Research Article

Received: 10 October 2008 Revised: 12 December 2008 Accepted: 22 December 2008 Published online in Wiley Interscience: 13 February 2009

(www.interscience.wiley.com) DOI 10.1002/jsfa.3528

Effects of incorporated amaranth albumins on

the functional properties of wheat dough
Mária Oszvald,a Cecı́lia Tamás,b Mariann Rakszegi,b Sándor Tömösközi,c
Ferenc Békésd and László Tamása,∗

Abstract
BACKGROUND: Recent developments in micro-scale testing methodology and in methods modelling the effects of native forms
of constituents by in vitro methods have provided a new approach to study the impact of added foreign proteins on dough
end-use quality. Amaranth (Amaranthus) is a member of the pseudo-cereal family, whose storage proteins have superior
nutritional quality due to their essential amino acid composition. The aim of this project was to study the effects of the
incorporated amaranth albumin proteins on the rheological properties of the wheat dough.

RESULTS: The mixing time requirements, dough strength and stability of the reconstructed dough increased proportionally
with the amount (1, 3 and 5%) of amaranth albumin proteins incorporated. These results were supported by measurements on
the non-extractable polymeric protein ratio of the dough indicating the change in polymer size distribution.

CONCLUSION: It was demonstrated that amaranth albumin proteins are capable of interacting with gluten proteins through
disulfide bonds, showing similar effects to the individual glutenin subunits of wheat flour proteins. Improvements in dough
strength and stability without a substantial increase in the mixing requirements are of great significance for developing energy
saving technologies in the baking industry.

c 2009 Society of Chemical Industry

Keywords: amaranth albumin; wheat dough; incorporation; functional properties; storage proteins

INTRODUCTION some biologically active albumins also serve as storage molecules

Amaranth (Amaranthus spp) is a member of the pseudo-cereal
family, with great potential for an increased level of consumption
in the near future. The nutritional quality of amaranth seed is
superior to that of most cereal grains (wheat, barley, rice), because
of the higher protein content and more balanced essential amino
acid composition.1 There are three main species in the Amaranthus
genus, A. caudatus, A. curentus and A. hypochondriacus. Their
lysine content is higher (3.2–6.4%) than that of most the cereal
grains (2.2–4.5%), and the difference is even greater compared
to cereal flours (0.25–0.45%). This high lysine concentration is
complemented with elevated levels of sulfur amino acid content
(2–5%), which is higher than that measured in the most important
legumes (1.4%), such as peas, beans and soybeans. This makes
amaranth a promising crop as a source of high-quality dietary
proteins.2,3
The major protein fractions of amaranth seeds are characterised
as albumins, globulins, and glutelins, according to the Osborne4
protein classification. Albumins are reported as the main protein
fraction the various different amaranth species with values around
50%. The ratio of the glutelin and globulin fractions (about 31 and
16%, respectively) is lower, while the prolamin proteins are present
only in trace amounts (0.9–2%).5 It is interesting to note that
although seed proteins generally serve as storage molecules for
the growing plantlets,6 in the case of amaranth seed, the albumins
which are usually biologically active, can be found in the highest
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in the amaranth seeds, as previously reported for the wheat
β-amylase inhibitor, which is an integral part of the polymeric
glutenin fraction.8 The polypeptides in the albumin fractions
are very heterogeneous in size, but the low molecular weight
components are the most abundant. These are characterised by
sedimentation coefficients of 1.4S–2S, while the 4.6S proteins are
minor components.
Several studies were conducted to investigate the potential of
using amaranth-based food additives,9 – 11 because of the superior
nutritional quality of the amaranth storage proteins. When a food
system is supplemented with amaranth proteins, they may alter
its functional properties, so this issue also has to be considered.
Burisova et al.12 found positive changes in the dough properties
∗ Correspondence to: László Tamás, Eotvos Lorand University, Department of
Plant Physiology and Molecular Plant Biology, Budapest, Hungary.
E-mail: tamasl@ludens.elte.hu
a Eotvos Lorand University, Department of Plant Physiology and Molecular Plant
Biology, Budapest, Hungary
b Agricultural Research Institute of the Hungarian Academy of Sciences,
Martonvásár, Hungary
c Budapest University of Technology and Economics, Department of Applied
Biotechnology and Food Technology, Budapest, Hungary

amount.7 One possible explanation for this phenomenon is that d CSIRO, Food Futures National Research Flagship, Canberra, Australia

