Accepted Manuscript

Effect of different iron compounds on rheological and

technological parameters as well as bioaccessibility of minerals in
whole wheat bread

Ana Paula Rebellato, Jéssica Bussi, Joyce Grazielle Siqueira

Silva, Ralf Greiner, Caroline Joy Steel, Juliana Azevedo Lima
Pallone

PII: S0963-9969(17)30026-1
DOI: doi: 10.1016/j.foodres.2017.01.016
Reference: FRIN 6572
To appear in: Food Research International
Received date: 10 November 2016
Revised date: 8 January 2017
Accepted date: 19 January 2017

Please cite this article as: Ana Paula Rebellato, Jéssica Bussi, Joyce Grazielle Siqueira
Silva, Ralf Greiner, Caroline Joy Steel, Juliana Azevedo Lima Pallone , Effect of different
iron compounds on rheological and technological parameters as well as bioaccessibility
of minerals in whole wheat bread. The address for the corresponding author was captured
as affiliation for all authors. Please check if appropriate. Frin(2017), doi: 10.1016/
j.foodres.2017.01.016

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 ACCEPTED MANUSCRIPT

EFFECT OF DIFFERENT IRON COMPOUNDS ON RHEOLOGICAL AND

TECHNOLOGICAL PARAMETERS AS WELL AS BIOACCESSIBILITY OF
MINERALS IN WHOLE WHEAT BREAD

Ana Paula Rebellato1, Jéssica Bussi1, Joyce Grazielle Siqueira Silva1, Ralf
Greiner3, Caroline Joy Steel2, Juliana Azevedo Lima Pallone1.

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1
Department of Food Science, School of Food Engineering, University of

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Campinas. 80 Monteiro Lobato Street, Zip Code: 13083-862, Campinas, São
Paulo, Brazil.

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2
Department of Food Technology, School of Food Engineering, University of
Campinas, 80 Monteiro Lobato Street, Zip Code: 13083-862, Campinas, São
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Paulo, Brazil.
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Department of Food Technology and Bioprocess Engineering, Max Rubner-
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Institut, Federal Research Institute of Nutrition and Food, Haid-und-Neu-

Straße 9, 76131 Karlsruhe, Germany.
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Corresponding author: jpallone@unicamp.br

Phone: +55 19 35212169
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ABSTRACT

This study aimed at investigating the effect of iron compounds used in whole
wheat flour (WWF) fortification, both on rheological properties of the dough
and on bread technological quality. Furthermore, bioaccessibility of iron (Fe),
zinc (Zn) and calcium (Ca) in the final breads was determined. Rheological

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properties (mainly dough development time, stability, mixing tolerance index,
resistance to extension and ratio number) of the dough and the technological

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quality of bread (mainly oven spring and cut opening) were altered. However,
producing roll breads fortified with different iron compounds was still possible.

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NaFeEDTA (ferric sodium ethylene diamine tetra acetic acid) proved to be the
most effective iron compound in the fortification of WWF, since it presented the
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highest levels of solubility (44.80%) and dialysability (46.14%), followed by
microencapsulated ferrous fumarate (FFm). On the other hand, the
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microencapsulated ferrous sulfate (FSm) and reduced iron presented the

lowest solubility (5.40 and 18.30%, respectively) and dialysability (33.12 and
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31.79%, respectively). Zn dialysis was positively influenced by NaFeEDTA,

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FSm, and ferrous fumarate. As for Ca, dialysis was positively influenced by
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FSm and negatively influenced by FFm. The data indicated that there is a
competitive interaction for the absorption of these minerals in whole wheat roll
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breads, but all studied minerals can be considered bioaccessible.

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Keywords: Whole wheat flour, roll bread, rheological properties, minerals, in

vitro methods.

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1. INTRODUCTION
In Brazil, roll bread is the most consumed type of bread, and the
incorporation of bran, with or without wheat germ, has been generally
accepted by the population (Brasil, 2005), because of the high fiber content
and presence of most of the nutrients, such as: vitamins, amino acids,
antioxidants, minerals (iron, zinc, calcium, magnesium, phosphorus,

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potassium) (Buri et al., 2004).
Although wheat flour contains iron (Fe), zinc (Zn), and calcium (Ca),

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it has been used as a carrier in fortification programs in order to improve
nutrient absorption, particularly in what concerns Fe, since the lack of this

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mineral in the body can cause serious health consequences (Brasil, 2002).
Iron deficiency anemia is currently considered a public health problem which
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occurs in about 25% of the population worldwide (WHO, 2006). Moreover, Zn
and Ca are also considered important for the proper functioning of the human
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body (Casé et al., 2005; Pereira & Hessel, 2009).

The fortification of wheat and corn flours with iron is carried out in
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many countries. Actually, in Brazil, it is performed since 2002 (Brasil, 2002).

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The Brazilian Legislation allows the addition of iron compounds (4.2 mg/100
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g), such as: dehydrated ferrous sulphate (dried) (FS), ferrous fumarate (FF),
reduced iron (RI), electrolytic iron, ferric sodium ethylene diamine tetra acetic
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acid (NaFeEDTA), ferrous glycinate chelate, and other bioavailable

compounds, that are not inferior in bioavailability to those permitted (Brasil,
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2002).
Some studies associate the use of different iron compounds with
possible alterations that may occur in food, such as sensory and technological
properties (dough rheology and final product quality), differences in
bioavailability, and the cost of each iron form. These factors must be taken into
consideration in the Fe fortification process (Akhtar et al., 2011; WHO, 2006).
The rheological properties of flour dough can provide information on
the behavior of dough manipulation during processing. They also serve as a

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quality indicator for the final product. Alterations in these parameters are not
desirable (Sollars & Rubenthaler, 1975).
Different iron compounds used for fortification may affect the mineral
bioavailability and bioaccessibility in different ways. It is noteworthy that iron
compounds such as: FS, FF, NaFeEDTA, and FSm are indicated for cereals
fortification, since they are more likely to be absorbed by the body (Akhtar et

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al., 2009; Hurrell et al., 2010). Furthermore, the microencapsulated iron
compound has the advantage of protecting the food from undesirable

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organoleptic alterations, besides preventing oxidation (Cocato et al., 2007).
There are studies that suggest replacing ferrous sulphate by

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microencapsulated ferrous sulphate, as a fortification alternative, considering
its advantages (Cocato et al., 2007; Gotelli et al., 1996; Lysionek et al., 2001)
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Several authors used in vitro methods to study the availability of iron
and other minerals in order to understand which compounds are the most
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effective in the fortification process (Benito & Miller, 1998; Cámara et al., 2005;
Kapsokefalou et al., 2005; Miller et al., 1981; Perales et al., 2006). Moreover,
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there are studies that associate low mineral absorption with the presence of
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antinutritional factors, such as phytates (Schons et al., 2011), which are

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present in plant seeds and grains and in high amounts in wheat bran. Phytate
[myo-inositol (1,2,3,4,5,6) hexakisphosphate] is the stored form of phosphate
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in plant seeds and the most abundant inositol phosphate in nature. In isolated
form myo-inositol (5, 6) being the most important for complexing minerals in
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foods (Greiner & Konietzny, 2006). Furthermore, its ability of chelating

multivalent cations (De Carli et al., 2006), such as Fe2+/3+, Cu2+, Zn2+ and Ca2+
(Ries, 2010), which are poorly soluble, reduces the bioaccessibility of the
bound cations (Carbonaro et al., 2001).
To the best of our knowledge, this is the first report which describes
the effect of different iron compounds used to fortify whole wheat flour on
dough rheological characteristics, quality properties of whole wheat roll bread
and bioaccessibility of Fe, Zn, and Ca in the final bread. In addition, myo-
inositol phosphate esters (phytates) were quantified in the final breads.
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2. MATERIAL AND METHODS

2.1 Preparation of whole wheat roll breads (WWRB)

To obtain whole wheat flour (100 kg), 6% of wheat bran were
incorporated into 94% of refined wheat flour (Moinho Guaçu Mirim, Brazil). The

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whole wheat flour (WWF) (control) was characterized for its composition. It
presented moisture, proteins, lipids, and ash contents of 13.22 ± 0.11, 12.95 ±

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0.42, 1.29 ± 0.01, 0.80 ± 0.02 g/100g, respectively (methods 44-15.02, 46-
13.01, 30-25.01, and 08-01.01, AACC (2010), respectively). The total dietary

