LWT - Food Science and Technology 151 (2021) 112156

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LWT
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Psyllium as an improver in gluten-free breads: Effect on volume, crumb

texture, moisture binding and staling kinetics
Bojana Filipčev , Milica Pojić *, Olivera Šimurina , Aleksandra Mišan , Anamarija Mandić
Institute of Food Technology, University of Novi Sad, Bulevar cara Lazara 1, 21000, Novi Sad, Serbia

A R T I C L E I N F O A B S T R A C T

Keywords: The effect of replacing commonly used hydrocolloids (i.e. xanthan (XTH), guar gum (GG), hydrox­
Gluten-free bread ymethylcellulose (HPMC)) with psyllium on crumb texture, distribution of water populations and staling kinetics
Staling in gluten-free (GF) bread based on a blend of buckwheat and carob flour was studied. The addition of psyllium in
Thermogravimetric analysis
GF formulation yielded bread with the softest and most resilient crumb. Psyllium was an effective anti-staling
Avrami model
agent which significantly decreased the crumb hardening rate. The GF breads with psyllium and the hydrocol­
loids showed similar distribution of water populations, though psyllium tended to slightly increase their binding
strength to the matrix. In the buckwheat/carob-based GF system, crumb firmness development was directly
correlated to the amount of easily removable water population and inversely to the peak temperatures of strongly
bound water which indicate higher bonding strength to the matrix. The results implied that psyllium and the
tested hydrocolloids (i.e. XTH, GG, HPMC) can be interchangeably used to effectively control crumb texture and
its evolution during storage of buckwheat/carob GF bread.

1. Introduction
The main challenges in gluten-free (GF) bread production are related
to overcoming their inferior structure, texture, shelf-life, sensory and
nutritional quality. These problems arise due to being based on starchy
ingredients and GF flours and the absence of raw materials that would be
able to adequately simulate the structure-building role of gluten. Many
approaches have been used to overcome these issues which include the
quest for novel ingredients, additives, process conditions and techno­
logical alternatives (Capriles & Arěas, 2014).
Among the alternative GF flours with high nutritional value, buck­
wheat and carob flours emerge as promising ingredients. Several studies
reported on their suitability in breadmaking (Miñarro, Albanell, Aguilar,
Guamis, & Capellas, 2012; Tömösközi & Langó, 2017; Tsatsaragkou
et al., 2012; Tsatsaragkou, Gounaropoulos, & Mandala, 2014; Turfani,
Narducci, Durazzo, Galli, & Carcea, 2017). The studies revealed the
enhancing effect of buckwheat on viscoelastic properties of GF batter
based on starch and/or GF flour (rice flour, corn/potato starch) that
resulted in higher loaf specific volume and crumb softness (Alvar­
ez-Jubete, Auty, Arendt, & Gallagher, 2010; Wronkowska, Troszyńska,
Soral-Śmietana, & Wolejszo, 2008). It was hypothesized that the positive
effects could be attributed to presence of natural emulsifiers (polar
lipids) that stabilise gas bubbles prior gelatinization during breadmak­
ing (Alvarez-Jubete, Auty, Arendt, & Gallagher, 2010). An observed
delay in GF bread staling upon buckwheat supplementation could be
also associated with the presence of emulsifiers that form complexes
with amylose and limit starch swelling, leaching and retrogradation
(Wronkowska, Haros, & Soral-Śmietana, 2013). Moreover, buckwheat
addition enhanced the nutritive profile of GF breads by increasing the
content of microelements and proteins (Krupa-Kozak, Wronkowska, &
Soral-Śmietana, 2011), total phenolics and antioxidants (Sakač, Torbica,
Sedej, & Hadnađev, 2011) and resistant starch (Wronkowska et al.,
2008). Carob flour is also a highly nutrious ingredient that contains
lysine-rich proteins, minerals, dietary fibres, lignans and phenolics
(Tsatsaragkou et al., 2012). Carob flour was found to improve the
quality of GF bread based on rice flour (Tsatsaragkou et al., 2012). Good
breadmaking performance of carob flour can be associated to the pres­
ence of polysaccharide galactomannan (locust bean gum) and protein
caroubin.
During storage, GF bread undergoes rapid staling compared to con­
ventional breads. GF bread crumb staling is manifested by the deterio­
ration of the bread sensory properties – structural and textural
properties, aroma and taste (Arslan, Rakha, Xiaobo, & Mahmood, 2019).
Although the staling mechanism has not been completely understood

* Corresponding author.
E-mail address: milica.pojic@fins.uns.ac.rs (M. Pojić).