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Amaranth albumin and wheat dough
and baking qualities when wheat flour was supplemented with
20% amaranth flour. The loaf volume of bread made of composite
flour (wheat flour substituted with 5–20% of amaranth flour)
decreased compared to the control sample.13 Rheological tests
based on farinograph and alveograph studies indicated that the
addition of purified amaranth albumins (1% and 3% relative to the
wheat flour) had a positive influence on the dough properties; the
mixing stability improved, and the bread also had better crumb
characteristics.14
The direct addition of isolated native or heterologously
expressed wheat storage proteins15 to a carefully chosen wheat
flour (called base flour) allows the role of monomeric prolamins on
rheological properties of the dough to be studied.16 – 19 However,
if the added proteins are able to form polymers, simple addition
does not show their real impact on the functional characteristics
of the dough, because they do not become part of the polymeric
network of the gluten.20 – 22 To overcome this problem an
in vitro incorporation technique, called dough reconstitution, was
developed, which is based on a reversible reduction/oxidation
procedure.23,24 The results gained from in vitro experiments
measuring the effects of the Ax1 and Dx5 HMW glutenin subunit
proteins have been confirmed by the use of transgenic wheat flour
expressing the same HMW subunits.25,26 The excellent agreement
between the results of these two approaches indicates the
feasibility of the in vitro studies to predict the effects of amaranth
albumin proteins on the bread making quality in transgenic wheat
flour samples.
Although albumins are the main fraction in amaranth seeds,
there are only a few reports on their functional characteristics.27
The aim of the present work was to provide further information
on how these proteins influence the rheological properties and
end-use quality of the wheat dough. Two approaches were used
to modify the protein composition of the flour: the simple addition
method and the incorporation technique.
MATERIALS AND METHODS
Materials
Flour produced from spring (Cadenza) and winter (Mv Emese)
wheat (Triticum aestivum L.) varieties, both with good bread-
making quality, were chosen for this study as base flours. Flours
were obtained from the grains by milling on a laboratory mill
(METEFEM Ltd, Budapest, Hungary). The protein content of the
wheat flours was determined by the Dumas method (%N × 5.7)
using an automated protein analyser (LECO FP-528; St. Joseph,
Michigan, USA) according to the AOAC official method.28 The
protein contents of Cadenza and Mv Emese were 16.54% and
9.95%, respectively.
Albumin protein fraction extraction
Albumins were extracted from amaranth seeds (A. hypochondri-
acus) adapting the method of Osborne.4 The seed was mixed in
a ratio of 1 : 5 (w/v) with distilled water containing 0.0001 mol
L−1 phenylmethylsulfonyl fluoride (PMSF) and was then ground
in an explosion-proof blender (GE Ltd, Gainesville, GA, USA) for
3 × 3 min. The solubilised proteins were separated by centrifu-
gation (5000 × g, 30 min, 4 ◦ C) and the supernatant was dialysed
against distilled water for 48 h at 4 ◦ C, changing the water fre-
quently. The supernatant carrying the albumin protein fraction was
separated from the residue by centrifugation (10 000 × g, 30 min,
4 ◦ C), after which it was freeze-dried and stored at 4 ◦ C for further
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Figure 1. SDS gel electrophoresis of Amaranth hypochondriacus storage
proteins extracted with water and DMSF (lane a) without reducing agent
(DTT), and (lane b) in the presence of reducing agent. M, molecular mass
marker.
studies. The protein components present in the isolated albumin
fraction were separated by sodium dodecyl sulfate polyacrylamide
gel electrophoresis (SDS–PAGE) according to Laemmli29 and mon-
itored (Fig. 1). The protein content of the purified fraction was
95%.
Mixing studies
The incorporation and addition types of experiments were carried
out in composite wheat flours containing 1, 3 and 5% amaranth
albumin proteins, relative to the protein content of the wheat
flour. The method applied to incorporate amaranth proteins into
the protein network of the gluten involves two consecutive steps,
as originally developed for dough reconstitution by Békés et al.30
Four grams of Cadenza flour with or without amaranth albumins
was mixed with 2.25 mL deionised water and 100 µL dithiothreitol
(DTT) solution (2 mg mL−1 ) for 30 s and allowed to react without
mixing for a further 4 min. Fifty microlitres of KIO3 (potassium
iodate) solution (2.5 mg mL−1 ) was added to the partially reduced
flour and mixed for 30 s. After 5 min resting the flour was further
mixed for 10 min and the mixing curve was registered. In case
of Mv Emese flour less deionised water (2.1 mL) was added,
while the solutions were added in the same volume but at lower
concentration (1 mg mL−1 of DTT and 1.25 mg mL−1 of KIO3 ). In
the addition type of experiments amaranth albumins were simply
mixed into the flour samples without the reduction/oxidation
steps. In this case, the DTT and KIO3 solutions were substituted by
water, as published earlier.31
Dough mixing studies were performed in a prototype of a
micro z-arm mixer (METEFEM).32 The following parameters were
determined from the mixing curves: dough development time
(DDT), time to reach the maximum consistency of the dough
(in seconds); peak resistance (PRmax), the resistance at DDT
Valorigraph Unit (VU); stability (ST), the time between the first
and last excursions over the 500 VU line (in seconds); resistance in
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breakdown (BD), the area enclosed by the midline of the recorded
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data for 10 min after the DDT and the 500 VU line, which crosses
the curve maximum (VU x sec). Dough samples were taken at
peak time from the micro z-arm mixer and freeze dried for further
protein analysis using size exclusion HPLC (SE-HPLC).

