Food Research International 44 (2011) 2806–2813

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Food Research International

j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / f o o d r e s

Influence of breadmaking on antioxidant capacity of gluten free breads based on rice

and buckwheat flours
Marijana Sakač, Aleksandra Torbica ⁎, Ivana Sedej, Miroslav Hadnađev
Institute for 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

Article history: In the present study new formulations for gluten-free bread based on mixtures of rice (RF) and buckwheat
Received 15 December 2010 flour (light buckwheat flour (LBF) or wholegrain flour (WBF)), in the proportions of 90:10, 80:20, and 70:30
Accepted 6 June 2011 were made. The gluten-free breads were investigated for their total phenolic content, rutin and quercetin
contents, antioxidant activity (AOA) by β-carotene bleaching method, reducing power, scavenging activity on
Keywords:
1,1-diphenyl-2-picrylhydrazyl (DPPH•) radicals, and chelating activity on Fe 2+. The increased amount of LBF
Gluten-free bread
Antioxidant capacity
or WBF in the dough formulation resulted in the final products with higher antioxidant properties. Baking
Breadmaking treatment expressed different influences on antioxidative properties of the final gluten-free product in terms
Wholegrain buckwheat flour of raw materials, applied recipe and antioxidative capacity in comparison to calculated values based on raw
Light buckwheat flour materials. Final gluten-free products were characterized by lower total phenolic and rutin contents, lower
antioxidative and reducing activity and on the other hand higher DPPH and chelating activity as well as
quercetin content in comparison to calculated values. Bread containing wholegrain buckwheat flour
expressed in most of the cases higher values of measured antioxidative parameters than bread prepared with
light buckwheat flour and thus contributes to their additional functional property.
© 2011 Elsevier Ltd. All rights reserved.

1. Introduction
Cereal products, especially breads, are the main part of a diet in many
countries. Elastic and extensible properties of dough, required to
produce good quality breads, are attributed to the gluten—the protein
fraction found in most cereals. However, celiac disease patients are
unable to consume bread and other food products that contain proteins
of gluten complex. The majority of gluten-free flours as well as gluten-
free products are based on wheat starch. Consequently, they can
threaten the health of celiac disease patients, since very small amount of
gluten might be still present (Katina et al., 2005). Different starches and
flours, such as rice, corn, cassava, and potato are used for the production
of gluten-free products (Sanchez, Osella, & De La Torre, 2002).
Many commercially available gluten-free breads are inferior in
quality to their gluten-containing counterparts (Gallagher, Kunkel,
Gormley, & Arendt, 2003). Numerous researchers have investigated
the substitution of gluten containing component by ingredients which
are able to mimic its properties in dough preparation. Good quality
gluten-free bread can be produced only if different gluten-free flours
and polymeric substances are included in the gluten-free formulation.
It is recommended to use a range of gluten-free flours rather than just
one flour type to achieve products with good sensory and textural
properties (Arendt, Morrissey, Moore, & Dal Bello, 2008).
⁎ Corresponding author. Tel.: + 381 21 485 3779; fax: + 381 21 450 725.
E-mail address: aleksandra.torbica@fins.uns.ac.rs (A. Torbica).
Rice flour is one of the most suitable cereal flour for gluten-free
products because it has a low level of prolamine. Besides, rice
possesses unique nutritional, hypoallergenic, colorless, and bland
taste properties (Sanchez et al., 2002). The manufacture of bread from
rice presents considerable technological difficulty due to the absence
of gluten (Sivaramakrishnan, Senge, & Chattopadhyay,2004).
Common buckwheat (Fagopyrum esculentum Moench) and tartary
buckwheat (Fagopyrum tataricum) have recently attracted much
interest due to their health benefits (Tang, Peng, Zheng, & Chen,2009).
Buckwheat has been reported to possess higher antioxidant activity
than other cereals, mainly due to high rutin content (Holasova et al.,
2002; Takahama, Tanaka, & Hirota, 2010). Rutin and its hemisynthetic
derivatives exert different biological effects like normalization of
increased vascular permeability and fragility, edema protection and
antioxidant and antihemorrhagic properties (Hung & Morita, 2008).
Quercetin, also identified in buckwheat is known as an inhibitor of low
density lipoprotein oxidation and potent antioxidant (Mikstacka,
Rimando, & Ignatowicz, 2010; Quettier-Deleu et al., 2000).