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fiber content was 6.91 ± 0.31 g/100g (method 985.29, AOAC (2000)).
The iron compounds used in the fortification were: monohydrated
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ferrous sulfate (FS), ferrous fumarate (FF), reduced iron (Nutrafine, RS, Brazil)
(RI), ferric sodium ethylene diamine tetra acetic acid (NaFeEDTA) (Vogler
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Ingredients, SP, Brazil), microencapsulated ferrous sulfate (FSm), and

microencapsulated ferrous fumarate (FFm) (Functional Mikron, Valinhos,
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Brazil). The quantification of iron content in WWF was performed before and
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after the fortification process, according to Rebellato et al. (2015). The iron
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concentration found in WWF was 1.38 ± 0.06 mg/100 g. In the fortification

process, each sample was homogenized using a V-blender (Tecnal
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Piracicaba, Brazil) for 30 min, in 5.0 kg portions. After adding the different iron
compounds to WWF, the obtained Fe concentrations ranged from 4.80 to 6.29
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mg/100 g.
The whole wheat roll breads (WWRB) were elaborated using the
Modified Straight Dough Method. The dry ingredients (100% whole wheat
flour, 1.5% instant dry yeast, 1.8% salt, 2.5% bread improver) and water
(614.80 ± 28.40 mL) were mixed in a HAE 10 mixer (Hyppolito, Ferraz de
Vasconcellos, Brazil), at slow speed for 4 min, then at high speed until full
development of the gluten network. The dough was divided into 65 ± 1 g
portions, rounded and left to rest for 15 min. Then, they were molded in a HM2
Hp 0.5 molder (Hyppolito, Ferraz de Vasconcellos, Brazil) and proofed in a
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20B proofing chamber (Super Freezer, Poços de Caldas, Brazil) for 60-70 min
at 27-30ºC with a relative humidity of 85-90%. After fermentation, a cut
opening was made on the surface of the dough pieces, and then the samples
were baked in a HF 4B oven (Haas, Curitiba, Brazil), with top and bottom
(hearth) temperatures of 170 °C and 180 °C, respectively, for 16 ± 3 minutes.
After cooling for ± 30 min, the technological quality of breads was determined

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in one portion of the samples. Another portion (10 breads) was stored in the
freezer (-20°C) for mineral content (Fe, Zn, and Ca) analysis and quantification

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of myo-inositol phosphate esters. We used solubility and dialysis assays to
estimate their bioaccessibility.

2.2 Rheological characterization

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The same samples were subjected to rheological analysis. In detail,
water absorption (WA), arrival time (AT), dough development time (DDT),
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stability (S), and mixing tolerance index (MTI) were evaluated using method
54-21.01 of the AACC (2010) and the Brabender Farinograph (Duisburg,
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Germany), model 827505. To evaluate the extensographic properties,

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resistance to extension (R), maximum resistance (Rm), extensibility (E) and

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ratio number (D = R/E) were determined using the Brabender Extensograph

(Duisburg, Germany), model 860703, and method 54-10.01 of the AACC
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(2010).
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2.3 Characterization of roll bread quality

The following parameters were determined in the final breads:
specific volume (SV), using method 10-05.01 of the AACC (2010); shape,
measuring height and width of the rolls according to Bodroza-Solarov et al.
(2008); oven spring according to Shittu et al. (2008); cut opening and cut
height according to Almeida and Chang (2012). All measurements were
performed in four replicates.

2.4 Determination of myo-inositol phosphate esters

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Myo-Inositol phosphate esters in the final breads were extracted

according to Sandberg et al. (1989) and quantified using method 986.11 of the
AOAC (2006).

2.5 Quantification of Fe, Zn, and Ca

The minerals were quantified using Flame Atomic Absorption

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Spectrometry, according to Rebellato et al. (2015). For Ca determination, a
lanthanum oxide solution was added to obtain a final concentration of 0.5% in

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order to eliminate the possible interferences. The method used for the Zn and
Ca quantification was validated based on parameters of precision, recovery,

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sensitivity, linearity, and limits of detection and quantification. The method was
validated according to the analytical method validation guidelines.
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The accuracy parameter set as the mean value of the variation
coefficient, was ± 10%. The recovery was performed by adding standard (Ca
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and Zn) to the control sample, at levels of 50 and 100%. Recovery

percentages close to 100% were considered satisfactory. The sensitivity was
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expressed by the slope of the linear regression equation. The limits of

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detection and quantification were calculated based on the calibration curve

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and both were expressed in mg/100 g of bread.

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2.6. Solubility and dialysis assay

The solubility assay was carried out according to Cámara et al.
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(2005), with modifications described by Rebellato et al. (2015). The volume of

enzyme solution used in the enteral step was adjusted to 6.0 mL (0.4 g of
porcine pancreatin, P-7545 and 2.5 g of bovine and sheep bile, B-8631, Sigma
Chemical Co., St. Louis, USA, in 100 mL of NaHCO3 0.1 mol/L).
The dialysis assay was performed according to Perales et al. (2006),
with modifications following Rebellato et al. (2015). In the enteral step, 30 mL
of a PIPES buffer solution, 0.30 M (piperazine-N, N0-bis [2-ethanesulfonic
acid] disodium salt), pH adjusted to 6.3 with concentrated HCl was used in the
dialysis membrane, instead of NaHCO3 solution, as reported by Kapsokefalou
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et al. (2005). The volume of enzyme solution (bile and pancreatic) was altered
to 6 mL, as described in the solubility assay.
Fe, Zn, and Ca contents in the fractions, soluble and dialyzed, were
determined by FAAS, according to Rebellato et al. (2015).

2.6 Data analysis

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All data were analyzed using variance analysis (ANOVA) and the
Tukey test (p<0.05) for mean comparison using the Statistica 7.0 software

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(StatSoft, USA).

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3 RESULTS AND DISCUSSION NU
3.1 Rheological characterization
The farinographic and extensographic parameters for the control and
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fortified whole wheat flour are shown in Tables 1 and 2, respectively.

“Table 1”
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Farinographic analysis assesses dough behavior during the mixing

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step. For water absorption (WA), only the sample fortified with NaFeEDTA
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showed significant difference (p<0.05) from the control. The different iron
compounds added to WWF showed no significant difference (p <0.05), i.e.,
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regardless of the iron form used in flour fortification, WA was not affected.
Akhtar et al. (2009) evaluated the effect of fortification on the rheological
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properties of whole wheat flour, finding that the addition of Fe and Zn affected
WA of fortified flour. However, the flour fortified with NaFeEDTA and ZnSO4
showed no effect on the WA parameter, a reverse effect to what we observed
in this study, where only flour fortified with NaFeEDTA had a lower WA, when
compared to the control sample.
The values obtained for the arrival time (AT) parameter ranged from
1.03 to 1.24 min, and the fortified samples showed no significant difference (p
<0.05) when compared to the control. Stojceska and Butler (2008) found AT
values of 1.3 min for refined wheat flour, while Schmiele et al. (2012) found AT
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values of 1.5 min for WWF. Regarding dough development time (DDT),
values ranged from 13.43 to 15.59 min, with samples fortified with RI and
NaFeEDTA showing no difference (p>0.05) from the control. The remaining
samples presented a small DDT increase, when compared to the control.
Besides, we verified that there was no significant difference among the iron
compounds used in flour fortification.

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The stability parameter (S) ranged from 20.61 to 27.90 min for
samples fortified with NaFeEDTA and FFm, respectively. We can verify that

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this parameter was increased by the iron compounds used in the fortification
process and it may indicate a strengthening of the flour. An exception occurred

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with FF and NaFeEDTA forms, which showed similarities to the control
sample. Likewise, Akhtar et al. (2009) reported an increased S when
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elemental iron was used, with values similar to the control when NaFeEDTA
was used in WWF fortification.
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Regarding the mixing tolerance index (MTI), the values ranged from
17.33 to 37.33 FU, for the sample fortified with FFm and the control,
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respectively. This parameter had its values reduced by the different iron
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compounds used. The sample with NaFeEDTA showed no significant

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difference in relation to the control. The MTI reduction may indicate that the
iron compounds used can strengthen the flour. In general, iron in NaFeEDTA
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form was the one which least altered the farinographic parameters of WWF,
when compared to the control. Nevertheless, all fortified flours, with different
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iron compounds, were considered suitable for producing breads.