SE-HPLC analysis
Size exclusion liquid chromatography was carried out to determine
the protein size distribution in both the unextractable and the
extractable protein fractions of the dough according to Larroque
et al.33 Freeze-dried dough samples were ground and the proteins
were extracted in two consecutive steps following the method
of Gupta et al.34 The first SDS soluble fraction represents the
extractable proteins, while the protein fraction recovered from
the residue after sonication in SDS–phosphate buffer consists
of unextractable proteins. Using a Phenomenex Biosep-SEC 4000
column (300 × 7.8 mm; 5 µm clone; Torrance, CA, USA) the first
three of the five fractions were considered to represent the
polymeric proteins. The proportion of unextractable polymeric
protein (UPP%) was calculated as reported earlier.35

Statistical analysis
All measurements were carried out in triplicate. Significant
differences between the samples were determined by ANOVA
analysis. The clustering of the samples was carried out using
multivariable exploratory techniques. The statistica 7.0 program
(StatSoft, Inc., 2006, Tulsa, OK, USA) was used for statistical
evaluation.

RESULTS
Addition and incorporation of amaranth albumins into wheat
flour
Measurements were made on the effects of added and incorpo-
rated amaranth albumin proteins on the functional properties of
different wheat flours. Dough samples were produced with or
without reduction/oxidation steps in the bowl of a micro z-arm
mixer and the mixing curves were analysed. The amount of exoge-
nous protein added to the composite flour in this study is given in
Table 1.
In the dough reconstitution experiments it was found that
200 µg and 100 µg DTT per 4 g of flour were optimal to partially
reduce the polymerised protein network of cv Cadenza and cv
Mv Emese flours, respectively. To properly restore the rheological
properties of the reduced control sample the proteins in the flour
were re-polymerised by 125 µg and 62.5 µg of KIO3 . Two different
experimental approaches were employed to study the effects of
the incorporated exogenous proteins, differing in the sequence
of the chemical reduction and the protein addition steps. In the
first type, called ‘Inc I’, the amaranth proteins were first mixed with
the base flour in the mixing bowl and then all the proteins were
reduced. In the second type of experiment (Inc II) the wheat flour
proteins were reduced first and the albumin fraction was added
later.
Three mixing curves, characteristic of the base flours and the
composite flours, are shown in Fig. 2. Closer examination of the
curves reveals that the incorporation of amaranth albumin proteins
(Fig. 2C) increased the mixing time requirement (the peak shifted
to the right) and produced a more stable dough, represented by a
wider mixing curve around the peak and by a slope which is flatter
after peaking. The simple addition of the protein also modified
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Figure 2. Mixing curves registered during dough making in the bowl
of the z-arm mixer. (A) control base flour from Cadenza wheat variety,
(B) composite wheat flour with the addition of 30 mg amaranth albumin,
(C) reconstituted dough after the incorporation of 30 mg amaranth
albumin. Dough development time (DDT), peak resistance (PRmax) and
resistance in breakdown (BD) are marked on panel A. VU, resistance at the
DDT.
The mixing parameters were measured in three replicates on both
flours in the addition and incorporation studies and the calculated
values are displayed in Table 1. For better comparison of the data
obtained in the different types of experiments, the measured
values have been expressed as the percentage of the appropriate
control sample.
The addition of 1 or 3% amaranth albumins into either Cadenza
or Mv Emese flours had no significant effect on the dough
development time; however, the highest amount (5%) of added
proteins caused a small but significant increase in DDT. Dough
stability was considerably improved (14%) in both doughs when
supplemented with 3% albumins, which was supported by the
decrease (around 7%) in BD values. It is interesting to note that the