On contrary, processing conditions can significantly affect chem-
ical composition of food product. There are many researches
concerning negative influence of thermal processing of fruits,
vegetables and cereals on flavonoid compounds (Dietrych-Szostak &
Oleszek, 1999). The type of the heated subtracts and processing
conditions are the main factors of the flavonoids loss (Şensoy, Rosen,
Ho, & Karwe, 2006). There are many investigations (Alvarez-Jubete,
Wijngaard, Arendt, & Gallagher, 2010b; Lindenmeier & Hofmann,
2004; Vogrinčič, Timoracka, Melichacova, Vollmannova, & Kreft, 2010;

0963-9969/$ – see front matter © 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.foodres.2011.06.026
 M. Sakač et al. / Food Research International 44 (2011) 2806–2813
Zieliński, Kozłowska, & Lewczuk, 2001; Zieliński, Michalska, Piskuła, &
Kozłowska, 2006) concerning the issue of baking and heating
treatment on the antioxidative properties and polyphenol composi-
tion. However, there is a lack of papers regarding the changes of
antioxidative properties of the final gluten-free products based on rice
and buckwheat flours.
In a previous study (Torbica, Hadnađev, & Dapčević, 2010),
rheological characteristics of the rice dough supplemented with
buckwheat flour for gluten-free bread production were investigated.
The results of mentioned study showed that optimal rheological and
textural as well as acceptable sensory properties of produced gluten-
free breads were obtained. The aim of this work was to provide the
insight in antioxidative capacity and level of its stability during the
baking process of gluten-free breads based on rice and buckwheat
flours.
2. Materials and methods
2.1. Chemicals
β-Carotene,1,1-diphenyl-2-picrylhydrazyl(DPPH),3-(2-pyridyl)-
5-6-bis(4-phenyl-sulfonicacid)-1,2,4-triazine(ferrozine),ferricchlo-
ride,Folin-Ciocalteu'sreagent,gallicacid,linoleicacid(99%),potassium
ferricyanide,quercetin,rutin,sodiumcarbonate,Tween40andtrichlor-
acetic acid (TCA) were obtained from Sigma (Sigma-Aldrich GmbH,
Sternheim,Germany).HPLCgrademethanolwasusedforchromatogra-
phy (Merck, Darmstadt, Germany). All other chemicals and solvents
(ethanol, methanol, chloroform, formic acid, ferrous sulfate, sodium
dihydrogen phosphate) were of analytical grade and purchased from
Merck,Darmstadt,Germany.WaterwaspurifiedusingMilliporeElix10UV
water purification system, and ultrapure water used in mobile phase
preparationforHPLCwasobtainedusingSimplicityUV,Millipore.
2.2. Materials
Rice flour — RF (moisture content: 9.09%, protein content
(N × 5.7): 7.31%, and starch content: 81.5%), wholegrain buckwheat
flour — WBF (moisture content: 9.76%, protein content (N × 5.7):
12.4%, and starch content: 67.4%) and light buckwheat flour — LBF
(moisture content: 10.1%, protein content (N × 5.7): 7.50%, and starch
content: 68.2%) were obtained from Hemija Komerc, Novi Sad, Serbia.
Vegetable fat was donated by Puratos, Belgium, while the other
ingredients (salt, sugar and fresh yeast) were purchased at the local
market.
2.3. Bread formulations
All formulations of enriched gluten-free bread were made
according to Torbica et al. (2010). Mixtures of RF and LBF as well as
of RF and WBF were prepared. The proportion of RF to buckwheat
flours was 90:10, 80:20, and 70:30, respectively.
Flour mixture (100 g), fresh yeast (8 g), salt (3 g), sugar (4 g),
hydrogenated vegetable fat (4 g), and appropriate amount of water
(180 mL for mixtures with LBF, and 190 mL for mixtures with WBF),
previously tempered at 30 °C, were used in all formulations.
2.4. Breadmaking process
Flour mixtures (RF and LBF/WBF) and the other ingredients were
mixed together in one step mixing process for 2 min. Consequently,
the obtained dough was left to rest in molds for 15 min at 30 °C.
The baking tests were carried out for 35 min at 220 ± 2 °C for
dough based on flour mixtures (RF and LBF/WBF). The breads were
cooled down to room temperature 23 ± 1 °C prior to analysis.
2807
2.5. Preparation of ethanolic extracts
Rice flour, buckwheat flour and gluten-free bread, respectively
(10 g) was mixed with 100 mL of 80% ethanol. Extraction was carried
out by shaking the mixture at room temperature 23 ± 1 °C for 1 h. The
extract was separated by filtering through the filter paper (Whatman,
Grade 4 Chr, UK), and procedure was repeated twice with 100 mL of
solvent. The extracts (3 × 100 mL) were combined and dried by
vacuum-evaporator. The dried extract was weighed and the yield was
calculated based on the wet mass of the samples. The dried extract
was redissolved in 80% ethanol to 25 mL volume for buckwheat flours
and breads and to 5 mL volume for rice flour. The extract obtained by
following this procedure was used for further investigation of
antioxidant activity.