“Table 2”
In the extensographic analysis, we assess dough behavior during
resting and proofing stages. For the resistance to extension parameter (R),
after 45 minutes, we verified that only the sample fortified with NaFeEDTA
showed significant difference (p<0.05) from the control. For the other periods,
90 and 135 minutes, the samples fortified with different iron compounds did
not differ significantly (p>0.05) from the control. The same effect was observed
for the maximum resistance to extension parameter (Rm) and the ratio number
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(D), with only the sample fortified with NaFeEDTA differing from the control
after 45 min; at other periods, the fortified samples showed no significant
difference (p>0.05) from the control samples.
For the extensibility parameter (E), the fortified samples showed no
significant difference (p>0.05) when compared to the control sample at
different time periods, i.e. any form of iron can be used without causing

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alterations to this parameter.
In general, the NaFeEDTA compound was the one that most

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affected the extensographic parameters evaluated. Notwithstanding, the
farinographic parameters were less affected when this compound was used,

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i.e. NaFeEDTA presented a delayed action when used, and compared to the
control sample.
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We also verified that the majority of iron compounds used in the
fortification of WWF had a more pronounced effect on the farinographic
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analysis than on the extensographic analysis.

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3.3 Characterization of roll bread technological quality

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We evaluated the following parameters in WWRB: specific volume

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(SV); shape, by measuring width and height of the rolls; oven spring; and cut
opening. The results are shown in Table 3.
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The specific volume values (SV) ranged from 4.94 to 6.30 cm3/g and
there was no significant difference (p<0.05) between fortified breads and the
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control. Similar values were reported by Esteller and Lannes (2005), who
assessed the SV of roll bread and obtained a mean value of 4.63 cm3/g. In
contrast, Gupta et al. (2012) obtained SV values ranging from 3.57 to 3.97
cm3/g.
Regarding shape, considering the height/width ratio, the values
ranged from 0.74 to 0.84, with no significant difference (p>0.05) between
fortified breads and the control. Regarding height, the sample fortified with
NaFeEDTA was higher than the control. However, among the iron compounds
used in the fortification of WWF, there was no significant difference (p>0.05).
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Differently, the width parameter was influenced by iron compounds and the
bread with FS was the one with the greatest width value, with breads fortified
with NaFeEDTA and FFm slightly differing from the control. Nogueira and
Steel (2016) studied the use of sourdough to reduce sodium in roll bread and
obtained a mean shape value of 0.72 with commercial flour.
“Table 3”

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The oven spring was negatively influenced by the different iron
compounds when compared to the control. This parameter ranged from 1.15 ±

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0.20 to 2.08 ± 0.10 cm, for bread fortified with FS and control, respectively.
Nogueira and Steel (2016) obtained oven spring mean value of 1.43 cm in roll

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bread.
Regarding cut opening, the breads fortified with FF, RI, FSm, and
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FFm showed better results when compared to the control, as shown in Table
3. The cut height was also evaluated; all WWRB showed the absence (zero) of
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this parameter. Possibly, the incorporation of fibers from wheat bran influenced
this parameter, since there is a smaller amount of gluten present in the flour.
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3.4 Determination of myo-inositol phosphate fractions

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The myo-inositol phosphate fractions, both in control and fortified

WWRB samples, with different iron compounds, were below the detection
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limits (8x10-9 mol L-1) of the analytical method, not being detected. It seems
that under the conditions applied, myo-inositol phosphates were
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dephosphorylated at least to myo-inositol bisphosphate. The applied method

for myo-inositol phosphate quantification is only capable of separating and
quantifying InsP6, InsP5, InsP4 and InsP3. Dephosphorylation is very likely
due to the activity of the intrinsic wheat phytase.
However, there are studies reporting detectable amounts of different
myo-inositol phosphates and their fractions in baked products derived from
refined wheat flour, whole wheat flour, and other cereals flour (Brune et al.,
1992; Frontela et al., 2011; Park et al., 2011; Sanz-Penella et al., 2012). This
difference might be due to the different conditions used in the baking process,
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as well as differences in the intrinsic phytase activities of the wheat flour used.
According to Frontela et al. (2011), fermentation and cooking steps
significantly reduce the phytate content in baking processes.

3.5 Quantification of Fe, Zn, and Ca in WWRB

For Ca analyses, a lanthanum oxide solution was added, obtaining a

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final concentration of 0.5%. To establish linearity, calibration curves were
constructed and the concentration varied, for Zn from 8.00 to 750.00 µg/L and

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for Ca from 0.50 to 5.00 mg/L. The correlation coefficients of the calibration
curves were considered adequate (r>0.99). The residual graphs showed a

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normal distribution, around zero, free of trends. The sensitivity was considered
adequate for determining Ca and Zn in WWRB. Detection and quantification
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limits of Zn ranged from 5.00 to 20.00 µg/100 g, and for Ca from 0.40 to 1.50
mg/100 g.
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For accuracy evaluation, the relative standard deviation (RSD) was

2.41% for Zn and 2.55% for Ca. The intermediate precision was evaluated by
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variance analysis (ANOVA), and F values calculated were lower than F

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tabulated (4.26) for Zn and Ca contents. Recovery rates, at levels of 50 and

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100%, were 115 and 114% for Zn; and 113 and 114% for Ca, respectively.
Therefore, using the parameters evaluated, we can affirm that the method for
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determining Zn and Ca in WWRB is suitable.

The Fe, Zn and Ca contents in all the samples are presented in
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Table 4. The control WWRB presented average concentrations of Fe, Zn, and
Ca of 2.86, 1.13, and 12.95 mg/100 g respectively. After fortification, bread
samples presented iron concentrations ranging from 4.83 to 6.34 mg/100 g, in
average 5.50 mg/100 g. This difference may be linked to the homogenization
of WWF fortified with iron, since the amount of mineral added to the flour was
equivalent for all iron compounds (4.2 mg/100 g flour).

3.6. Solubility and dialysis assay

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The estimated bioaccessibility of Fe, Zn, and Ca was evaluated in

WWRB, using in vitro methods, solubility and dialysis, and the values obtained
are shown in Table 4.
“Table 4”
For the solubility assay, the concentration of soluble iron showed no
significant difference (p>0.05) between the control and the WWRB fortified

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with FSm (0.34 mg/100 g). The remaining iron compounds showed significant
differences (p<0.05) when compared to the control. The NaFeEDTA presented

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the highest soluble fraction (2.56 mg/100 g) and the highest solubility
percentage. WWRB samples, with different iron compounds, showed a

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solubility percentage ranging from 5.40 (FSm) to 44.80% (NaFeEDTA). For the
other compounds of Fe, solubility percentage values ranged from 14.80 to
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21.99%. Frontela et al. (2011) discovered that bread prepared with 80% of
refined wheat flour and 20% of whole wheat flour, had an iron solubility
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percentage of 18.63%.
In iron dialysis, the control showed 1.06 mg/100g of dialyzable
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mineral, while the fortified WWRB ranged from 1.68 to 2.64 mg/100 g. All iron
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compounds used in the fortification showed significant differences (p<0.05)

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when compared to the control. Dialysis percentages ranged from 31.79 (RI) to
46.14% (NaFeEDTA) in WWRB; the control showed dialysis percentage
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(37.17%) higher than reduced Fe used in fortification. The dialyzed Fe

percentage obtained in this study was higher than the values reported by
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Frontela et al. (2011) who studied bioaccessibility of naturally occurring

minerals in bread and obtained 26% of iron dialysis. Kiskini et al. (2007)
studied iron dialysis (%) of gluten-free breads fortified with different iron
compounds and concluded that gluten-free breads fortified with ferric
pyrophosphate, NaFeEDTA, ferrous bis-glycinate, ferrous gluconate, and
ferrous sulfate presented dialysis levels higher than the gluten-free breads
fortified with electrolytic iron, ferrous lactate, and ferric pyrophosphate.
However, the results obtained in this study, for the dialysis assay, showed that

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the microencapsulated compounds (FS, FF) showed no significant difference

(p>0.05) when compared to non-microencapsulated compounds.
Additionally, Zn and Ca solubility and dialysis were evaluated in
samples of WWRB fortified with different iron compounds.
Soluble Zn and solubility percentage ranged from 0.08 to 0.19
mg/100 g and from 7.88 to 15.30%, respectively. The control WWRB did not

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differ significantly (p>0.05) from the samples fortified with FF and RI.
Additionally, samples fortified with FS, NaFeEDTA, and FFm were the ones

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that showed the highest soluble Zn content and solubility percentage. They
presented no significant difference (p>0.05) between them.