the properties of the dough, but to a smaller degree (Fig. 2B). highest protein concentration (5%) had a substantially larger effect

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Table 1. Effects of addition/incorporation procedures on the mixing properties of different wheat flours

Wheat variety Peak resistance (%) Dough development time (%) Resistance in breakdown (%) Stability (%)

Cadenza
Control 100.00 100.00 100.00 100.00
Add – 1% (6 mg) 99.61 100.95 99.35 106.03
Add – 3% (18 mg) 96.43 101.54 93.24 114.42
Add – 5% (30 mg) 94.61 102.62 78.01 147.00
Inc I – 1% (6 mg) 100.62 102.53 88.04 112.68
Inc I – 3% (18 mg) 102.86 104.56 83.92 117.76
Inc I – 5% (30 mg) 103.38 106.81 72.48 155.27
Inc II – 1% (6 mg) 105.56 102.00 97.66 110.93
Inc II – 3% (18 mg) 103.94 106.89 95.19 155.98
Inc II – 5% (30 mg) 104.99 108.00 93.50 180.00
Mv Emese
Control 100.00 100.00 100.00 100.00
Add – 1% (6 mg) 99.30 100.72 98.20 103.07
Add – 3% (18 mg) 98.90 101.00 93.05 114.00
Add – 5% (30 mg) 97.00 102.82 89.12 123.00
Inc I – 1% (6 mg) 101.97 101.38 75.71 102.00
Inc I – 3% (18 mg) 102.09 103.68 73.95 131.00
Inc I – 5% (30 mg) 102.47 105.92 69.52 145.00
Inc II – 1% (6 mg) 103.32 101.00 93.32 121.00
Inc II – 3% (18 mg) 104.15 104.60 96.15 156.00
Inc II – 5% (30 mg) 105.49 106.56 84.49 176.00
Least significant difference (P < 0.05) 2.00 2.00 3.70 5.60

in the case of Cadenza flour (47%) than for Mv Emese flour (23%).
The resistance in breakdown (BD) data showed similar differences
at the highest protein concentration (22% and 11% reduction,
respectively). Small decreases in PRmax values were also observed
in simple addition experiments, indicating the change in dough
strength.
Both types of incorporation experiments had a positive effect on
the dough mixing properties, even when the amaranth albumins
made up as little as 1% of the flour protein content (see Table 1).
The course of changes detected in these types of experiment
was similar to those measured after simple addition, but at a
significantly higher level. The dough development time increased
by 8% and 6%, and the dough stability by 80% and 76% in
composite flours based on Cadenza and Mv Emese, respectively.
Not only did stability-related mixing parameters (ST and BD)
improve during incorporation, but the strength-related PRmax Figure 3. SE-HPLC chromatogram of the proteins extracted by sonication
parameter as well. This was the only parameter that changed from the control dough produced from Cadenza flour. Regions used
for evaluation of the dough mixing experiments are marked on the
in opposite directions in the simple addition and incorporation chromatogram. P-I = large polymeric protein, P-II and P-III = small
studies. It was also observed that the impact of amaranth proteins polymers, P-IV = monomeric prolamins and P-V = albumin/globulin
on the dough characteristics was more pronounced if the amaranth fraction.
albumins were added into the mixing bowl after the partial
reduction of the base flour proteins (Inc II method).
albumins/globulins. The SE-HPLC results were statistically analysed
using the normalised data as in the case of the mixing study
Size exclusion HPLC analysis analysis. Significant changes could only be detected in two
In order to monitor changes within the protein network fractions, peak I (P-I) and peak V (P-V), consisting of the large
structure of the gluten after the addition or incorporation of polymeric proteins and the monomeric proteins (albumins and
amaranth albumins, proteins were extracted from the dough globulins), respectively. The changes in these two fractions as
and separated by SE-HPLC. Five fractions were distinguished the result of amaranth protein addition and incorporation are
on the chromatograms and the peak areas were quantified shown in Fig. 4. The proportion of the large polymeric fraction
(Fig. 3). Based on SDS–PAGE analysis (data not shown) the P-I (P-I) was substantially increased when the albumin fraction was
fraction corresponded to large polymers, P-II and P-III to smaller incorporated into the protein matrix of the dough. This increase
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polymers, P-IV to monomeric wheat prolamin proteins and P-V to was greater in the case of Cadenza flour than for Mv Emese flour.