https://doi.org/10.1016/j.lwt.2021.112156
Received 13 May 2021; Received in revised form 13 July 2021; Accepted 14 July 2021
Available online 15 July 2021
0023-6438/© 2021 Elsevier Ltd. All rights reserved.
B. Filipčev et al.
due to its complexity, it has been related to changes in starch fraction
(formation of rigid, crystalline starch network due to amylopectin
retrogradation), and in gluten network (dehydration of gluten network
due to moisture migration) (Gray & Bemiller, 2003). Moisture loss
additionally aggravates the undesirable quality changes but does not
affect the onset of starch retrogradation. Staling in GF bread is essen­
tially associated with starch retrogradation (Schober, 2009). Hager,
Bosmans, and Delcour (2014) suggested that amylopectin retrograda­
tion and water migration to retrograded starch region are main con­
tributors to staling in oat and rice GF bread. Masure, Fierens, and
Delcour (2016) studied the anti-staling effect of buckwheat flour in
substituted starch-based GF bread and observed that amylopectin
retrogradation enthalpy did not change over 6-day storage but there
were significant differences in the distribution of water, emphasizing the
importance of water redistribution in the staling mechanism of GF
bread. Water transfers in various directions seem to be an important
factor in staling mechanism of both glutenous and non-glutenous
matrices (water loss, crumb-to-crust migration, redistribution at mo­
lecular level between and among various regions of bread components)
but the exact role remains unrevealed (Choi et al., 2008; Gray &
Bemiller, 2003). The staling rate is dependent on the amount of free and
bound water - a rapid staling rate related to water release at about 70 ◦ C,
and slower staling rate related to water release at 90 ◦ C associated to a
maximum in the crumb elastic modulus as well as a maximum water
activity value (Schiraldi & Fessas, 2018). Deconvolution of first deriv­
ative data of thermogravimetric (TG) traces has been found useful in
discriminating and quantifying different water populations. According
to Fessas and Schiraldi (2005), these populations are characterized by
variable binding strengths to the macromolecular matrix of a sample,
which is consequently reflected in different kinematic energy states of
water in the sample. Weakly bound water populations evaporate at
lower temperature ranges, whereas strongly bound populations evapo­
rate at higher temperatures. The area under each peak of the deconvo­
luted curve is proportional to the amount of water in each population
(Schiraldi, Piazza, & Riva, 1996).
Although a number of studies utilized thermogravimetric analysis
(TGA) to study the water distribution in wheat dough, bread and water-
flour slurries (Dimitrios Fessas & Schiraldi, 2001, 2005; Lapčík, Vašina,
Lapčíková, & Valenta, 2016; Liu et al., 2014; Lodi & Vodovotz, 2008;
Simmons, Smith, & Vodovotz, 2012), research on thermogravimetric
behaviour of GF systems is scarce. Witek, Maciejaszek, and Surówka
(2020) utilized TGA to perceive the effects of GF pasta enrichment with
freeze-dried egg white, yolk and whole egg on water behaviour in fresh
pasta dough, dried and cooked rice pasta. The potential of TGA to
perceive the roles of polysaccharides and proteins of buckwheat flour
was demonstrated by Fessas et al. (2008), while Crockett, Ie, and
Vodovotz (2011) utilized the same methodology to monitor the water
status in a model GF dough containing xanthan and hydroxypropyl
methylcellulose.
In practice, the most successful way to delay staling is the usage of
additives such as hydrocolloids, emulsifiers, enzymes etc. (Schober,
2009). Most commonly GF breads and bakery products contain guar
gum (E412), xanthan gum (E415), hydroxypropylcellulose (E463),
hydroxypropylmethylcellulose (E464), but also other types of gums, for
example locust bean gum (E410) (Salehi, 2019). The anti-staling effects
of hydrocolloids are associated with their ability to retain water and
limit its redistribution in starchy matrices (Gray & Bemiller, 2003;
Sozer, Bruins, Dietzel, Franke, & Kokini, 2011) but the overall perfor­
mance is dependent on type, source and molecular weight of hydro­
colloids (Guarda, Rosell, Benedito, & Galotto, 2004; Gujral, Haros, &
Rosell, 2004). There is a lack of comprehensive research works on the
mechanisms of GF crumb staling and impact of additives and ingredients
(Masure, Wouters, Fierens, & Delcour, 2019).
Dietary fibres are other ingredients that may positively influence GF
bread quality and staling process. Tsatsaragkou, Protonotariou, and
Mandala (2016) reviewed different studies on the implementation of
2
LWT 151 (2021) 112156
fibres from various sources and of various forms in GF breads and
summarized that a delay in starch retrogradation and staling in GF
breads was exerted by isolated flour dietary fibres, soluble isolated fibres
and resistant starch (Tsatsaragkou et al., 2016). High water binding
capacity of fibres and their possible hydrogen bonding to starch may be
involved in the mechanism of delaying staling in GF crumb. The
anti-staling effect of fibres depends on the ratio of soluble and insoluble
fibres. Soluble fibres seem to have better ability to delay staling by
dissolving in the aqueous part of dough, enfolding starch granules thus
decreasing the amount of water absorbed and consequently the degree
of starch gelatinization/retrogradation (Tsatsaragkou et al., 2016). One
of the emerging dietary fibre source for GF bread is psyllium – Plantago
ovata seed husk, which can also be used as a hydrocolloid replacer due to
water-binding/holding, gel-forming and structure-building properties
(Cappa et al., 2013; Mancebo, San Miguel, Martínez, & Gómez, 2015;
Fratelli, Muniz, Santos, & Capriles, 2018; Santos, Aguiar, Centeno,
Rosell, & Capriles, 2020; Ziemichód, Wójcik, & Różyło, 2019). GF baked
products formulated with psyllium as a structural agent received good
sensory acceptance from both celiac and non-celiac individuals (Zan­
donadi, Botelho, & Araújo, 2009). The studies implemented wide range
of psyllium levels (1.5–2.5%, 3%, 4%, 4.5–12.5%, 2.86–17.14%) in
complex GF formulations with very different ingredient composition
(combined starch and GF flours-based formulations, with or without
eggs, with or without chick pea flour, in combination with one or more
hydrocolloids or fibres, enzymes, dairy ingredients, oil, etc.). Exception
is the study of Belorio and Gómez (2020) which elaborated the func­
tionality of psyllium as a stand-alone improver at 2% level in rice flour i.
e. maize starch-based GF bread. Apart from being able to improve GF
dough handling during processing and overcome most quality de­
ficiencies of GF breads, psyllium offers additional nutritional and health
benefits that arise from its nature as a bioactive soluble fibre. The ben­
efits include elevation of fibre content in the baked product, alleviation
of gastro-intestinal and diabetic symptoms, facilitation of cholesterol
and satiety control. The objective of this study was to 1) evaluate the
potential of psyllium husk as an improver and anti-staling agent in GF
bread which is not based on starchy ingredients but on buckwheat/carob
flour blend and 2) to compare the effects of psyllium with commonly
used hydrocolloids (guar gum, xanthan gum and
hydroxy-propyl-methyl-cellulose) in the buckwheat/carob matrix. The
performance of these compounds was evaluated on crumb texture,
moisture content and water population distribution as well as on crumb
staling kinetics during 10 days of storage. Comparison was made to the
bread baked from a commercial mixture, as a representative of an
equivalent product type at disposal for every-day nutrition of consumers
adhering GF diet. It was considered important to simultaneously observe
the quality changes during storage of experimental and commercial
bread despite the differences in their formulations because there is little
observation in the literature related to the behaviour of commercial GF
breads and their comparison with research breads (Roman, Belorio, &
Gómez, 2019).
2. Materials and methods
2.1. Materials
The materials used in this study were buckwheat flour (Hemija
Commerce, Novi Sad, Serbia), carob flour (Aroma začini, Futog, Serbia),
psyllium husk (Beyond, Niš, Serbia), guar gum, xanthan and hydrox­
ypropylmethylcellulose (all purchased from LG Hemija, Belgrade,
Serbia), dry yeast, margarine, sugar and salt (all purchased in a local
supermarket), Na-propionate (purchased from Puratos, Belgrade,
Serbia) and commercial GF mixture comprising maize starch, millet and
rice flour, guar gum and psyllium (Aleksandrija, Čurug, Serbia).
B. Filipčev et al. LWT 151 (2021) 112156