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After the dialysis assay, the Zn content ranged from 0.21 to 0.49
mg/100 g. The WWRB used as control only showed a significant difference
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(p<0.05) to microencapsulated iron compounds (FSm and FFm). Regarding
the dialysis percentage, the breads fortified with NaFeEDTA, FSm, and FFm
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presented the highest values (35.96, 33.06 and 36.42%, respectively) for zinc.
Hemalatha et al. (2007) studied the bioaccessibility of zinc in cereal grains and
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found a wheat dialysis percentage of 8.93 ± 0.44%.

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Data indicate that the Fe compounds used in flour fortification

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interfere with the possibility of Zn absorption, naturally occurring in whole

wheat breads. Frontela et al. (2011) found Zn solubility value (15.73%) similar
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to that found in this study. As for the dialysis assays, these authors found
values (4.13%) lower than those obtained in this work.
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Using the data obtained, we could verify that the zinc naturally
occurring in WWRB can be positively influenced by the absorption of iron
compounds used in fortification. In addition, NaFeEDTA compounds, FSm,
and FFm promoted the highest percentages of solubility and dialysis. There
are studies which report the positive effect of using Na2EDTA and/or
NaFeEDTA on the absorption of iron and zinc (Hettiarachchi et al., 2004;
Moretti et al., 2014; Zhu et al., 2009).
The soluble Ca content ranged from 3.18 to 11.84 mg/100 g. The
control showed no significant difference (p>0.05) compared to samples
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fortified with RI and NaFeEDTA. We verified that Ca solubility was positively

influenced in the samples fortified with FS and FF, with the highest content of
soluble Ca, and the samples fortified with compounds of microencapsulated
iron being negatively impacted. The solubility percentage ranged from 24.07 to
82.87%, for samples fortified with FSm and FS, respectively. However, the
concentration of dialysate Ca ranged from 5.55 to 8.42 mg/100 g, which

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corresponds to 38.29 and 63.77% of dialysis, respectively. The control sample
showed significant difference (p<0.05) only in the dialysis assay, with samples

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fortified with microencapsulated compounds. Frontela et al. (2011) obtained a
percentage of solubility and dialysis for Ca in whole wheat bread of 10.57 and

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4.27%, respectively. The values reported by the authors are lower than those
obtained in this study
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The in vitro simulation (solubility and/or dialysis) does not represent
the complexity of the human digestive process, since it is a one-time process
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that does not suffer the influence of physiological factors, which may affect the
bioavailability of minerals, but these in vitro methods, with or without chemical
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modifications, have been widely used seeking to predict a trend on the

E

availability of nutrients present in foods, since they have been well correlated
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with in vivo models (Hur et al., 2011).

Data on bioaccessibility of Fe minerals (in different compounds used
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in fortification), Zn, and Ca naturally occurring in WWRB are important for

verifying the estimated potential absorption of these essential minerals when
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consuming bread.

4. CONCLUSION
The results showed that the different iron compounds used in the
WWF fortification for producing whole wheat roll bread (WWRB) influenced
both the rheological characteristics of the dough, mainly dough development
time, stability, mixing tolerance index, resistance to extension and ratio
number and bread quality, mainly oven spring and cut opening, when
compared to the control, but these parameters did not affect bread production.
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We found no myo-inositol phosphate fractions in WWRB samples.

Iron content and fractions, soluble and dialysable, in WWRB were
determined, and NaFeEDTA and FSm proved to be the most efficient for WWF
fortification, since they have the highest soluble and dialysate Fe percentages,
contributing more effectively to the reduction of iron deficiency anemia.
Besides, they allow the naturally occurring Zn, present in wheat flour, to be

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more readily available for absorption in formulations that contain NaFeEDTA,
FSm and FF. Regarding Ca, we found that the FSm compound positively

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influenced the bioaccessibility of this mineral.
Finally, we concluded that all samples fortified with different iron

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compounds contained bioaccessible Fe, Ca and Zn.
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5. REFERENCES
AACC. (2010). Approved Methods of Analysis (11 ed.). St. Paul, MN,
U.S.A: AACC International.
Akhtar, S., Anjum, F. M., & Anjum, M. A. (2011). Micronutrient
fortification of wheat flour: Recent development and strategies.
Food Research International, 44(3), 652-659. doi:
10.1016/j.foodres.2010.12.033
Akhtar, S., Anjum, F. M., Rehman, S.-U., & Sheikh, M. A. (2009). Effect

PT
of mineral fortification on rheological properties of whole wheat
flour. Journal of Texture Studies, 40(1), 51-65. doi: 10.1111/j.1745-
4603.2008.00169.x

RI
Almeida, E. L., & Chang, Y. K. (2012). Effect of the addition of enzymes
on the quality of frozen pre-baked French bread substituted with

SC
whole wheat flour. LWT - Food Science and Technology, 49(1), 64-
72. doi: http://dx.doi.org/10.1016/j.lwt.2012.04.019
AOAC. (2000). Association of Official Analytical Chemists. Official
NU
methods of analysis of Association of Official Analytical Chemists.
Gaithersburg: AOAC International.
Official methods of analysis. Association of Official Analytical Chemists.,
MA

(2006).
Benito, P., & Miller, D. (1998). Iron absorption and bioavailability: An
updated review. Nutrition Research, 18(3), 581-603. doi:
D

http://dx.doi.org/10.1016/S0271-5317(98)00044-X
Bodroza-Solarov, M., Filipcev, B., Kevresan, Ž., Mandic, A., & Simurina,
E

O. (2008). Quality of bread supplemented with popped amaranthus

PT

cruentus grain. Journal of Food Process Engineering, 31(5), 602-

618. doi: 10.1111/j.1745-4530.2007.00177.x
Brasil. (2002). Agência Nacional de Vigilância Sanitária (ANVISA). RDC
CE

344/2002 (pp. www.anvisa.org.br).

Brasil. (2005). Regulamento técnico de identidade e qualidade da farinha
AC

de trigo. IN 8/2005. Brasília, DF: Diário Oficial da República

Federativa do Brasil.
Brune, M., Rossander-Hulten, L., Hallberg, L., Gleerup, A., & Sandberg,
A. S. (1992). Iron absorption from bread in humans: inhibiting
effects of cereal fiber, phytate and inositol phosphates with
different numbers of phosphate groups. The Journal of nutrition,
122(3), 442-449.
Buri, R. C., von Reding, W., & Gavin, M. H. (2004). Description and
Characterisation of Wheat Aleurone. Cereal Foods World, 49(5), 8.
Cámara, F., Amaro, M. A., Barberá, R., & Clemente, G. (2005).
Bioaccessibility of minerals in school meals: Comparison between

17
 ACCEPTED MANUSCRIPT

dialysis and solubility methods. Food Chemistry, 92(3), 481-489.

doi: 10.1016/j.foodchem.2004.08.009
Carbonaro, M., Grant, G., Mattera, M., Aguzzi, A., & Pusztai, A. (2001).
Investigation of the mechanisms affecting Cu and Fe bioavailability
from legumes. [journal article]. Biological Trace Element Research,
84(1), 181-196. doi: 10.1385/bter:84:1-3:181
Casé, F., Deliza, R., & Rosenthal, A. (2005). Produção de 'leite' de soja
enriquecido com cálcio. Food Science and Technology
(Campinas), 25, 86-91.

PT
Cocato, M. L., Ré, M. I., Trindade Neto, M. A., Chiebao, H. P., & Colli, C.
(2007). Avaliação por métodos in vitro e in vivo da

RI
biodisponibilidade de sulfato ferroso microencapsulado. Revista de
Nutrição, 20, 239-247.