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Figure 4. Effects of the addition and incorporation of amaranth albumin on the molecular size distribution of the gluten proteins in Cadenza and Mv
Emese flour doughs, using the results of SE-HPLC. (A) P-I fraction (large polymeric proteins), (B) P-V fraction (albumins and globulins) of Cadenza dough,
(C-D) P-I and P-V fraction of Mv Emese dough, respectively. (
), addition; (), incorporation type I; (), incorporation type II.

Simple addition did not alter the P-I area significantly in either
of the wheat varieties. In contrast, simple addition increased the
amount of the P-V fraction to a similar extent for both base flours.
When the results of the two different incorporation approaches
(Inc I and Inc II) were compared to each other, only slight differences
(at p = 0.1 level) were detected in the data for the P-I and P-V
fractions.
The unextractable polymeric protein ratio (UPP%), a widely used
parameter to characterise the size distribution of polymerised
proteins, was also determined for each wheat dough sample
(Fig. 5). While the addition of albumin proteins to the flour did
not alter the UPP% in the dough during mixing, the incorporation
procedure resulted in a significant increase in the size distribution
of the polymeric proteins. This increase was demonstrated to be
proportional to the amount of amaranth albumins incorporated.
Depending on the source of the base flour, slight differences were
also observed in the UPP% values.
Relationships between the mixing properties of the composite
flours and the SE-HPLC data were also studied. Correlation coeffi-
cients are shown in Table 2 and confirm that the amount of large
polymers (P-I) and the size distribution of the polymeric proteins
(UPP%) substantially influenced the mixing time requirement of
the dough, regardless of the base flour.
Figure 5. Effects of the addition and incorporation of amaranth albumins
on the UPP% values using cv Cadenza (A) and cv Mv Emese (B) wheat
DISCUSSION flours. Add: addition; Inc I: incorporation type I; Inc II: incorporation type II.
Strength is a rheological property of the dough, which means the
resistance to extension. Dough strength is highly influenced by
the polymeric protein quantity and the size distribution of it. The optimal dough development time, is in close correlation with the
parameter characteristic for this property is called Rmax, which dough strength and can be related to bread quality.36
can be measured by the Mixograph. In our study we did not use One of the practical limitations to improving dough strength
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a Mixograph but PRmax, as measured by the z-arm mixer at the and stability is that these properties are strongly related to

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mixing parameters were measured to characterise the effects of