2.2. Bread making procedure packed in polyethylene foil and kept on room temperature. The breads
contained Na-propionate to prevent moulding during storage. Crumb
Four GF bread were prepared by mixing wholegrain buckwheat/ firmness and resilience were measured at 0, 1, 3, 5, 7 and 10 days of
carob flour blend (90.7 g/100 g:7.3 g/100 g) with psyllium husk (2 g/ storage, and obtained data were fitted to the Avrami’s non-linear
100 g) (formulation PSY), guar gum (0.5 g/100 g) (formulation GG), regression equation (Angioloni & Collar, 2009; Armero & Collar,
xanthan (0.5 g/100 g) (formulation XTH) and HPMC (0.5 g/100 g) 1996). Each crumb attribute was fitted to two Avrami models: a general
(formulation HPMC), and 2 g/100 g margarine, 2 g/100 g dry yeast, 2 g/ model with estimated n and k parameters and a restricted model with n
100 g salt, and 1 g/100 g sugar, based on flour weight. Water was added fixed to 1. The curve fitting was performed by Origin 8 Lab software
at constant level of 149.6 g/100 g to obtain batter-like dough. The water (OriginLab Corporation).
level for commercial GF mixture (formulation COM) was 100%, as
recommended by the manufacturer. The level of psyllium (2 g/100 g
flour basis) in PSY bread formulation was chosen on the basis of pre­ 2.6. Statistical analysis
liminary experimental design which showed that 2 g/100 g psyllium
level was optimal for GF bread based on buckwheat/carob blend. The Statistical analysis was performed by one-way analysis of variance
selected hydrocolloids used instead of psyllium in formulations GG, XTH (ANOVA) followed by Tukey’s post-hoc test (Statistica 13, TIBCO, USA).
and HPMC were applied at 0.5 g/100 g level following manufacturer’s Pearson coefficient of correlation was used to calculate correlation
recommendations. coefficients.
All dry ingredients were premixed before water addition for 1 min at
low speed using a hand mixer (Gorenje, Slovenia). After water and 3. Results and discussion
margarine addition, mixing was continued for 5 min at the same speed.
Batters were divided into portions of 50 g and placed into a muffin pan 3.1. Characterization of GF breads
with round cups (16 cups) and proofed for 50 min (30 ◦ C, 85% RH). The
breads were baked for 30 min at 230 ◦ C in a deck oven (MIWE, Ger­ 3.1.1. Specific volume and crumb texture
many) with steaming. Baked breads were cooled to ambient temperature GF breads within the current study were developed on the basis of
and packed in polyethylene foil. From every dough type, 3 sets, each batter-like dough from buckwheat and carob flour. Due to the absence of
containing 16 loaves were baked. One set of experimental breads was gluten-forming proteins, buckwheat flours are not able to yield visco­
immediately analysed 2 h after baking while the rest of the samples were elastic dough that can expand and retain gas. Despite the lack of struc­
left for storage analysis. Storage regime is explained in detail below, tural proteins, substitution of starch with buckwheat flour positively
section 2.5. influenced GF bread volume and crumb texture observed as decreased
crumb firmness and delayed staling (Wronkowska et al., 2013). On the
2.3. Bread quality other hand, suitability of carob flour is associated to the water-insoluble
protein caroubin, able to form fibrillar structures and sheets when hy­
2.3.1. Bread volume drated, exhibiting the similar rheological properties to gluten (Wang
Specific bread volume was recorded using a Volscan Profiler (Stable et al., 2001).
Micro Systems, UK). The measurements were run on at least 5 loaves per Specific volume and crumb textural properties determined 2 h after
type of bread. baking of different GF formulations are presented in Table 1. Formula­
tions XTH and GG exhibited significantly higher volume, followed by
2.3.2. Crumb texture PSY, COM and HPMC bread. Loaf volumes of COM and HPMC were
Bread crumb texture (firmness, resilience) was determined using a significantly lower whereas PSY volume significantly differed only from
texture analyzer TA.XTplus (Stable Micro Systems, UK) equipped with a that of XTH. The crumb hardness was the highest for HPMC that had
25.4 mm stainless probe according to AACC 74-10.02 (AACC Interna­ significantly firmer crumb in comparison to other formulations except
tional 2010). The reported results are average of 5 measurements. COM, but with better resilience than COM, comparable to those of GG
and XTH. PSY was characterized with significantly highest crumb
2.3.3. Moisture content and water activity resilience and the lowest crumb hardness, although significant differ­
Moisture content was obtained from TGA data, while water activity ence in latter was reached only in comparison to HPMC. The positive
(aw) was measured using an Aw-meter Testo 650 (Testo AG, Germany) textural properties of PSY (highest crumb resilience and low crumb
at 24 ◦ C. Determinations were conducted in triplicates.
Table 1
2.4. Thermogravimetric analysis (TGA) Specific volume and crumb textural attributes determined 2 h after baking of
different GF formulations.
Bread crumb samples (≈0.80 g) were heated from ambient temper­ Formulation Moisture Aw value Specific Hardness Resilience
ature (≈25 ◦ C) to 180 ◦ C using a TG analyzer TGA701 LECO (Leco content (g/ volume (N) (%)
100 g) (l/kg)
Corporation, Michigan, US) in dynamic air atmosphere at the rate 1 ◦ C/
min and held isothermally for 5 min. The resulting TGA trace of mass PSY 56.72 ± 0.960 ± 2.0 ± 2.97 ± 46.40 ±
loss, attributed to water loss, and its first derivative (DTG) were 1.13b 0.0100c 0.064a,b 0.30a 1.25c
COM 44.25 ± 0.935 ± 1.97 ± 3.29 ± 32.99 ±
considered for the analysis. The DTG curve was deconvoluted to a sum of 0.94a 0.0110a 0.043a 0.28a,b 1.39a
3 Gaussian peak functions to determine the presence of water pop­ GG 53.86 ± 0.9476 2.18 ± 3.07 ± 39.27 ±
ulations in the sample and the evaporation temperature range of each 0.96a,b ± 0.103b,c 0.30a 1.82b
water population. Deconvolution of DTG data was performed using 0.0070b
XTH 56.82 ± 0.9602 2.25 ± 3.05 ± 38.02 ±
Origin 8 Lab software (OriginLab Corporation). The reported measure­
0.84b ± 0.058c 0.29a 1.58b
ments are average of duplicate measurements on crumb portions ob­ 0.0070c
tained from the centre of at least 2 loaves of bread per type. HPMC 55.35 ± 0.9568 1.92 ± 4.01 ± 36.50 ±
0.65b ± 0.062a 0.32b 1.11a,b
2.5. Storage study and Avrami modelling 0.0030b,c
a,b,c
Means denoted with different letters within a column are significantly
A storage study was conducted over 10 days. The samples were different (p < 0.05, HSD Tukey’s test).