SC
De Carli, L., Rosso, N. D., Schnitzler, E., & Carneiro, P. I. B. (2006).
Estudo da estabilidade do complexo ácido fítico e o íon Ni(II). Food
Science and Technology (Campinas), 26, 19-26.
NU
Esteller, M. S., & Lannes, S. C. d. S. (2005). Parâmetros
complementares para fixação de identidade e qualidade de
produtos panificados. Food Science and Technology (Campinas),
MA

25, 802-806.
Frontela, C., Ros, G., & Martínez, C. (2011). Phytic acid content and
“in vitro” iron, calcium and zinc bioavailability in bakery products:
The effect of processing. Journal of Cereal Science, 54(1), 173-
D

179. doi: 10.1016/j.jcs.2011.02.015

E

Gotelli, C. A., Gotelli, M. J., Boccio, J. R., Zubillaga, M. B., Caro, R. A.,
PT

Garcia del Rio, H., & Weill, R. (1996). Bioavailability of

microencapsulated ferrous sulfate in fluid milk studies in human
beings. Acta Physiol Pharmacol Ther Latinoam, 46(4), 239-245.
CE

Greiner, R., & Konietzny, U. (2006). Phytase for food application. Food
Technology and Biotechnology, 44(2), 123-140.
Gupta, S., Shimray, C. A., & Venkateswara Rao, G. (2012). Influence of
AC

organic acids on rheological and bread-making characteristics of

fortified wheat flour. International journal of food sciences and
nutrition, 63(4), 411-420. doi: 10.3109/09637486.2011.631522
Hemalatha, S., Platel, K., & Srinivasan, K. (2007). Zinc and iron contents
and their bioaccessibility in cereals and pulses consumed in India.
Food Chemistry, 102(4), 1328-1336. doi:
http://dx.doi.org/10.1016/j.foodchem.2006.07.015
Hettiarachchi, M., Hilmers, D. C., Liyanage, C., & Abrams, S. A. (2004).
Na2EDTA enhances the absorption of iron and zinc from fortified
rice flour in Sri Lankan children. The Journal of nutrition, 134(11),
3031-3036.

18
 ACCEPTED MANUSCRIPT

Hur, S. J., Lim, B. O., Decker, E. A., & McClements, D. J. (2011). In vitro
human digestion models for food applications. Food Chemistry,
125(1), 1-12. doi: 10.1016/j.foodchem.2010.08.036
Hurrell, R., Ranum, P., de Pee, S., Biebinger, R., Hulthen, L., Johnson,
Q., & Lynch, S. (2010). Revised recommendations for iron
fortification of wheat flour and an evaluation of the expected impact
of current national wheat flour fortification programs. Food and
nutrition bulletin, 31(1 Suppl), S7-21.
Kapsokefalou, M., Alexandropoulou, I., Komaitis, M., & Politis, I. (2005).

PT
In vitro evaluation of iron solubility and dialyzability of various iron
fortificants and of iron-fortified milk products targeted for infants

RI
and toddlers. International journal of food sciences and nutrition,
56(4), 293-302. doi: 10.1080/09637480500146515

SC
Kiskini, A., Argiri, K., Kalogeropoulos, M., Komaitis, M., Kostaropoulos,
A., Mandala, I., & Kapsokefalou, M. (2007). Sensory characteristics
and iron dialyzability of gluten-free bread fortified with iron. Food
NU
Chemistry, 102(1), 309-316. doi: 10.1016/j.foodchem.2006.05.022
Lysionek, A. E., Zubillaga, M., Salgueiro, J., Caro, R., Ettlin, E., &
Boccio, J. (2001). Bioavailability studies of a new iron source by
MA

means of the prophylactic-preventive method in rats. Biological

trace element research, 84(1-3), 123-128. doi: 10.1385/bter:84:1-
3:123
Miller, D. D., Schricker, B. R., Rasmussen, R. R., & Van Campen, D.
D

(1981). An in vitro method for estimation of iron availability from

E

meals. The American Journal of Clinical Nutrition, 34(10), 2248-

PT

2256.
Moretti, D., Biebinger, R., Bruins, M. J., Hoeft, B., & Kraemer, K. (2014).
Bioavailability of iron, zinc, folic acid, and vitamin A from fortified
CE

maize. Annals of the New York Academy of Sciences

1312, 54-65. doi: 10.1111/nyas.12297
Nogueira, A. C., & Steel, C. J. (2016). Process Parameters and
AC

Technological Quality of French Rolls Produced with Dry

Sourdough to Reduce Sodium. Cereal Chemistry Journal, 93(2),
138-144. doi: 10.1094/cchem-05-15-0095-r
Park, Y. J., Park, J., Park, K. H., Oh, B. C., & Auh, J. H. (2011).
Supplementation of alkaline phytase (Ds11) in whole-wheat bread
reduces phytate content and improves mineral solubility. Journal of
food science, 76(6), C791-794. doi: 10.1111/j.1750-
3841.2011.02206.x
Perales, S., Barbera, R., Lagarda, M. J., & Farre, R. (2006). Fortification
of milk with calcium: effect on calcium bioavailability and
interactions with iron and zinc. Journal of agricultural and food
chemistry, 54(13), 4901-4906. doi: 10.1021/jf0601214
19
 ACCEPTED MANUSCRIPT

Pereira, T. C., & Hessel, G. (2009). Deficiência de zinco em crianças e

adolescentes com doenças hepáticas crônicas. [artigo de revisão].
Revista Paulista de Pediatria, 27(3), 7.
Rebellato, A. P., Pacheco, B. C., Prado, J. P., & Lima Pallone, J. A.
(2015). Iron in fortified biscuits: A simple method for its
quantification, bioaccessibility study and physicochemical quality.
Food Research International, 77, 385-391. doi:
10.1016/j.foodres.2015.09.028
Ries, E. F. (2010). Estudo da produção, caracterização e aplicação de

PT
nova fitase de Saccharomyces cerevisiae. (Tese (Doutorado em
Ciência de Alimentos) ed., pp. 130). Engenharia de Alimentos.

RI
Universidade Estadual de Campinas, Campinas, SP.
Sandberg, A. S., Carlsson, N. G., & Svanberg, U. (1989). Effects of

SC
Inositol Tri-, Tetra-, Penta-, and Hexaphosphates on In Vitro
Estimation of Iron Availability. Journal of Food Science, 54(1), 159-
161. doi: 10.1111/j.1365-2621.1989.tb08591.x
NU
Sanz-Penella, J. M., Laparra, J. M., Sanz, Y., & Haros, M. (2012).
Assessment of iron bioavailability in whole wheat bread by addition
of phytase-producing bifidobacteria. Journal of agricultural and
MA

food chemistry, 60(12), 3190-3195. doi: 10.1021/jf205048r

Schmiele, M., Jaekel, L. Z., Patricio, S. M. C., Steel, C. J., & Chang, Y.
K. (2012). Rheological properties of wheat flour and quality
characteristics of pan bread as modified by partial additions of
D

wheat bran or whole grain wheat flour. International Journal of

E

Food Science & Technology, 47(10), 2141-2150. doi:

PT

10.1111/j.1365-2621.2012.03081.x
Schons, P. F., Ries, E. F., Battestin, V., & Macedo, G. A. (2011). Effect
of enzymatic treatment on tannins and phytate in sorghum
CE

(Sorghum bicolor) and its nutritional study in rats. International

Journal of Food Science & Technology, 46(6), 1253-1258. doi:
10.1111/j.1365-2621.2011.02620.x
AC

Shittu, T. A., Dixon, A., Awonorin, S. O., Sanni, L. O., & Maziya-Dixon, B.
(2008). Bread from composite cassava–wheat flour. II: Effect of
cassava genotype and nitrogen fertilizer on bread quality. Food
Research International, 41(6), 569-578. doi:
http://dx.doi.org/10.1016/j.foodres.2008.03.008
Sollars, W. F., & Rubenthaler, G. L. (1975). Flour fractions affecting
farinograph absorption [Water-holding properties of wheat flours].
Cereal Chemistry (USA).
Stojceska, V., & Butler, F. (2008). Digitization of farinogram plots and
estimation of mixing stability. Journal of Cereal Science, 48(3),
729-733. doi: http://dx.doi.org/10.1016/j.jcs.2008.04.001

20
 ACCEPTED MANUSCRIPT

WHO. (2006). Guidelines on food fortification with micronutrientes.