2.6. Total phenolic content
Total phenolic content of rice or buckwheat flour and gluten-free
bread extracts was determined spectrophotometrically by using
Folin-Ciocalteu's reagent (Singleton, Orthofer, & Lamuela-Raventos,
1999). Gallic acid was used as a reference standard and results were
expressed as gallic acid equivalents (GAE) (μg GAE/g of sample on dry
mass basis). The extract (0.1 mL) of rice or buckwheat flour or gluten-
free bread was diluted with pure water (7.9 mL). Folin-Ciocalteu's
reagent (0.5 mL) and sodium carbonate solution (1.5 mL; concentra-
tion 20 g/100 mL) were added, and the reaction mixture was mixed
thoroughly. The mixture was allowed to stand for 120 min with
intermittent shaking, and the absorbance at 750 nm was measured
(Jenway, 6405 UV/VIS, Bibby Scientific Ltd, Stone, UK).
2.7. Antioxidant activity (AOA) by β-carotene bleaching method
Oxidative loss of β-carotene in a β-carotene/linoleic acid emulsion
was used to assess the antioxidant activity of the examined extracts
(Moure et al., 2001). β-Carotene (2 mg) was dissolved in 10 mL of
chloroform and 1 mL of β-carotene solution was mixed with 20 mg of
purified linoleic acid and 200 mg of Tween 40 in a round-bottomed
flask. Chloroform was removed by purging with nitrogen. Pure water
(50 mL) was added into a β-carotene/linoleic acid emulsion and
mixed well by using a vortex mixer (V1 plus BOECO, Hamburg,
Germany). The extracts of rice or buckwheat flour or gluten-free
bread at various concentrations (0.50–20.0 mg/mL) (0.2 mL) and
aliquots (5 mL) of the β-carotene/linoleic acid emulsion were placed
in capped culture tubes and mixed thoroughly. The tubes were
immediately placed in a water bath and incubated at 50 °C. Oxidation
of the β-carotene/linoleic acid emulsion was monitored spectropho-
tometrically by measuring the absorbance at 470 nm after 120 min
(Jenway, 6405 UV/VIS). A control was prepared by using 0.2 mL of 80%
ethanol instead of the extract.
Degradation rate of the extracts was calculated according to the
first order kinetics using the equation (Al-Saikhan, Howard, & Miller,
1995):
lnða = bÞ⋅1 = t = sample degradation rate ð1Þ
where ln is natural log, a is initial absorbance (470 nm) at time zero,
b is absorbance (470 nm) at 120 min, and t is time (min).
The antioxidant activity (AOA) was expressed as inhibition (in %)
relative to the control using the equation:
AOA = ðdro –drÞ⋅100 = dro ð2Þ
where dro and dr are the degradation rates of the control and the
sample, respectively.
2808 M. Sakač et al. / Food Research International 44 (2011) 2806–2813
The IC50 value (mg/mL) was defined as effective concentration at
which the AOA was 50% under the experimental conditions. It was
obtained by interpolation from linear regression analysis.
2.8. Reducing power
Reducing power of the 80% ethanolic extracts was measured
according to the method of Oyaizu (1986). Various concentrations
(0.50–20.0 mg/mL) of the ethanolic extracts (0.5 mL) were mixed with
2.5 mL of phosphate buffer (0.2 M, pH= 6.6) and 2.5 mL of potassium
ferricyanide (1%). The mixtures were incubated at 50 °C for 20 min, and
after that TCA (10%, 2.5 mL) was added. The mixtures were centrifuged
at 650 g for 10 min. The supernatant (2.5 mL) was mixed with 2.5 mL of
pure water and 1 mL of 0.1% ferric chloride and the absorbance was
measured at 700 nm (Jenway, 6405 UV/VIS). Higher absorbance of the
reaction mixture indicates greater reducing power.
The IC50 value (mg/mL) was defined as an effective concentration
of extract at which the absorbance of reaction mixture reaches 0.5 for
reducing power. It was obtained by interpolation from linear
regression analysis.
2.9. DPPH radical scavenging activity
Effect of the 80% ethanolic extracts on the scavenging of 1,1-
diphenyl-2-picrylhydrazyl radicals (DPPH•) was estimated according
to the modified method of Hatano, Kagawa, Yasuhara, and Okuda
(1988). The concentration of the DPPH• solution used in the assay was
90 μM (22.5 mL of 0.4 mM DPPH• solution (0.01577 g DPPH• in
100 mL methanol) was diluted with 95% methanol to 100 mL). An
aliquot (1.0 mL) of the DPPH• solution (90 μM) was diluted in 2.9 mL
methanol, and 0.1 mL of the extracts at various concentrations (0.50–
20.0 mg/mL for buckwheat flours and breads, and 0.50–30.0 mg/mL
for rice flour) was added. The mixture was shaken vigorously and left
to stand for 60 min in the dark, then the absorbance was measured at
517 nm (Jenway, 6405 UV/VIS) against the blank. The blank consisted
of 80% ethanol and the reagent solution without 80% ethanolic extract
added and the procedure was carried as described above.
The IC50 value (mg/mL) was defined as the concentration of an
antioxidant extract which was required to scavenge 50% of the initial
amount of DPPH• under the experimental conditions given. It was
obtained by interpolation from linear regression analysis.