3
B. Filipčev et al.
hardness) can be associated to the presence of psyllium fibres in the
formulation and their ability to form films, bind and hold water
throughout the baking process (Cappa, Lucisano, & Mariotti, 2013a).
Better textural properties may also be related to the improvement of the
volume of the breads. Fratelli et al. (2021) reported significant
improvement in the specific volume and crumb texture with increasing
levels of psyllium in GF breads based on rice flour and cassava starch and
observed significant negative correlations between specific volume and
crumb firmness in the GF breads showing that loaves with higher vol­
ume had softer and more porous crumb. This is contrasting to the
findings of Ren, Linter, Linforth, and Foster (2020), Santos et al. (2020)
and Mancebo et al. (2015) that evidenced a detrimental effect of psyl­
lium on specific volume and hardness of GF breads. However, Mancebo
et al. (2015) noticed that the negative effect of psyllium on volume was
lessened when hydration levels increased. Indeed, Cappa et al. (2013a)
pinpointed that psyllium inclusion to GF bread formulation should be
carefully adjusted in order to fully benefit from its functionalities
because inadequate combination of psyllium and hydration levels may
lead to development of excessively hard crumb. Santos et al. (2020)
commented that the observed detrimental effect of psyllium on specific
volume and firmness may be related to fixed dough hydration which
limited the water plasticizing effect. The study of Belorio and Gómez
(2020) also confirmed the importance of hydration optimization in GF
bread made with psyllium husk and hydrocolloids to achieve high spe­
cific volumes. They noted a similar behaviour of psyllium and xanthan
in GF matrices based on rice flour and maize starch in relation to specific
volume and loaf weight loss. Their study revealed that the dependence
on hydration level was more evident in GF breads with HPMC than in
those with psyllium and xanthan. Ren et al. (2020) emphasized the
complexity of influences between hydrocolloids (psyllium, methylcel­
lulose) and water amounts on loaf volume and crumb texture. They
observed that higher hydration levels may not necessarily lead to higher
specific volumes due to complicated interaction of many factors such as
water absorbability of ingredients, effect of hydrocolloids on pasting
viscosity, dough stability, gas trapping ability and void formation. On
the other hand, after compiling the results from various other studies,
Matos and Rosell (2015) concluded that low dough consistency and low
gelling values were correlated to development of soft GF crumb but
could not correlate visco-elastic moduli of GF doughs with specific
volume and crumb texture.
The above mentioned studies all agree that psyllium is the ingredient
with significant influence on GF bread development, which effect is
dependent on the water content, psyllium-water interaction and its
strong ability to form complexes with systems proteins through ionic
and nonionic interactions, thus affecting dough strength and conse­
quently crumb texture (Pejcz, Spychaj, Wojciechowicz-Budzisz, & Gil,
2018; Santos et al., 2020). Therefore, making comparison with the re­
sults of other authors that utilized psyllium as GF ingredient is difficult,
since those studies differ in the amount of used water, the type of main
GF flour ingredient, the amount of psyllium and breadmaking conditions
(bread shape and dimensions).
3.1.2. Moisture content and water activity (Aw)
Moisture content of bread is an important quality indicator which
impacts its physical, microbial and sensory properties. The fixed hy­
dration was chosen to be able to recognize the cause of improvement
effect on staling properties (water content or the ingredients) as sug­
gested by Santos et al. (2020). The moisture content of buckwheat-carob
breads (Table 1) was high and ranged over 53.86 ± 0.96 g/100 g
(GG)-56.82 ± 0.84 g/100 g (XTH). XTH and PSY breads were amongst
the highest in moisture content, followed by HPMC bread although
significant difference among them did not exist. However, the COM
bread was significantly lower in moisture (44.25 ± 0.94 g/100 g). This
result is expected since the buckwheat/carob breads were mixed with
higher and fixed amount of water than the bread made from the com­
mercial GF mixture. Similarly distributed were water activities (Aw)
4
LWT 151 (2021) 112156
which ranged from ≈0.9600 ± 0.003 for PSY and XTH to 0.9476 ±
0.007 for GG, whereas COM was significantly the lowest in Aw (0.9348
± 0.011). PSY, XTH and HPMC did not significantly differ regarding Aw
value.
3.1.3. Water distribution by thermogravimetric analysis (TGA)
Deconvolution with 3 Gaussian peaks of the TGA mass loss first de­
rivative data (DTG) was performed on the GF bread samples (PSY, COM,
GG, XTH, HPMC) to differentiate water populations (the examples are
included in Fig. 1). The temperature at peak maximum and area under
the peak are presented in Fig. 2 (A, B), respectively. As it can be seen, the
obtained DTG records for GF breads show a single broad peak which is a
characteristic trace for matrices without gluten (see Fig. 1). Earlier
findings for DTG traces of doughs prepared from various GF ingredients
(cereals, pseudocereals and legumes) showed similar curve shapes
which reflected easy migration of water from core to surface due to
absence of real competition for water between starch polymers and non-
gluten proteins (Fessas et al., 2008). Most of the water present in the
developed GF breads (up to ≈90 g/100 g) evaporated at temperatures
below 100 ◦ C indicating weakly bound water (two populations near
75 ◦ C (W1) and 90–95 ◦ C (W2)) (Fig. 2). At temperatures over 100 ◦ C (i.
e. 100-107 ◦ C), more tightly bound water evaporated (W3), accounting
for a portion of around 10 g/100 g of total water. In GF systems based on
starch, usually the presence of two water populations was observed
(Fessas et al., 2008; Dimitrios Fessas & Schiraldi, 2001) that evaporate at
temperatures below 100 ◦ C. These can be attributed to easily removable
water that diffuses through starch gel and water associated with
non-gluten proteins undergoing reticulation (Fessas et al., 2008).
However, the presence of water-binding agents in GF systems may lead
to the formation of third water subspecies as demonstrated by Crockett
et al. (2011), who reported a third water population associated with
HPMC, soy protein isolate or egg white added to cassava starch-rice flour
based doughs. The third water population can be explained by the
presence of fiber-rich constituents in the GF formulations capable of
altering the distribution of free and bound water in the system as
demonstrated by Roozendaal, Abu-hardan, and Frazier (2012) who
showed a presence of uniform multiple peaks after deconvolution of
DTG traces associated with 100% bran. In our samples, the presence of
third water population can be attributed to the presence of either psyl­
lium or hydrocolloids (xanthan, guar gum, HPMC).
The DTG records demonstrated that there was a similar temperature
distribution for peaks for all samples in the case of water populations up
to 95 ◦ C (W1+W2) (Fig. 2). For the third water population (W3), GF
buckwheat/carob breads (PSY, GG, XTH, HPMC) exhibited more clus­
tered at higher temperature (>103 ◦ C) than the bread made of the
commercial GF mixture (COM) (99.8 ◦ C), showing their ability to better
entrap water (Fig. 2A). The amount of water contributing to each water
population is displayed as % of total area (Fig. 2B). The first peak water
contribution (W1) was the greatest for sample COM, followed by HPMC,
PSY, XTH, and GG, but significant difference existed only between COM
(64.6%) and GG (58.6%). Water in the middle peak temperature (W2)
was the lowest for sample COM (23.1%) while the strongest bound water
population (W3) was mostly similarly distributed across the samples
(between 8 and 12%) except for GG and COM that had significantly
higher third water contribution (12% and ≈17%, respectively). Meng
and Kim (2020) observed that addition of various hydrocolloids to GF
high-moisture rice fermented cake lowered the water 3 content. By
contrast, in cassava starch-based GF dough, addition of HPMC or XTH
caused changes in the distribution of water populations by increasing
the amount of more physically entrapped water (W2 and W3) (Crockett
et al., 2011).
The distribution of water populations showed that COM and GG
bread had weaker water association owing to lower peak temperatures
for all three water populations. In addition, COM lost the highest
amount of water evaporating at low temperatures (W1), showing that
hydrophilic structures of COM batter formed the weakest COM network
B. Filipčev et al. LWT 151 (2021) 112156