Table 2. Linear correlation coefficients between the quantified peak
areas of the SE-HPLC chromatograms and the dough property the exogenous protein fraction on dough rheological properties.
parameters According to previous experiments,24 the incorporation technique
is a realistic model to mimic the in vivo situation, when the
Dough Resistance in
Wheat Peak development breakdown Stability genes of interest are introduced by wheat transformation and
variety resistance time (min) (VU) (min) the storage proteins are synthesised together during endosperm
development.
Cadenza The conditions of the reduction/oxidation procedure had to be
P-I −0.153 0.799∗∗∗ −0.383 0.752∗∗∗ optimised separately for the flours derived from wheat varieties
P-II −0.141 −0.566∗∗ 0.741∗∗∗ −0.572∗∗ Cadenza and Mv Emese. The amounts of reducing agents used
P-III −0.087 −0.679∗∗ 0.041 −0.629∗∗ in these studies were higher than those found to be optimal for
P-IV −0.19 −0.476 0.558∗∗ −0.372 mixing in a pin-mixer, called the 2g Mixograph.30
P-V −0.524∗∗ −0.039 −0.432 0.086 Before carrying out incorporation-type experiments, the effects
UPP% −0.573∗∗ 0.708∗∗∗ −0.478 0.663∗∗ of simple protein addition on the dough quality were investigated.
Mv Emese The addition process had multiple effects on the end-use quality
P-I −0.688∗∗ 0.756∗∗∗ −0.617∗∗ 0.83∗∗∗ of the doughs, for example a slight positive alteration in the
P-II −0.707∗∗∗ 0.629∗∗ −0.829∗∗∗ 0.563∗∗ mixing time requirement and a significant improvement in dough
P-III −0.687∗∗ 0.604∗∗ 0.182 −0.169 stability. Sanchez et al.14 also reported an improvement in dough
P-IV −0.597∗∗ 0.582∗∗ 0.154 0.182 mixing stability measured with a Brabender farinograph, although
P-V 0.442 0.565∗∗ 0.541∗∗ −0.567∗∗ they used a much higher amount (1 and 3% relative to the wheat
UPP% −0.275 0.721∗∗∗ −0.587∗∗ 0.841∗∗∗ flour quantity) of exogenous proteins. In the present studies only
1, 3 and 5% of amaranth albumins, relative to the protein content
Marked correlation coefficients are significant at the ∗∗ 0.05 and ∗∗∗ 0.01
levels. of the wheat flour were added into the mixing bowl. Our results
VU, XXXXX. indicated that about one tenth of the protein used in earlier studies
had positive effects on the dough mixing parameters.
The simple addition of different wheat prolamin proteins to
wheat flour usually has similar effects on its functional properties,
the mixing time requirement of the dough, thus involving regardless of the number and distribution of the cysteine residues
increased energy costs. To break this tight relationship between in the molecule.22 Addition generally increases the monomeric
mixing requirement and the strength and stability of the dough, to polymeric protein ratio, which makes the dough weaker and
new wheat varieties, new technologies and/or new ingredients less stable.5,23 There are, however, reports on spontaneous in-
would be advantageous and a preferred solution for the baking corporation (without partial reduction) of the added glutenin
industry. The structure–function relationship has been studied subunit proteins into the polymeric glutenin network.38,39 It was
by altering the type and the amount of proteins in model suggested that incorporation occurred because of damage in the
systems.21,23,24 In vitro methods provide a powerful technique polymer structure during the mixing action. This spontaneous in-
to study the quantitative effects of specific proteins on wheat flour corporation had similar but smaller effects on the dough properties
quality. The structure–function relationships observed in in vitro compared to chemical incorporation through reduction/oxidation
studies provide key information to predict the results of in vivo steps. These results indicate the potential to use amaranth proteins
experiments, such as predicting the effects of a particular protein as food additives in the baking industry, because it could improve
in transgenic wheat flour.24,25 During the last 15 years, new micro the quality of certain flour types even in small quantities.
scale equipment and techniques have been developed to test the The mixing time requirement, dough strength and stability
functional properties of as little as 5–10 mg of wheat prolamin changed proportionally to the increase in the amount of polymeric
proteins. In conjunction with the development of molecular protein fraction.38,39 To study whether the added albumin proteins
biology, these techniques are beginning to cast a new light were able to become incorporated into the dough, SE-HPLC
on the relationship between the processing quality of the flour analyses were carried out. No significant changes were observed
and the structure of various gluten proteins along with any non- in the size distribution of the polymeric proteins. Therefore, it
wheat proteins introduced by either supplementation or genetic was hypothesised that the modification of the dough properties
engineering methods. was probably not the result of the spontaneous incorporation of
The albumin type storage proteins in amaranth seed represent the added amaranth albumins, but the consequence of certain
around 50% of the total protein content. They can be classified non-covalent interactions between the polymeric glutenin and
into a few major polypeptides with molecular masses of 52, 54 the amaranth proteins. This is in good agreement with previous
and 56 kDa, as described by Martinez et al.37 These polypeptides observations, when the effects of non-covalent interactions on the
were divided into several subunits after reduction with DTT, surface characteristics of large polymeric proteins were studied.40
characterised by molecular masses between 19 and 38 kDa Hydration and mixing properties of the flour changed, because
(Fig. 1). It was also observed that lyophilised albumin was highly they became less hydrophobic due to the addition of various water
polymerised, with a molecular mass of around 300 kDa and that soluble proteins.
these polymers were also stabilised by intermolecular S-S bonds.37 To estimate the impact of exogenous proteins containing free
Based on previous studies on amaranth albumin proteins it was SH groups on the end-use quality of the dough it is necessary to
hypothesised that it was possible to incorporate them into the chemically incorporate them into the polymeric network of the
protein matrix of the wheat gluten through disulfide bonds. gluten. Our data demonstrated that amaranth albumins were able
To prove this concept, dough reconstitution studies were carried to interact with the glutenin type subunits of the wheat storage
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out in the mixing bowl of the micro z-arm mixer and several proteins through disulfide bonds. The effects were very similar