2.10. Chelating activity on Fe 2+
Chelating activity of the ethanolic extracts on Fe 2+ was measured
according to the method of Decker and Welch (1990). Aliquots of
1 mL of different concentrations of ethanolic extracts of rice or
buckwheat flour or gluten-free bread (0.50–20.0 mg/mL for buck-
wheat flours and breads, and 0.50–40.0 mg/mL for rice flour) were
mixed with 3.7 mL of pure water. The mixture was left to react with
ferrous sulfate (2 mM, 0.1 mL) and ferrozine (5 mM, 0.2 mL) for
10 min at room temperature 23 ± 1 °C, and then the absorbance was
measured at 562 nm by Jenway 6405 UV/VIS spectrophotometer. A
lower absorbance indicates a higher chelating power.
Chelating activity (in %) was calculated according to the following
equation:
Chelating activity = 100 – ðA⋅100Þ =Ao
where Ao and A are absorbances of the control and the sample,
respectively at 562 nm.
The IC50 value (mg/mL) was defined as the concentration of an
antioxidant extract which chelates 50% of present Fe 2+ under the
experimental conditions. It was obtained by interpolation from linear
regression analysis.
2.11. HPLC determination of rutin and quercetin
Five grams of rice or buckwheat flours and ground gluten-free
breads was extracted with 20 mL methanol/water (90/10, v/v) at
room temperature 23 ± 1 °C during 24 h, ultrasonicated for 10 min
and filtered through 0.45 μm pore size nylon filter (Rotilabo-
Spritzenfilter 13 mm, Roth, Karlsruhe, Germany) before injection
into the HPLC system.
HPLC analysis was performed by using a liquid chromatograph
(Agilent 1200 series), equipped with a diode array detector (DAD),
Chemstation Software (Agilent Technologies), on an Agilent, Eclipse
XDB-C18, 1.8 μm, 4.6 × 50 mm column, at a flow-rate of 1.000 mL/min.
Based on the previously used method (Ćetković, et al., 2008), new
chromatographic conditions were developed. The solvent linear
gradient program was created by varying the proportion of solvent
A (methanol) to solvent B (1% formic acid in water (v/v)) as follows:
initial 10% A; 0–10 min, 10–25% A; 10–20 min, 25–60% A; 20–30 min,
60–70% A. The run time and post-run time were 45 and 10 min,
respectively. The column was operated at 30 °C. The injected volume
of samples and standards was 5 μL and it was done automatically,
using autosampler. The spectra were acquired in the range of 210–
400 nm and chromatograms plotted at 330 and 350 nm with
reference wavelength set at 550/100 nm.
Phenolic components in a sample extract were identified by
matching the retention time and their spectral characteristics against
those of the standards. The purity of the peaks was determined to
ensure the identification. The external standard method was a
technique used for quantification. For each component (rutin and
quercetin) a stock solution was made from the commercial standards
which were dissolved in methanol and the obtained concentration
was 1.00 mg/mL. The diluted stock solutions were used for calibration.
The final concentrations were in the range of 0.005–0.20 mg/mL.
2.12. Statistical analysis
All analyses were performed in triplicates, and the mean values
with the standard deviations (S.D.) were reported. Analysis of
variance and Duncan's multiple range test were used. Statistical
data analysis software system STATISTICA (StatSoft, Inc. (2008),
version 9.0. www.statsoft.com) was used for analysis. P values b 0.05
were regarded as significant.
3. Results and discussion
In this paper values of parameters of antioxidant activity for flour
samples were investigated and summarized in Table 1. The values of
the same parameters for the enriched gluten-free breads are
graphically illustrated using histograms. Thus are simultaneously
Table 1
Total phenolic content, AOA, reducing activity, scavenging activity on DPPH• and
chelating activity on Fe2+ of ethanolic extract (80% v/v) of rice flour (RF), light (LBF)
and wholegrain buckwheat flour (WBF).
RF LBF WBF
Total phenolic content 9.98 ± 0.253a 332.43 ± 4.76b 415.08 ± 13.8c
ð3Þ (mg GAE/100 g dmb)
AOA, IC50 (mg dmb/mL) 13.09 ± 0.20a 8.4 ± 0.31b 6.91 ± 0.13c
Reducing activity, IC50 13.76 ± 0.04a 3.03 ± 0.01b 2.59 ± 0.08c
(mg dmb/mL)
DPPH. scavenging activity, IC50 31.1 ± 3.22a 1.36 ± 0.01b 1.26 ± 0.09b
(mg dmb/mL)
Chelating activity on Fe2+, IC50 37.92 ± 1.72a 2.13 ± 0.04b 1.73 ± 0.15c
(mg dmb/mL)
Values are means of three determinations ± standard deviation.
Values of the row with the same superscript are not statistically different (P b 0.05).