Fig. 1. Examples of DTG thermographs of GF breads at day 1 of storage. A) GF bread with psyllium husk (PSY); B) Commercial GF bread (COM); C) GF bread with
hydrocolloid (GG).

Fig. 2. (A) Peak temperatures of different water populations (W1, W2, W3) and (B) percent of total area under the curve of peaks obtained by deconvolution of DTG
data into 3 Gaussian distributions. PSY is GF bread with psyllium husk; COM is GF bread made of commercial GF mixture; GG is GF with guar gum; XTH is GF bread
with xanthan; HPMC is GF bread with HPMC. Error bars represent ±1 standard deviation (sample size (n) = 2 for each bread type).

from which water freely migrated.
3.2. Storage study
3.2.1. Changes in water content and Aw during storage
Fig. 3 shows the changes in moisture content and water activity (Aw)
of bread crumb during storage over 10 days. According to expectations,
both moisture and Aw of the samples gradually decreased during stor­
age. After 10 days of storage, the highest ability to retain moisture was
observed for GF bread formulated with psyllium husk (PSY), followed by
XTH and GG. The highest loss of moisture was recorded for COM (4.8 g/
100 g) and HPMC (4 g/100 g), whereas it ranged from 1.5 to 3.5 g/100 g
for the rest of bread samples. By the end of storage moisture content
followed the order PSY>XTH>GG>HPMC>COM. It seems that the
highest ability to retain moisture was exhibited by psyllium and
xanthan, while the performance of guar gum and HPMC was somewhat
weaker. Psyllium and xanthan had similar hydration properties with
prominent water binding capacities (WBC 4.82 g H2O/g and 4.98 H2O/
g, respectively) (Belorio, Marcondes, & Gómez, 2020) which by far
exceed the capacities of starch, sugar beet fibre and rice bran (Sánches,
Bartholomai, & Pilosof, 1995; Cappa et al., 2013a,b; Belorio et al.,
2020). Moreover, xanthan is known to be the best water binder among
commonly used food hydrocolloids such as guar gum, alginates and
locust bean gum (Sánches et al., 1995). In mixture with corn starch,
psyllium and xanthan positively affected the water absorption capacity
of starch in a similar way and promoted similar changes in gel rheology
(Belorio et al., 2020).
When Aw values of bread crumbs were compared after 10 days of
storage, only guar gum showed lower ability to hold water in compar­
ison to psyllium, xanthan and HPMC. The correlation coefficient

Fig. 3. Changes in moisture content (A) and water activity (B) of different GF bread formulations. PSY is GF bread with psyllium husk; COM is GF bread made of
commercial GF mixture; GG is GF bread with guar gum; XTH is GF bread with xanthan; HPMC is GF bread with HPMC. Error bars represent ±1 standard deviation
(sample size (n) = 3 for each bread type).