Geneva: World Health Organization.
Zhu, L., Glahn, R. P., Nelson, D., & Miller, D. D. (2009). Comparing
soluble ferric pyrophosphate to common iron salts and chelates as
sources of bioavailable iron in a Caco-2 cell culture model. Journal
of agricultural and food chemistry, 57(11), 5014-5019. doi:
10.1021/jf900328t

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Table 1. Farinograph parameters of whole wheat flour, control and fortified with the different iron compounds.
WWF WA (%) AT (min) DDT (min) S (min) MTI (FU)
Control 56.93 ± 0.58 a 1.11 ± 0.10 a,b 13.43 ± 0.67 b 21.18 ± 1.94 c 37.33 ± 2.89 a
FS 56.23 ± 0.32 a,b 1.07 ± 0.01 b 15.59 ± 0.45 a 27.89 ± 0.37 a 20.67 ± 1.15 d,e
FF 56.30 ± 0.26 a,b 1.24 ± 0.03 a 15.01 ± 0.54 a 22.62 ± 1.65 b,c
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30.00 ± 2.00 b,c

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RI
NaFeEDTA
FSm
56.57 ± 0.06 a,b
55.97 ± 0.45 b
56.23 ± 0.25 a,b
1.03 ± 0.04 b 14.73 ± 0.49 a,b 26.79 ± 2.42 a,b
1.12 ± 0.05 a,b 14.17 ± 0.90 a,b 20.61 ± 1.51 c
1.17 ± 0.10 a,b 15.31 ± 0.22 a 25.73 ± 1.75 a,b R I 23.39 ± 1.21 d
34.33 ± 2.31 a,b
26.00 ± 2.00 c,d
FFm 56.10 ± 0.17 a,b 1.11 ± 0.03 a,b 15.45 ± 0.15 a
C
27.90 ± 0.82 a

S
17.33 ± 1.15 e
Mean ± standard deviation. WWF: whole wheat flour. WA: water absorption; AT: arrival time; DDT: dough development time; S: stability; MTI: mixing tolerance

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index; FU: farinographic units. Control: whole wheat flour without addition of iron; FS: ferrous sulfate; FF: ferrous fumarate; RI: reduced iron; NaFeEDTA:
sodium iron ethylenediaminetetraacetic acid; FSm: microencapsulated ferrous sulfate; FFm: microencapsulated ferrous fumarate. Averages with different
letters in the same column indicate significant difference between samples (p<0.05).

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Table 2. Extensographic parameters of whole wheat flour, control and fortified with
the different iron compounds.
Time
Parameter WWF
45 min 90 min 135 min
Control 694.33 ± 26.63 b,c 1088.67 ± 34.99 a,b 1011.00 ± 85.77 a
FS 664.33 ± 30.50 b,c 1128.67 ± 38.66 a 955.67 ± 22.50 a
Resistance to FF 637.00 ± 6.24 c 1067.00 ± 55.56 a,b 989.67 ± 62.50 a
extension, R RI 723.67 ± 26.50 a,b 965.67 ± 43.68 b 1155.00 ± 123.04 a
(EU) NaFeEDTA 779.33 ± 44.38 a 1060.00 ± 24.43 a,b 1018.00 ± 21.66 a

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FSm 675.67 ± 19.35 b,c 1145.33 ± 48.60 a 1005.33 ± 18.01 a
FFm 735.00 ± 23.58 a,b 1156.33 ± 64.66 a 1088.00 ± 25.24 a
Control 796.00 ± 7.50 a 1107.57 ± 32.19 a 1084.80 ± 27.50 a,b

RI
FS 746.93 ± 23.30 a 1110.00 ± 6.33 a 985.53 ± 21.03 b
Maximum FF 720.43 ± 11.59 a 1074.13 ± 57.93 a 1042.87 ± 29.20 a,b

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resistance to
extension, Rm RI 800.20 ± 25.13 a 1078.70 ± 74.06 a 1159.17 ± 105.67 a
(UE) NaFeEDTA 883.50 ± 54.71 b 1066.50 ± 21.04 a 1069.80 ± 71.50 a,b
FSm 741.83 ± 34.57 a 1150.17 ± 50.09 a 1066.43 ± 44.70 a,b
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FFm 791.47 ± 25.48 a 1159.30 ± 68.51 a 1117.40 ± 9.90 a,b
Control 107.03 ± 2.97 a 80.13 ± 6.61 a 71.87 ± 2.10 a
FS 105.87 ± 4.32 a 7487 ± 2.69 a 71.33 ± 7.84 a
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FF 105.63 ± 1.12 a 77.70 ± 4.97 a 66.30 ± 2.46 a

Extensibility,
E (mm) RI 102.53 ± 1.46 a 69.20 ± 1.11 a 70.37 ± 3.25 a
NaFeEDTA 99.03 ± 7.28 a 73.93 ± 4.41 a 70.83 ± 3.91 a
D

FSm 102.53 ± 0.40 a 75.77 ± 0.40 a 70.13 ± 5.49 a

FFm 99.50 ± 6.51 a 73.20 ± 4.47 a 68.70 ± 4.27 a
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Control 6.5 ± 0.37 b,c 13.65 ± 1.12 a 15.01 ± 0.48 a,b

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FS 6.29 ± 0.54 c 15.09 ± 0.63 a 13.50 ± 1.34 b

FF 6.03 ± 0.09 c 13.07 ± 0.87 a 14.92 ± 0.40 a,b
Ratio number, D
RI 7.06 ± 0.32 a,b,c 14.02 ± 0.57 a 15.78 ± 0.89 a
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(EU/mm)
NaFeEDTA 7.88 ± 0.14 a 14.36 ± 0.74 a 15.51 ± 0.44 a,b
FSm 6.59 ± 0.20 b,c 15.11 ± 0.71 a 14.39 ± 0.98 a,b
FFm 7.42 ± 0.74 a,b 14.89 ± 0.32 a 15.86 ± 0.62 a
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Mean ± standard deviation. R: resistance to extension; Rm: maximum resistance to extension; E:

extensibility; D: ratio number (R / E); EU: extensographic units. WWF: whole wheat flour. Control:
whole wheat flour without addition of iron; FS: ferrous sulfate; FF: ferrous fumarate; RI: reduced iron;
NaFeEDTA: sodium iron ethylenediaminetetraacetic acid; FSm: microencapsulated ferrous sulfate;
FFm: microencapsulated ferrous fumarate. Averages with different letters in the same column indicate
significant difference between samples (p<0.05).

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Table 3. Quality parameters of the whole wheat roll breads.

WWRB SV (cm3/g) Height (cm) Width (cm)
Control 5.54 ± 0.56 a,b 5.36 ± 0.37 b 6.72 ± 0.26 c
FS 6.18 ± 0.39 a 5.68 ± 0.14 a,b 7.66 ± 0.19 a
FF 6.30 ± 0.36 a 5.63 ± 0.09 a,b 7.16 ± 0.10 b
RI 6.09 ± 0.22 a 5.83 ± 0.16 a,b 7.19 ± 0.07 b
NaFeEDTA 4.94 ± 0.19 b 5.89 ± 0.03 a 7.01 ± 0.09 b,c
FSm 5.10 ± 0.29 b 5.73 ± 0.38 a,b 7.25 ± 0.03 b
FFm 5.17 ± 0.12 b 5.52 ± 0.11 a,b 7.05 ± 0.24 b,c
WWRB Shape Oven spring (cm) Cut opening (cm)

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Control 0.80 ± 0.08 a,b 2.03 ± 0.18 a 0.65 ± 0.10 c
FS 0.74 ± 0.02 b 1.15 ± 0.20 b 0.76 ± 0.14 b,c
FF 0.79 ± 0.01 a,b 1.18 ± 0.05 b 1.06 ± 0.12 a
RI 0.81 ± 0.02 a,b 1.52 ± 0.11 b 1.04 ± 0.02 a

RI
NaFeEDTA 0.84 ± 0.01 a 1.50 ± 0.12 b 0.88 ± 0.03 a,b,c
FSm 0.79 ± 0.05 a,b 1.28 ± 0.14 b 1.02 ± 0.08 a

SC
FFm 0.78 ± 0.02 a,b 1.28 ± 0.08 b 0.97 ± 0.02 a,b
Mean ± standard deviation (n = 4). SV: specific volume; WWRB: whole wheat roll breads. FS: ferrous
sulfate; Control: whole wheat roll breads without addition of iron; FF: ferrous fumarate; RI: reduced
iron; NaFeEDTA: sodium iron ethylenediaminetetraacetic acid; FSm: microencapsulated ferrous
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sulfate; FFm: microencapsulated ferrous fumarate. Averages with different letters in the same column
indicate significant difference between samples (p<0.05).
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Table 4. Bioaccessibility of Fe, Zn and Ca in whole wheat roll breads.