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to those measured after the incorporation of wheat glutenin
subunits into the polymeric matrix of the dough.21,41,42 The mixing
requirement increased due to the stronger and more stable dough.
The extent of this effect was influenced by the quality of the base
flour and the amount of albumin. Two different incorporation
approaches were tested to find out if it was necessary to reduce
the amaranth proteins before the oxidation step. The small
differences detected in the dough development time between
the two different procedures indicated that the initial reduction of
the amaranth albumins was not required for incorporation. This
confirms that amaranth albumins have free SH groups in their
original forms, available for inter-molecular S-S bond formation.
The observations discussed above were supported by the results
of SE-HPLC analyses. In both incorporation procedures the value of
UPP% was higher in the reconstituted dough samples compared
to the control sample, indicating the increased polymer size of the
polymeric proteins after the incorporation process.
The amaranth albumin incorporation experiments showed a
higher impact on dough strength and stability when the base
flour was produced from the wheat variety Cadenza. This difference
might be explained by the higher protein content of the flour of
cv Cadenza and by the different high molecular weight glutenin
subunit allelic composition of the storage proteins in the two
varieties (Cadenza: n 14 + 15, 5 + 10; Mv Emese: 2∗ , 7∗ + 8, 5 + 10).
CONCLUSIONS
This is the first report on the effects of the chemical incorpo-
ration of amaranth storage proteins into the protein matrix of
wheat dough. The experiments clearly demonstrated that the re-
duction/oxidation method developed earlier for wheat glutenin
subunits was also suitable for amaranth albumin incorporation
into the polymeric protein fraction of the gluten. Albumins are
able to form inter-molecular disulfide bonds with wheat glutenin
subunits, due to the presence of free SH groups in the molecules.
The effects of these proteins on the reconstituted dough properties
were similar to the effects of wheat glutenin subunits, although to
different extents. The improvement of dough strength and stabil-
ity without a substantial increase in the mixing time requirement
can be exploited by the baking industry to develop energy saving
technologies. Both simple addition and partial reduction/oxidation
procedures offer an option to improve the nutritional and baking
quality of the amaranth–wheat composite flour.
The supplemented amaranth proteins have beneficial effects on
the properties of wheat flour dough, so they can be used as food
additives. Another possible approach is to introduce the genes
encoding amaranth proteins into wheat by genetic engineering.
The expressed amaranth proteins are able to interact with the
wheat storage proteins due to their free SH groups, as proved
in this study. Based on in vitro studies it is hypothesised that the
functional properties of wheat flour can be improved through
the gene technology approach if around 3% or more amaranth
albumin protein is expressed in the starchy endosperm, relative to
its protein content.
ACKNOWLEDGEMENTS
Maria Oszvald is in receipt of a grant from the Hungarian
Foundation Scientia Amabilis. The authors wish to acknowledge
the excellent technical assistance of Mrs Timea J. Bodnár and
888
Györgyi Balogh.
www.interscience.wiley.com/jsfa

c 2009 Society of Chemical Industry
M. Oszvald et al.
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