5
B. Filipčev et al.
between the moisture content and Aw values of the samples was r2Pearson
= 0.686 (p = 0.0002).
3.2.2. Changes in water population distribution during storage (TGA data)
The changes of the amount of water populations during storage
among the samples are depicted in Fig. 2 whereas Table 2 presents the
summary of the distribution in the temperature and water contributions
within the GF breads by the end of storage time. In spite of lack of
consistent trend, it could be observed an increase in the amount of
easily-removable water population (W1) within most of the samples
except in PSY. Similar increase in the amount of easily-removable water
population during storage was observed by Liu et al. (2014) and Lodi
and Vodovotz (2008) in gluten-containing systems. In contrast, Meng
and Kim (2020) reported a consistent decrease in the amount of W1
population during a 4 day-storage in high-moisture fermented rice cake.
In this study, as indicated by the highest peak area, the most noticeable
increase in the amount of the least strongly bound water population
(W1) could be observed in the commercial COM bread (Table 2). During
storage, COM had a significant shift to lower temperatures across all
water populations, meaning earlier evaporation i.e. less ability to bind
water (Table 2, Fig. 3A) which led to the highest progressive weakening
of water interactions with matrix in comparison to the buckwheat/carob
breads with psyllium and hydrocolloids. This is probably due to lower
hydration level of COM and presumably lower concentrations of psyl­
lium and guar present in the commercial GF mixture.
During storage, the changes in W3 water populations were not
consistent among the bread samples: an increase was observed for PSY
and XTH, and a decrease for COM and GG. At the end of storage time, the
amount of strongest bound water population (W3) was similar among
buckwheat/carob breads with psyllium and the hydrocolloids (ranging
from 8 to 11%), particularly among those with psyllium, GG and XTH
(≈11%) whereas COM was significantly lower in this fraction (7%)
(Table 2, Fig. 2B). In contrast, a decrease in the strongest bound water
population was reported during storage of wheat bread supplemented
with konjac glucomannan and its derivative (Liu et al., 2014), mixed
wheat-soy bread enriched with almonds (Lodi & Vodovotz, 2008), and
GF high-fermented rice cake with added hydrocolloids (Meng & Kim,
2020). Different results may be attributed to variations in experimental
designs. In this study, the experimental GF breads were not starch based
but buckwheat/carob flour based and longer storage period was
observed. Hydrocolloids’ behaviour may vary in conjunction with other
proteins, thus changing the water redistribution in the system (Crockett
et al., 2011).
The increase in W3 population during storage may be interpreted as
an indicator of increased rate of starch retrogradation as being caused by
the migration of freezable water from the amorphous matrix to the
crystalline hydrate of retrograded starch (Hallberg & Chinachoti, 2002)
and may point to starch (amylopectin) retrogradation as the main
mechanism of staling. However, a prominent decrease in W3 population
Table 2
Trends in the water peak distributions and peak temperatures of the deconvo­
luted DTG curves after 10 days of storage within the studied GF bread
formulations.
Water Peak temperature Peak area (% of total area)
population
W1 XTHa>PSYa>GGa>HPMCa>COMb COMa>HPMCb>XTHb,
c
>GGb,c>PSYc
W2 PSYa>HPMCa,b>XTHb, PSYa>GGa,b>HPMCa,
c
>GGc>COMd b
>XTHb>COMc
W3 PSYa>HPMCa>XTHa>GGa>COMb XTHa>PSYa>GGa>HPMCa,
b
>COMb
PSY is GF bread with psyllium husk; COM is GF bread made from commercial GF
mixture; GG is GF bread with guar gum; XTH is GF bread with xanthan; HPMC is
GF bread with HPMC; W1-3 are water populations.
a,b, …
Different superscripts denotes significant difference (p < 0.05).
6
LWT 151 (2021) 112156
observed in COM bread could be a consequence of excessive drying out.
3.2.3. Kinetic of staling
Fast staling is an important issue in GF bread and a quest for anti-
staling alternative ingredients is ongoing. Loss of freshness in GF
bread is mostly perceived through increased crumb firmness and
crumbliness. The simplest analytical tool to measure crumb staling is the
measurement of crumb textural properties (firmness, resilience). Kinetic
modelling enables better understanding of the staling process by
providing means to quantitatively describe the rates of crumb firming
which can be later used to optimize and predict the shelf-life of bread.
Crumb firming during storage is an example of a process-limited kinetics
(Sehn & Steel, 2020). The evolution of crumb properties (firmness,
resilience) during storage is displayed in Fig. 4. Buckwheat/carob breads