 M. Sakač et al. / Food Research International 44 (2011) 2806–2813
compared measured and calculated values (calculation was estimated
using the obtained antioxidative activity parameters of rice and
buckwheat flours). Based on a comparison of calculated and observed
values of tested parameters, the impact of breadmaking process on
the extent of the antioxidative stability was estimated and reported in
several different aspects.
3.1. Total phenolic content
Total phenolic contents of the extracts of RF, LBF and WBF are
shown in Table 1. The amount of extractable matters differed between
rice and buckwheat flours and resulted in different extraction yields,
while the yields of enriched gluten-free breads were in the range of
5.30–6.42%.
The total phenolic content of buckwheat flours was found to be
significantly higher (P b 0.05) than of RF (Table 1). Therewith, higher
concentrations of phenolic compounds in buckwheat were found in
outer layers of the grain (Holasova et al., 2002) and, therefore, the LBF,
as the refined one, contained less phenolic compounds in comparison
to WBF (Table 1). According to Şensoy et al. (2006) the obtained
values of total phenolic content in white and dark buckwheat flours
were 1.79 and 10.47 mg/g respectively, and in raw tartary buckwheat
flour it was 0.823 mg/g (Zhang, Chen, Li, Pei, & Liang,2010).Total
phenolic content of enriched gluten-free breads, expressed by
comparing the calculated and analytically determined values is
expressed in Fig. 1.
There are many references indicating that wheat based products
have been enriched with buckwheat flour in order to achieve better
functionality of the final product (Fessas et al., 2008; Lin, Liu, Yu, Lin, &
Mau, 2009). The possibility for upgrading gluten-free products such as
bread, pasta and confectionary products by application of pseudocer-
eals has been reported by Gallagher (2008) and Alvarez-Jubete,
Arendt, and Gallagher (2010a). Buckwheat was investigated as a
component for high-quality gluten-free breads production (Moore,
Schober, Dockery, & Arendt, 2004; Renzetti, Dal Bello, & Arendt,
2008). According to Alvarez-Jubete et al. (2010) the obtained total
phenolic content of buckwheat bread was 64.5 mg GAE/100 g dmb.
Our investigations showed that increased portion of LBF or WBF in
mixture for gluten-free breads (10–30%) resulted in breads with
Fig. 1. Total phenolic content of ethanolic extract (80% v/v) of enriched gluten-free
breads; LBFc — calculated content in light buckwheat bread; LBFd — determined
content in light buckwheat bread; WBFc — calculated content in wholegrain buckwheat
bread; WBFd — determined content in wholegrain buckwheat bread; and b/N% — mean
loss/yield%.
2809
increased total phenolic contents (Fig. 1). Therefore, the bread with
30% of WBF in the mixture possessed the highest total phenolic
content, i.e. 115.5 mg GAE/100 g dmb.
The current findings suggest on the fact that processing conditions
can modify the polyphenol content of foods in several ways (Manach,
Scalbert, Morand, Remesy, & Jimenez, 2004), and that thermal
processing not necessarily detrimentally effect on TPC (Zieliński et
al., 2001). Our results had shown the changes in total polyphenol
content in all investigated gluten-free bread samples during the
baking process. It is noticeable that the baking process results in a
higher percentage of reducing the amount of TPC in samples of bread
supplemented with LBF, while there were only minor changes in TPC
in samples of bread with 10% and 20% of the WBF.
3.2. Antioxidant properties
There are numerous studies showing the marked antioxidant
activity of buckwheat mainly due to high rutin content (Holasova et al.,
2002), which is known as a potent antioxidant (Yang, Guo, & Yuan,
2008). Therefore, the LBF and WBF extracts demonstrated high
potential in AOA assay, while RF extract exhibited lower activity to
suppress lipid peroxidation in β-carotene/linoleic acid system
(Table 1). There are some references concerning antioxidant properties
of rice, but they indicate that high potency of rice bran (Abdul-Hamid,
Raja Sulaiman, Osman, & Saari, 2007) is due to the presence of
tocopherols, tocotrienols, γ-oryzanol and polyphenol compounds
mainly concentrated in the outer layers of rice (Fardet, Rock, &
Rémésy, 2008). According to Lindenmeier and Hofmann (2004),
thermal processing of cereals, such as baking, can also result in the
synthesis of substances with antioxidant properties, such as some
Maillard reaction products in bread crust (Şensoy et al., 2006) also
registered changes in non-polar and polar compounds in buckwheat
after roasting and extrusion using HPLC technique.The antioxidant
properties of RF, LBF and WBF are presented in Table 1.
AOA of enriched gluten-free breads, expressed by comparing the
calculated and analytically determined values is expressed in Fig. 2.