Total Fe Soluble Fe Solubility Dialyzed Fe Dialysis
WWRB
(mg/100g) (mg/100g) (%) (mg/100g) (%)
Control 2.86 ± 0.03 e 0.34 ± 0.02 d 11.75 1.06 ± 0.08 d 37.17
FS 5.61 ± 0.25 b,c 1.05 ± 0.05 b 18.80 1.88 ± 0.20 b,c 33.50
FF 4.83 ± 0.06 d 1.07 ± 0.07 b 21.99 1.80 ± 0.18 b,c 37.33
RI 5.28 ± 0.12 b,c,d 0.96 ± 0.04 b,c 18.30 1.68 ± 0.15 c 31.79
NaFeEDTA 5.71 ± 0.12 b 2.56 ± 0.13 a 44.80 2.64 ± 0.14 a 46.14
FSm 6.34 ± 0.10 a 0.34 ± 0.03 d 5.40 2.10 ± 0.24 b 33.12

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FFm 5.21 ± 0.42 c,d 0.82 ± 0.11 c 14.80 1.80 ± 0.07 b,c 34.65
Total Zn Soluble Zn Solubility Dialyzed Zn Dialysis
WWRB
(mg/100g) (mg/100g) (%) (mg/100g) (%)

RI
Control 1.13 ± 0.02 c 0.09 ± 0.01 c 7.88 0.26 ± 0.02 b 23.39
FS 1.21 ± 0.02 b,c 0.17 ± 0.01 a 14.41 0.26 ± 0.03 b 21.05

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FF 1.20 ± 0.02 b,c 0.08 ± 0.01 c 6.75 0.28 ± 0.03 b 23.27
RI 1.15 ± 0.01 b,c 0.11 ± 0.01 b,c 9.67 0.21 ± 0.01 b 18.40
NaFeEDTA 1.21 ± 0.02 b,c 0.18 ± 0.02 a 15.30 0.44 ± 0.02 a 35.96
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FSm 1.47 ± 0.14 a 0.14 ± 0.02 b 9.62 0.49 ± 0.06 a 33.06
FFm 1.28 ± 0.01 b 0.19 ± 0.02 a 14.99 0.47 ± 0.04 a 36.42
Total Ca Soluble Ca Solubility Dialyzed Ca Dialysis
WWRB
MA

(mg/100g) (mg/100g) (%) (mg/100g) (%)

Control 12.95 ± 0.57 b 7.71 ± 0.71 c 63.33 6.88 ± 0.75 b,c 53.01
FS 14.31 ± 0.27 a 11.84 ± 0.99 a 82.87 8.12 ± 0.36 a,b 56.75
FF 14.14 ± 0.55 a 10.17 ± 0.57 b 72.09 7.42 ± 0.89 a,b,c 52.35
D

RI 12.54 ± 0.03 b 7.50 ± 0.68 c 59.86 6.60 ± 0.23 c,d 52.61

NaFeEDTA 12.92 ± 0.10 b 7.85 ± 0.46 c 60.57 6.36 ± 0.26 c,d 49.23
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FSm 13.20 ± 0.27 b 3.18 ± 0.01 d 24.07 8.42 ± 0.41 a 63.77

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FFm 14.47 ± 0.30 a 4.26 ± 0.49 d 29.50 5.55 ± 0.66 d 38.29

Mean ± standard deviation (n = 4). Fe: iron, Zn: zinc, Ca: calcium. WWRB: whole wheat roll breads.
Control: whole wheat roll breads without addition of iron; FS: ferrous sulfate; FF: ferrous fumarate; RI:
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reduced iron; NaFeEDTA: sodium iron ethylenediaminetetraacetic acid; FSm: microencapsulated

ferrous sulfate; FFm: microencapsulated ferrous fumarate. Averages with different letters in the same
column indicate significant difference between samples (p<0.05).
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Table 1. Farinograph parameters of whole wheat flour control and fortified with the
different iron compounds.
WWF WA (%) AT (min) DDT (min) S (min) MTI (FU)
Control 56.93 ± 0.58 a 1.11 ± 0.10 a,b 13.43 ± 0.67 b 21.18 ± 1.94 c 37.33 ± 2.89 a
FS 56.23 ± 0.32 a,b 1.07 ± 0.01 b 15.59 ± 0.45 a 27.89 ± 0.37 a 20.67 ± 1.15 d,e
FF 56.30 ± 0.26 a,b 1.24 ± 0.03 a 15.01 ± 0.54 a 22.62 ± 1.65 b,c 30.00 ± 2.00 b,c
RI 56.57 ± 0.06 a,b 1.03 ± 0.04 b 14.73 ± 0.49 a,b 26.79 ± 2.42 a,b 23.39 ± 1.21 d
NaFeEDTA 55.97 ± 0.45 b 1.12 ± 0.05 a,b 14.17 ± 0.90 a,b 20.61 ± 1.51 c 34.33 ± 2.31 a,b
FSm 56.23 ± 0.25 a,b 1.17 ± 0.10 a,b 15.31 ± 0.22 a 25.73 ± 1.75 a,b 26.00 ± 2.00 c,d

PT
FFm 56.10 ± 0.17 a,b 1.11 ± 0.03 a,b 15.45 ± 0.15 a 27.90 ± 0.82 a 17.33 ± 1.15 e
Mean ± standard deviation. WWF: whole wheat flour. WA: water absorption; AT: arrival time; DDT:
dough development time; S: stability; MTI: mixing tolerance index; FU: farinographic units. Control:
whole wheat flour without addition of iron; FS: ferrous sulfate; FF: ferrous fumarate; RI: reduced iron;

RI
NaFeEDTA: sodium iron ethylenediaminetetraacetic acid; FSm: microencapsulated ferrous sulfate;
FFm: microencapsulated ferrous fumarate. Averages with different letters in the same column indicate
significant difference between samples (p<0.05).

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Table 2. Extensographic parameters of whole wheat flour control and fortified with
the different iron compounds.
Time
Parameter WWF
45 min 90 min 135 min
Control 694.33 ± 26.63 b,c 1088.67 ± 34.99 a,b 1011.00 ± 85.77 a
FS 664.33 ± 30.50 b,c 1128.67 ± 38.66 a 955.67 ± 22.50 a
Resistance to FF 637.00 ± 6.24 c 1067.00 ± 55.56 a,b 989.67 ± 62.50 a
extension, R RI 723.67 ± 26.50 a,b 965.67 ± 43.68 b 1155.00 ± 123.04 a

PT
(EU) NaFeEDTA 779.33 ± 44.38 a 1060.00 ± 24.43 a,b 1018.00 ± 21.66 a
FSm 675.67 ± 19.35 b,c 1145.33 ± 48.60 a 1005.33 ± 18.01 a
FFm 735.00 ± 23.58 a,b 1156.33 ± 64.66 a 1088.00 ± 25.24 a

RI
Control 796.00 ± 7.50 a 1107.57 ± 32.19 a 1084.80 ± 27.50 a,b
FS 746.93 ± 23.30 a 1110.00 ± 6.33 a 985.53 ± 21.03 b

SC
Maximum FF 720.43 ± 11.59 a 1074.13 ± 57.93 a 1042.87 ± 29.20 a,b
resistance to
extension, Rm RI 800.20 ± 25.13 a 1078.70 ± 74.06 a 1159.17 ± 105.67 a
NaFeEDTA 883.50 ± 54.71 b 1066.50 ± 21.04 a 1069.80 ± 71.50 a,b
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(UE)
FSm 741.83 ± 34.57 a 1150.17 ± 50.09 a 1066.43 ± 44.70 a,b
FFm 791.47 ± 25.48 a 1159.30 ± 68.51 a 1117.40 ± 9.90 a,b
Control 107.03 ± 2.97 a 80.13 ± 6.61 a 71.87 ± 2.10 a
MA

FS 105.87 ± 4.32 a 7487 ± 2.69 a 71.33 ± 7.84 a

FF 105.63 ± 1.12 a 77.70 ± 4.97 a 66.30 ± 2.46 a
Extensibility,
E (mm) RI 102.53 ± 1.46 a 69.20 ± 1.11 a 70.37 ± 3.25 a
D