(PSY, GG, HPMC, XTH) exhibited the lower increase in crumb firmness
during storage in comparison to the COM bread that exhibited dramatic
crumb hardening. Since the firmness is mainly influenced by water
content, the observed phenomena is probably due to the less amount of
water used for COM formulation as well as different ingredient
composition (starch-based ingredients) (Tsatsaragkou et al., 2012).
Among the buckwheat/carob breads, PSY, XTH and GG showed similar
behaviour whereas HPMC developed the firmer crumb, which is also
related to the amount of water present in bread crumb. Belorio and
Gómez (2020) noticed that the quality of GF breads made with HPMC
was more dependent on the level of hydration than of those made with
xanthan and psyllium. The least resilient crumb was developed by COM
bread. GF bread (PSY) exhibited the most elastic crumb during storage
while other buckwheat/carob GF breads showed similar crumb
resilience.
In order to provide a more detailed insight into the staling kinetics of
the observed GF breads, the Avrami model was employed to examine the
change in crumb firmness and resilience during storage over 10 days.
Although the interpretation of parameters from Avrami’s model in
complex food matrices is complicated due to lack of physical meaning,
the model is still considered to be adequate mathematic tool to fit the
observed data (Angioloni & Collar, 2009). In a simplistic way, it has
been used to explain bread staling as it was affected only by starch
retrogradation (Armero & Collar, 1996). The kinetic parameters are
presented in Table 3. The samples exhibited rates of crumb firming (k) in
the range 0.2066–0.2600, following the order PSY­
<XTH<HPMC<GG<COM, indicating that GF bread with psyllium
exhibited the lowest rate of developing firm crumb. This is in agreement
with the findings of Cappa et al. (2013) who observed notable
anti-staling effect of psyllium at 1.5 and 2.5% addition levels during 72 h
of storage. Moreover, Fratelli et al. (2021) reported 65–75% softer
crumb in psyllium-supplemented GF breads during 72 h of storage with
the longest delay in staling observed for the maximal level of psyllium
addition (17.14%) which is a much higher level that used in this study.
Both groups of authors agreed that the anti-staling effect of psyllium
may be associated to its high water binding capacity which decreases
starch hydration and gelatinization, thus reducing the amount of ret­
rograded starch. On the other hand, high water-binding capacity of
psyllium may be involved in limiting water mobility which also in­
fluences starch retrogradation and crumb firming kinetics. Involvement
of water mobility in the staling kinetics of experimental breads is dis­
cussed by studying the water population distribution. The crumb firming
rate within the breads coincides with the trends observed in peak tem­
perature for water populations W2 and W3 after 10 days of storage
(Table 2) showing that slower crumb firming can be associated with
higher water-binding strengths of W2 and W3 populations reflected
through higher peak temperatures. The value of the Avrami exponent
(n) was closely approaching unity, especially for the buckwheat/carob
breads (Table 3, general Avrami model) which suggest that starch
crystallization is the main cause of firmness development (Das, Ray­
chaudhuri, & Chakraborty, 2015). Bread staling mechanism cannot be
explained by single phenomena such as moisture redistribution, starch
B. Filipčev et al. LWT 151 (2021) 112156

Fig. 4. Evolution of crumb firmness (A) and resilience (B) of different GF bread formulations during 10-day storage. PSY is GF bread with psyllium husk; COM is GF
bread made from commercial GF mixture; GG is buckwheat-carob bread with guar gum; XTH is buckwheat-carob bread with xanthan; HPMC is buckwheat-carob
bread with HPMC. Error bars represent ±1 standard deviation (sample size (n) = 5 for each bread type).

Table 3
Staling kinetics parameters according to Avrami model for crumb firmness and resilience in buckwheat/carob based GF breads-effect of psyllium replacement with
xanthan, guar gum and HPMC.
General Avrami model Restricted Avrami model (n = 1)

Sample F0 (N) F∞ (N) F0– F∞(N) k (days-n) n adjR

2
k (days− 1) adjR
2

Firmness
PSY 2.97 3.95 0.98 0.2066 1.0620 0.9558 0.2278 0.9638
COM 3.19 27.48 24.29 0.2600 1.1920 0.9594 0.3061 0.9653
GG 3.01 4.85 1.84 0.2488 0.9954 0.9636 0.2457 0.9707
XTH 2.93 3.86 0.92 0.2260 1.0225 0.9547 0.2313 0.9636
HPMC 4.01 7.92 3.90 0.2329 1.0716 0.9903 0.2589 0.9911
Resilience R0 (%) R∞ (%) R0– R∞(%)
PSY 42.40 34.49 7.91 0.3337 0.7352 0.8845 0.2162 0.8859
COM 32.99 22.10 10.89 0.3802 1.1489 0.9974 0.4380 0.9950
GG 39.72 28.61 17.62 0.3490 0.8770 0.9871 0.2956 0.9859
XTH 38.02 29.58 8.44 0.2557 0.8516 0.8536 0.1987 0.8780
HPMC 36.50 28.46 8.04 0.0533 1.5380 0.8265 0.1446 0.8374