The increased portion of both buckwheat flours in flour mixtures
from 10% to 30% in the mixtures for gluten-free bread formulations
resulted in breads with increased antioxidant properties, expressed as
IC50. Also, gluten-free breads containing WBF expressed higher AOA in
comparison to LBF containing products which is in accordance with
Fig. 2. AOA of ethanolic extract (80% v/v) of enriched gluten-free breads; LBFc — calculated
content in light buckwheat bread; LBFd — determined content in light buckwheat bread;
WBFc — calculated content in wholegrain buckwheat bread; WBFd — determined content
in wholegrain buckwheat bread; and b/N% — mean loss/yield%.
2810 M. Sakač et al. / Food Research International 44 (2011) 2806–2813

the results of Lin et al. (2009) who showed that addition of

wholegrain buckwheat flour in wheat bread resulted in a product
with more improved antioxidant properties than the addition of light
buckwheat flour.
It is noticeable that the breadmaking process affects a loss of AOA
in samples of bread containing LBF as well as WBF. This loss of AOA
decreases with increasing the both buckwheat flour contents and was
more expressed for LBF containing products. Although, some
antioxidative compounds are likely to be heat labile (Stevenson et
al., 2008), it seems that some reactions of synthesis during the heat
treatment had occurred, i.e. formation of Mailard reaction products
(Gawlik-Dziki, Dziki, Baraniak, & Lin, 2009; Manzocco, Calligaris,
Mastrocola, Nicoli, & Lerici, 2001). Therefore, the formation of Mailard
reaction products may mask real decrease of total phenolic content
and AOA as well as loss of AOA in bread samples during the heat
treatment (Zhang et al., 2010). IC50 values for AOA of all investigated
bread extracts significantly differed (P b 0.05) except gluten-free
bread with addition of 10% LBF. Gluten-free bread with addition of
10% of LBF or WBF was not statistically different.
The results of AOA assay only indicate total antioxidant activity of
investigated material. However, lipid peroxidation could be inhibited
by decreasing concentration of peroxyl radicals via hydrogen atom
transfer from the antioxidant to the lipid peroxyl radical, deactivating
lipid peroxyl radicals by single electron transfer and chelating
transition metals to suppress the initiation of radical formation
during metal-catalyzed lipid peroxidation (Shahidi & Wanasundara,
1992). Hence, reducing activity as an indicator of electron-donating
capacity and DPPH test as an indicator of radical scavenging ability on
DPPH• were also performed (Table 1, Figs. 3 and 4).
Reducing activity of ethanolic extract (80% v/v) of enriched gluten-
free breads, expressed by comparing the calculated and analytically
determined values is expressed in Fig. 3.
All extracts of gluten-free breads, except with additional 10% and
20% LBF demonstrated significant differences (P b 0.05) in reducing
activity assay concerning their IC50 values. Experimentally obtained
values of reducing activity expressed as IC50 indicate that the addition
of 30% of LBF and WBF to gluten-free bread achieves the highest value
reducing activities. In most of the cases, it can be observed that the
increase of both buckwheat flour amounts resulted in decrease of
reducing activity loss. The loss of reducing activity was more
pronounced for LBF containing products in comparison to WBF
containing products. This is in accordance with Zieliński et al. (2001)
Fig. 4. Scavenging activity on DPPH• of ethanolic extract (80% v/v) of enriched gluten-free
breads; LBFc — calculated content in light buckwheat bread; LBFd — determined content in
light buckwheat bread; WBFc — calculated content in wholegrain buckwheat bread; WBFd —
determined content in wholegrain buckwheat bread; and b/N% — mean loss/yield%.
who reported that hydrothermal processing of grains may liberate
phenolic acids and other compounds from cell walls, thus resulting in
higher antioxidant potential. In this way WBF containing product
could express lower loss of reducing activity.
Scavenging activity on DPPH• of ethanolic extract (80% v/v) of
enriched gluten-free breads, expressed by comparing the calculated
and analytically determined values is expressed in Fig. 4.
According to Alvarez-Jubete et al. (2010b) buckwheat extract was
previously noted as a potent source of reducing agents. This finding
explains the evident increases of reducing activity after the flour
mixture was enriched with LBF or WBF (Table 1).
The ability to scavenge DPPH• by the extracts of rice and
buckwheat flours was in the following order: wholegrain buckwheat
flour ~ light buckwheat flour ≫ rice flour (Table 2). The DPPH•
scavenging activity of buckwheat graded milling flours was investi-
gated by Hung and Morita (2008). Their results illustrated that the
outer layer of buckwheat grains containing higher total phenolic and
flavonoid contents possessed significantly higher antioxidant capac-
ities than the inner fractions when the DPPH assay was used. This
finding is not in agreement with our IC50 values on DPPH• for
buckwheat flours, since LBF and WBF had similar IC50 values (Table 1).
Strong DPPH• scavenging activity of buckwheat flour extracts could
have been partly due to the presence of rutin which possesses strong
ability to scavenge DPPH• (Hsu, Chiang, Chen, Yang, & Liu, 2008). The
results presented in Table 1 indicate that IC50 values on DPPH• for
buckwheat flours are more than 20-fold lower than the IC50 value for
rice flour. Stronger scavenging effect on DPPH• of buckwheat flours
contributed to the increase of scavenging activity on DPPH• of gluten-
free breads enriched with LBF or WBF. The effectiveness of WBF was
better than of LBF. This is in agreement with the finding of Lin et al.