NaFeEDTA 99.03 ± 7.28 a 73.93 ± 4.41 a 70.83 ± 3.91 a

FSm 102.53 ± 0.40 a 75.77 ± 0.40 a 70.13 ± 5.49 a
E

FFm 99.50 ± 6.51 a 73.20 ± 4.47 a 68.70 ± 4.27 a

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Control 6.5 ± 0.37 b,c 13.65 ± 1.12 a 15.01 ± 0.48 a,b

FS 6.29 ± 0.54 c 15.09 ± 0.63 a 13.50 ± 1.34 b
FF 6.03 ± 0.09 c 13.07 ± 0.87 a 14.92 ± 0.40 a,b
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Ratio number, D
(EU/mm) RI 7.06 ± 0.32 a,b,c 14.02 ± 0.57 a 15.78 ± 0.89 a
NaFeEDTA 7.88 ± 0.14 a 14.36 ± 0.74 a 15.51 ± 0.44 a,b
FSm 6.59 ± 0.20 b,c 15.11 ± 0.71 a 14.39 ± 0.98 a,b
AC

FFm 7.42 ± 0.74 a,b 14.89 ± 0.32 a 15.86 ± 0.62 a

Mean ± standard deviation. R: resistance to extension; Rm: maximum resistance to extension; E:
extensibility; D: ratio number (R / E); EU: extensographic units. WWF: whole wheat flour. Control:
whole wheat flour without addition of iron; FS: ferrous sulfate; FF: ferrous fumarate; RI: reduced iron;
NaFeEDTA: sodium iron ethylenediaminetetraacetic acid; FSm: microencapsulated ferrous sulfate;
FFm: microencapsulated ferrous fumarate. Averages with different letters in the same column indicate
significant difference between samples (p<0.05).

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Table 3. Quality parameters of the whole wheat roll breads.

WWRB SV (cm3/g) Height (cm) Width (cm)
Control 5.54 ± 0.56 a,b 5.36 ± 0.37 b 6.72 ± 0.26 c
FS 6.18 ± 0.39 a 5.68 ± 0.14 a,b 7.66 ± 0.19 a
FF 6.30 ± 0.36 a 5.63 ± 0.09 a,b 7.16 ± 0.10 b
RI 6.09 ± 0.22 a 5.83 ± 0.16 a,b 7.19 ± 0.07 b
NaFeEDTA 4.94 ± 0.19 b 5.89 ± 0.03 a 7.01 ± 0.09 b,c
FSm 5.10 ± 0.29 b 5.73 ± 0.38 a,b 7.25 ± 0.03 b
FFm 5.17 ± 0.12 b 5.52 ± 0.11 a,b 7.05 ± 0.24 b,c

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WWRB Shape Oven spring (cm) Cut opening (cm)
Control 0.80 ± 0.08 a,b 2.03 ± 0.18 a 0.65 ± 0.10 c
FS 0.74 ± 0.02 b 1.15 ± 0.20 b 0.76 ± 0.14 b,c

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FF 0.79 ± 0.01 a,b 1.18 ± 0.05 b 1.06 ± 0.12 a
RI 0.81 ± 0.02 a,b 1.52 ± 0.11 b 1.04 ± 0.02 a
NaFeEDTA 0.84 ± 0.01 a 1.50 ± 0.12 b 0.88 ± 0.03 a,b,c

SC
FSm 0.79 ± 0.05 a,b 1.28 ± 0.14 b 1.02 ± 0.08 a
FFm 0.78 ± 0.02 a,b 1.28 ± 0.08 b 0.97 ± 0.02 a,b
Mean ± standard deviation (n = 4). SV: specific volume; WWRB: whole wheat roll breads. FS: ferrous
NU
sulfate; Control: whole wheat roll breads without addition of iron; FF: ferrous fumarate; RI: reduced
iron; NaFeEDTA: sodium iron ethylenediaminetetraacetic acid; FSm: microencapsulated ferrous
sulfate; FFm: microencapsulated ferrous fumarate. Averages with different letters in the same column
indicate significant difference between samples (p<0.05).
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Table 4. Bioaccessibility of Fe, Zn and Ca in whole wheat roll breads.

Total Fe Soluble Fe Solubility Dialyzed Fe Dialysis
WWRB
(mg/100g) (mg/100g) (%) (mg/100g) (%)
Control 2.86 ± 0.03 e 0.34 ± 0.02 d 11.75 1.06 ± 0.08 d 37.17
FS 5.61 ± 0.25 b,c 1.05 ± 0.05 b 18.80 1.88 ± 0.20 b,c 33.50
FF 4.83 ± 0.06 d 1.07 ± 0.07 b 21.99 1.80 ± 0.18 b,c 37.33
RI 5.28 ± 0.12 b,c,d 0.96 ± 0.04 b,c 18.30 1.68 ± 0.15 c 31.79

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NaFeEDTA 5.71 ± 0.12 b 2.56 ± 0.13 a 44.80 2.64 ± 0.14 a 46.14
FSm 6.34 ± 0.10 a 0.34 ± 0.03 d 5.40 2.10 ± 0.24 b 33.12
FFm 5.21 ± 0.42 c,d 0.82 ± 0.11 c 14.80 1.80 ± 0.07 b,c 34.65

RI
Total Zn Soluble Zn Solubility Dialyzed Zn Dialysis
WWRB
(mg/100g) (mg/100g) (%) (mg/100g) (%)

SC
Control 1.13 ± 0.02 c 0.09 ± 0.01 c 7.88 0.26 ± 0.02 b 23.39
FS 1.21 ± 0.02 b,c 0.17 ± 0.01 a 14.41 0.26 ± 0.03 b 21.05
FF 1.20 ± 0.02 b,c 0.08 ± 0.01 c 6.75 0.28 ± 0.03 b 23.27
NU
RI 1.15 ± 0.01 b,c 0.11 ± 0.01 b,c 9.67 0.21 ± 0.01 b 18.40
NaFeEDTA 1.21 ± 0.02 b,c 0.18 ± 0.02 a 15.30 0.44 ± 0.02 a 35.96
FSm 1.47 ± 0.14 a 0.14 ± 0.02 b 9.62 0.49 ± 0.06 a 33.06
MA

FFm 1.28 ± 0.01 b 0.19 ± 0.02 a 14.99 0.47 ± 0.04 a 36.42

Total Ca Soluble Ca Solubility Dialyzed Ca Dialysis
WWRB
(mg/100g) (mg/100g) (%) (mg/100g) (%)
Control 12.95 ± 0.57 b 7.71 ± 0.71 c 63.33 6.88 ± 0.75 b,c 53.01
D

FS 14.31 ± 0.27 a 11.84 ± 0.99 a 82.87 8.12 ± 0.36 a,b 56.75

FF 14.14 ± 0.55 a 10.17 ± 0.57 b 72.09 7.42 ± 0.89 a,b,c 52.35
E

RI 12.54 ± 0.03 b 7.50 ± 0.68 c 59.86 6.60 ± 0.23 c,d 52.61

PT

NaFeEDTA 12.92 ± 0.10 b 7.85 ± 0.46 c 60.57 6.36 ± 0.26 c,d 49.23
FSm 13.20 ± 0.27 b 3.18 ± 0.01 d 24.07 8.42 ± 0.41 a 63.77
FFm 14.47 ± 0.30 a 4.26 ± 0.49 d 29.50 5.55 ± 0.66 d 38.29
CE

Mean ± standard deviation (n = 4). Fe: iron, Zn: zinc, Ca: calcium. WWRB: whole wheat roll breads.
Control: whole wheat roll breads without addition of iron; FS: ferrous sulfate; FF: ferrous fumarate; RI:
reduced iron; NaFeEDTA: sodium iron ethylenediaminetetraacetic acid; FSm: microencapsulated
ferrous sulfate; FFm: microencapsulated ferrous fumarate. Averages with different letters in the same
AC

column indicate significant difference between samples (p<0.05).

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Graphical abstract

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Highlights
The fortification of whole wheat flour with iron did not affect bread quality.

NaFeEDTA and FSm proved to be the most efficient for whole wheat flour
fortification.
Zinc bioaccessibility was positively influenced by NaFeEDTA and FSm.
Calcium bioaccessibility in whole roll breads was positively influenced by
FSm.

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