PSY-formulation with psyllium; GG-formulation with 0.5 g/100 g guar gum instead of psyllium; XTH-0.5 g/100 g xanthan; HPMC-0.5 g/100 g hydrox­
ypropylmethylcellulose; COM-commercially available GF bread.
F0; R0 = crumb firmness/resilience of fresh bread.
F∞; R∞ = final crumb firmness/resilience.
k = rate constant.
n = Avrami exponent.
adj R2 = adjusted regression coefficient.

retrogradation, local dehydration, and water-biopolymer interactions
(Choi et al., 2008). In the case of resilience evolution, n was differing
from unity among the samples and showed how the sample approaches
to the full potential of crumb elasticity deterioration. COM and HPMC
breads showed the fastest loss of crumb resilience according to n, but in
the case of HPMC it was not supported by the value of k. To enable better
comparisons, the Avrami fitting using a fixed exponent (n = 1) was
reiterated as suggested by Rojas, Rosell, and Benedito de Barber (2001).
By analysing the restricted Avrami model (Table 3), it was evident that
GF bread with psyllium husk (PSY) exhibited the slowest rate of firmness
development, while the HPMC bread had the lowest loss of crumb
elasticity probably due to initially firmer crumb. COM bread had the
highest rate of staling in both terms of crumb firmness increase and loss
of crumb resilience. Low staling tendency and better performance was
observed for XTH bread in comparison to GG and HPMC breads. Nega­
tive correlation coefficients were determined between the crumb mois­
ture and staling kinetic parameters (r2Pearson = -0.96 (p = 0.000) for
k’firmness and r2Pearson = − 0.88 (p = 0.000) for k’resilience) being in
agreement with the statement of He and Hoseney (1990) that the higher
the moisture, the lower the staling rate. Higher crumb staling rates were
positively correlated with the amount of easily removable water (W1)
(r2Pearson = 0.71 (p = 0.000065) for k’firmness and r2Pearson = 0.81 (p =
0.000001) for nfirmness) and inversely correlated with peak temperatures
of stronger bound water populations (W2 and W3) (r2Pearson = − 0.78 for
k’resilience (p = 0.000005) and kfirmness (p = 0.000004); r2Pearson = − 0.88
(p = 0.000) for k’resilience, respectively). This shows that water binding
strengths affect the staling rate phenomena.
Moreover, the staling effects within this study can also be attributed
to the buckwheat and carob flour used as main GF ingredients. In this
regard, Wronkowska et al. (2013) observed a sudden and significant
increase in crumb firmness of buckwheat supplemented GF breads at up
to 30 g/100 g buckwheat content after one day of storage whereas
within the next 48 h no significant increase was noted. In contrast, at 40
g/100 g buckwheat supplementation, crumb firmness increased slowly
and gradually during 72 h of storage being consistent with our obser­
vations. The presence of carob flour in GF formulation also affects the
textural properties of bread crumb due to its fibre content and other
hydrophilic polymers (galactomannans) whose hydroxyl groups interact
with water via hydrogen bonds, but also a strong hydrophilic character
of caroubin exhibiting water absorption of almost 3 g of water/g at 25 ◦ C
(Turfani et al., 2017; Wang et al., 2001). Tsatsaragkou et al. (2012)
reported more pronounced water influence on the characteristics of GF
breads than that of carob flour.
4. Conclusions
This study evaluated the performance of psyllium as a single
improver in the GF formulation based on buckwheat/carob flour

7
B. Filipčev et al.
mixture - a formulation less addressed in the literature unlike those
commonly composed of starchy ingredients, rice flour or their combi­
nations. Performance of psyllium was compared to that of customarily
used hydrocolloids as well as a commercial GF mixture based on starch/
GF flour with a combination of psyllium and guar gum as improvers. In
this way, the study additionally contributed to documenting on the
existing differences in quality and shelf-life between commercial GF and
experimental breads, thus filling the gap present in the literature. It was
shown that the use of psyllium is technologically justified since psyllium
is capable of controlling the texture of GF bread comparable to
commonly used hydrocolloids and can be interchangeably used with
them. The addition of psyllium contributed to the softest and most
elastic crumb in the buckwheat/carob-based GF bread. Furthermore,
psyllium exhibited anti-staling properties and effectively retarded
crumb hardening during 10-day storage.
TGA provides useful information on water-solids interaction in food
matrices but is less commonly used to describe GF matrices. TGA
demonstrated that psyllium did not significantly alter the distribution of
water populations in the buckwheat/carob-based GF bread in compar­
ison to XTH, GG and HPMC; actually it contributed to the presence of
somewhat fewer amounts of easily removable water populations and to
higher retention of strongly bound water.
In buckwheat/carob-based GF matrix, the crumb firming rate
depended on the amount of easily removable water (W1) and the
binding strength of stronger bound water populations (W2 and W3).
Psyllium appeared to be a suitable ingredient for free-from bakery
products, thus contributing to the recent consumption requirements for
free-from foods.
Funding
This study was financially supported by the Republic of Serbia,
Ministry of Education, Science and Technological Development (con­
tract No. 451-03-9/2021-14/200222).
CRediT authorship contribution statement
Bojana Filipčev: Conceptualization, Data curation, Writing – orig­
inal draft. Milica Pojić: Supervision, Writing – review & editing. Oli­
vera Šimurina: Methodology, Investigation. Aleksandra Mišan:
Investigation. Anamarija Mandić: Investigation.
Declaration of competing interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.
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