
Safety, 16, 1101–1122.
Naqash, F., Gani, A., Gani, A., & Masoodi, F. A. (2017). Gluten-free baking: Combating the
challenges - a review. Trends in Food Science & Technology, 66, 98–107.
Noznick, P. P., Merritt, P. P., & Geddes, W. F. (1946). Staling studies on breads containing
waxy maize starch. Cereal Chemistry, 23, 297–305.
Purhagen, J. K., Sjöö, M. E., & Eliasson, A. C. (2011). Starch affecting anti-staling agents
and their function in freestanding and pan-baked bread. Food Hydrocolloids, 25(7),
1656–1666.
Pycia, K., Juszczak, L., Gałkowska, D., & Witczak, M. (2012). Physicochemical properties
of starches obtained from Polish potato cultivars. Starch Staerke, 64(2), 105–114.
Šárka, E., & Dvořáček, V. (2017a). Waxy starch as a perspective raw material (a review).
Food Hydrocolloids, 69, 402–409.
Šárka, E., & Dvořáček, V. (2017b). New processing and applications of waxy starch (a
review). Journal of Food Engineering, 206, 77–87.
Segura, M. E. M., & Rosell, C. M. (2011). Chemical composition and starch digestibility of
different gluten-free breads. Plant Foods for Human Nutrition, 66(3), 224–230.
Witczak, M., Juszczak, L., Ziobro, R., & Korus, J. (2012). Influence of modified starches on
properties of gluten-free dough and bread. Part I: Rheological and thermal properties
of gluten-free dough. Food Hydrocolloids, 28(2), 353–360.
Witczak, T., Juszczak, L., Ziobro, R., & Korus, J. (2017). Rheology of gluten-free dough
and physical characteristics of bread with potato protein. Journal of Food Process
M. Witczak et al.
Engineering, 40(3), e12491.
Witczak, M., Ziobro, R., Juszczak, L., & Korus, J. (2016). Starch and starch derivatives in
gluten-free systems - a review. Journal of Cereal Science, 67, 46–57.
Zhang, D., Mu, T., & Sun, H. (2017). Comparative study of the effect of starches from five
different sources on the rheological properties of gluten-free model doughs.
Carbohydrate Polymers, 176, 345–355.
482
LWT - Food Science and Technology 99 (2019) 476–482
Ziobro, R., Juszczak, L., Witczak, M., & Korus, J. (2016). Non-gluten proteins as structure
forming agents in gluten free bread. Journal of Food Science & Technology, 53(1),
571–580.
Ziobro, R., Korus, J., Witczak, M., & Juszczak, L. (2012). Influence of modified starches on
properties of gluten-free dough and bread. Part II: Quality and staling of gluten-free
bread. Food Hydrocolloids, 29(1), 68–74.