(2009) who found out that wheat bread enriched with wholegrain

Table 2
Correlations of total phenolic content, antioxidative activity, reducing activity, DPPH
activity and chelating activity of gluten-free bread extracts.

TPC TPC TPC TPC RED RED DPPH AOA AOA AOA
vs. vs. vs. vs. vs. vs. vs. vs. vs. vs.
Fig. 3. Reducing activity of ethanolic extract (80% v/v) of enriched gluten-free breads; LBFc — AOA RED DPPH CHEL DPPH CHE CHE RED DPPH CHE
calculated content in light buckwheat bread; LBFd — determined content in light
LBF 0.992 0.829 0.999 0.995 0.901 0.878 0.999 0.943 0.993 0.987
buckwheat bread; WBFc — calculated content in wholegrain buckwheat bread; WBFd —
WBF 0.908 0.994 0.989 0.911 0.999 0.97 0.98 0.973 0.978 0.999
determined content in wholegrain buckwheat bread; and b/N% — mean loss/yield%.
 M. Sakač et al. / Food Research International 44 (2011) 2806–2813
buckwheat flour was more effective concerning antioxidant activity in
comparison to wheat bread enriched with light buckwheat flour.
Results obtained by Lin et al. (2009) showed that IC50 value of
scavenging activity on DPPH• for husked and unhusked buckwheat
enhanced wheat breads were 20.29 and 9.75 mg/ml, respectively.
According to results presented in Fig. 4 similar IC50 values of
scavenging activity on DPPH• for gluten-free bread were expressed.
In comparison to results of Lin et al. (2009) which DPPH values
originate from phenolic compounds of both buckwheat as well as
wheat flour, our results for gluten-free bread samples expressed
similar values. However as rice flour is known to possess less phenolic
compounds than wheat flour, the obtained results are mainly
influenced by the antioxidative potential of used buckwheat flours.
Considering the ratio of calculated and determined IC50 values, trend
of yield instead of loss for IC50 values was observed. This percentage of
yield of IC50 values of scavenging activity on DPPH• increased with
increase of both buckwheat flour contents and it was more expressed
for WBF containing products. This phenomenon can be explained by
previously mentioned reactions of synthesis during the heat treat-
ment (Gawlik-Dziki et al., 2009; Manzocco et al., 2001; Zieliński et al.,
2001). The compounds with chelating capacity may significantly
inhibit the lipid peroxidation in biological and food systems. Distinct
differences in chelating activity on Fe 2+ were observed between RF
extract and LBF and WBF extracts (Table 1). Rutin as the most potent
antioxidant in buckwheat was evidenced as a strong antioxidant in
the Fenton reaction in which it could act as metal chelator and/or
radical scavenger (Yang et al., 2008). Thus, it is likely that the
enrichment of gluten-free breads with buckwheat flours in the range
of 10–30% in the mixture for the formulations contributed to the
increase of metal chelating activity on Fe 2+ (Fig. 5).
In addition to previous discussion correlations between the
performed measurements were estimated (Table 2). In Table 2
significant correlations between TPC and all antioxidative parameters
for LBF as well for WBF were found. According to Alvarez-Jubete et al.
(2010b) these high correlations could be ascribed to chemistry
mechanisms of used methods which are based on the same principles
(redox properties) thus having influence on obtaining high correla-
tions between values obtained by all these methods. The observed
differences of correlation values between gluten-free bread samples
with LBF and WBF could be explained by the presence of interfering
substances.
Fig. 5. Chelating activity on Fe2+ of ethanolic extract (80% v/v) of enriched gluten-free
breads; LBFc — calculated content in light buckwheat bread; LBFd — determined
content in light buckwheat bread; WBFc — calculated content in wholegrain buckwheat
bread; WBFd — determined content in wholegrain buckwheat bread; and b/N% — mean
loss/yield%.
2811
3.3. Phenolic compounds
Phenolic compounds identified in buckwheat are flavonoids: rutin,
orientin, vitexin, quercetin, isovitexin, isoorientin, kaempferol-3-
rutinoside, and catechins (Dietrych-Szostak & Oleszek, 1999; Watanabe,
1998). In the paper of Dietrych-Szostak and Oleszek (1999) determined
rutin content in dehulled buckwheat seeds and hulls was 17.86 and
33.3 mg/100 g dmb, respectively. In the same paper the findings of the
other researchers are presented. Accordingly, in buckwheat varieties
rutin content may range from 12.6 to 35.9 mg/100 g dmb, in buckwheat
seeds rutin content was 47 mg/100 g dmb, in hulls 4.3 mg/100 g dmb
and in buckwheat flours 17.86 mg/100 g dmb, Published data concern-
ing quercetin content are limited and the values range from 0.21 mg/g in
buckwheat sprouts (Hsu et al., 2008) to 0.63 mg/g found in buckwheat
flour (Vogrinčič et al., 2010). In our investigation the contents of rutin
which was the major phenolic compound found in LBF and WBF and
quercetin in both buckwheat flours are presented in Table 3.
WBF was characterized with higher rutin and quercetin contents
since buckwheat hull contains more phenolic compounds than other
buckwheat milling fractions (Quettier-Deleu et al., 2000). The
increasing amount of buckwheat flour in the mixtures for gluten-
free bread resulted in increase of rutin and quercetin in produced
breads. Their contents were significantly higher (P b 0.05) in gluten-
free breads containing WBF compared with breads with LBF (Table 3).
The same results were obtained by Lin et al. (2009) who also found
higher rutin and quercetin contents in wheat bread enriched with
wholegrain buckwheat flour (1.75 and 0.03 mg/100 g dmb) than in
wheat bread enriched with light buckwheat flour (0.90 and
0.04 mg/100 g dmb).
3.4. Rutin and quercetin contents during the breadmaking process
It is well known that thermal processing causes chemical changes
in food products. Data concerning loss of some functional components
in buckwheat due to thermal treatment could be found in the
literature (Şensoy et al., 2006). Rutin and quercetin contents in
gluten-free breads are changed in comparison to flours during
breadmaking process (Fig. 6). Changes in rutin and quercetin contents
in breads are calculated based on their calculated content according to
determined content in flours and determined content in breads. It can
be observed that 10% LBF containing products had higher rutin
content in comparison to calculated value and that gluten-free bread
with 10% of WBF had slightly lower rutin content in comparison to
calculated values. However, loss of rutin amount in breads with 20%
and 30% of LBF and WBF was in the range from 26% to 40%. This is in
agreement with the results of Zieliński, Michalska, Amigo-Benavent,
del Castillo, and Piskuła (2009) and Zhang et al. (2010) who reported
that thermal tretment of buckwheat flour caused a decrease in total
phenolics and total flavonoids. The heating time had the greatest
effect on rutin content, followed by the heating temperature and
tempering moisture (Im, Huff, & Hsieh, 2003). On the contrary,
quercetin contents in investigated gluten-free breads were increased
probably due to hydrolysis of rutin to quercetin (Fig. 6).
Table 3
Contents of rutin and quercetin in rice flour (RF), light (LBF) and wholegrain buckwheat
flours (WBF).
Extracts Rutin (mg/100 g dmb) Quercetin (mg/100 g dmb)
RF n.d.a n.d.a
LBF 8.71 ± 0.12b 0.54 ± 0.05b
WBF 21.34 ± 0.05c 0.58 ± 0.075b
Values are means of three determinations ± standard deviation.
Values of the same column with the same superscript are not statistically different
(P b 0.05).
n.d. — not detected.
2812 M. Sakač et al. / Food Research International 44 (2011) 2806–2813
Fig. 6. Changes in rutin and quercetin contents in gluten-free breads; LBFc — calculated
content in light buckwheat bread, LBFd — determined content in light buckwheat
bread, WBFc — calculated content in wholegrain buckwheat bread, WBFd — determined
content in wholegrain buckwheat bread; and b/N% — mean loss/yield%.
4. Conclusions
Buckwheat flours expressed significantly higher AOA, reducing
power, scavenging activity on DPPH• and chelating activity on Fe 2+
than rice flour when their IC50 values were compared. During the
breadmaking process final gluten-free products expressed different
changes of antioxidative parameters. Samples containing LBF exhib-
ited decrease in TPC value in comparison to calculated values, while
determined values of TPC for WBF containing samples remained
unchanged for breads with 10% and 20% of WBF. However, sample
prepared with 30% of WBF expressed loss of about 17% TPC. In
comparison to calculated values, antioxidative and reducing activities
are showing trend of activity decrease by the addition of both
buckwheat flours at 10% and 20%. These activities are slightly lower
for samples with 30% LBF (0.25 and 3.7%) and practically unchanged
for samples with 30% WBF. In comparison to calculated values, an
increase in chelating and DPPH scavenging activity for all investigated
samples was recorded. It was estimated that breads containing WBF
showed twice higher activity than LBF containing samples. There was
a loss in rutin content regarding to calculated content and that loss
increased with the increase of both buckwheat flours in the range of
4.57–40.4%. The opposite trend was observed for quercetin content
which increased 1.5 to 7 times. Taking all these data into consider-
ation, it can be concluded that thermal treatment not necessarily has
negative influence on nutritive value of gluten-free bakery product.
Improved functional properties of these bakery group products are
strongly dependent on their recipe, too.
Acknowledgments
This study was supported by the Ministry of Science and Techno-
logical Development, The Republic of Serbia (Project No: TR-20068).
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