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Effect of iron-enrichment on the antioxidant properties of wheat flour and bread
Accepted Manuscript Effect of iron-enrichment on the antioxidant properties of wheat flour and bread Akram Ranjbar, Ali Heshmati, Javad Karami Momtaz, Aliasghar Vahidinia PII:
S0733-5210(18)30891-9
DOI:
https://doi.org/10.1016/j.jcs.2019.03.010
Reference:
YJCRS 2735
To appear in:
Journal of Cereal Science
Received Date: 22 November 2018 Revised Date:
7 March 2019
Accepted Date: 12 March 2019
Please cite this article as: Ranjbar, A., Heshmati, A., Momtaz, J.K., Vahidinia, A., Effect of ironenrichment on the antioxidant properties of wheat flour and bread, Journal of Cereal Science (2019), doi: https://doi.org/10.1016/j.jcs.2019.03.010. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT
Effect of iron-enrichment on the antioxidant properties of wheat flour and bread
1 2
Akram Ranjbara, Ali Heshmatia*, Javad Karami Momtaza, , Aliasghar Vahidiniaa Nutrition Health Research Center, Hamadan University of Medical Sciences,
4
Hamadan, Iran
5
RI PT
a
3
SC
.
6 7 8 9
[email protected] (Ali Heshmati)
10
Tel: +98 8138381821 Fax: +98 8138381822
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EP
TE D
M AN U
*Corresponding author:
12 13 14 15 16
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17 18 19 20 21 22 23 1
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24
The enrichment of wheat flour with iron attracted notable attention in some
25
countries in order to prevent anaemia. For the first time, the current study was
26
aimed to investigate the effects of iron enrichment on change trend of the
27
antioxidant compounds and total antioxidant
wheat flour
28
the Folin–Ciocalteu and
29
aluminium chloride methods were used to measure the phenolic acid and
30
flavonoid concentrations, respectively. Also, the TAC was evaluated by using
31
the
no
32
significant change in the concentrations of flavonoid and phenolic acid in the
33
unenriched
TAC
34
reduction of the iron-enriched flour samples was higher than that of the iron-
35
unenriched samples. Moreover, no change in antioxidant attributes as a result
36
DPPH
and
methods.
iron-enriched
In
this
According
regard,
to
results,
flour
samples
was
during
observed.
storage,
However,
TE D
and
FRAP
baking.
of
SC
storage and bread
capacity (TAC)
M AN U
during
RI PT
Abstract
37
flavonoid, and TAC during bread baking, while in the iron-enriched samples,
38
the observed decrease was more pronounced probably due to further losses in
39
the
power
of
antioxidant
components
as
well
as
pro-oxidant
40
properties of iron. Obtained findings could be useful for making decisions on
41
the continuation of iron enrichment.
42
Keywords: wheat flour, bread, antioxidant capacity, phenolic acid, flavonoid
43
AC C
reducing
EP
of fermentation was noted. There was a decline in levels of phenolic acid,
1. Introduction Nowadays,
consumers
44 have
become
more
nutritional
45
values of the consumed foods. In this regard, they prefer to consume healthier
46
2
aware
regarding
the
ACCEPTED MANUSCRIPT
47
as important staple foods in many countries are considered as good sources
48
for energy and nutrients (Mousavi Khaneghah et al., 2018; Mousavi et al.,
49
2019; Zhou et al., 2015; Žilić et al., 2010). Because of the health benefits of
50
wholegrain cereals and antioxidant-rich cereal bread, the demands for such
51
products
Khaneghah,
52
Martins, et al., 2018; Rao et al., 2018; Zhou et al., 2015) While they have
53
captured attentions of both consumers and researchers due to their important
54
roles in the prevention of several diseases (Abdel-Aal and Rabalski, 2013; Rao
55
et al., 2018). In this context, their rich antioxidant contents can prevent the
56
accumulation
Brumaghim,
57
2009) consequently protect the body through a balance between pro-oxidants
58
and
The
59
production of ROS occurs when the levels of pro-oxidants and antioxidants is
60
imbalanced; a condition called oxidative stress (Qingming et al., 2010).
61
Cereals are foods
While among the,
62
phenolic compounds, especially phenolic acids, are the main types of available
63
antioxidants in cereals (Zhou et al., 2015). In several countries, especially in
64
low- and middle-income countries, wheat bread is the most consumed type of
65
cereal products (Khaneghah, Fakhri, Raeisi, et al., 2018). Although there is a
66
decline in the antioxidant content during processing and storage.
67
growing
(Khaneghah,
Fakhri
and
Sant'Ana,
2018;
reactive
and/or
dietary
species
(ROS)
antioxidants
(Perron
(Masisi
et
with considerable antioxidant content
and
al.,
2016).
AC C
EP
endogenous
oxygen
TE D
of
M AN U
SC
are
RI PT
foods to keep them away from the disease. Cereal products, particularly bread,
Due to the high prevalence of iron deficiency anaemia in most of under
68
devolving countries such as Iran, as a routine approach, wheat flour is enriched
69
3
ACCEPTED MANUSCRIPT
iron.
However,
incorporation
the
70
antioxidant capacity (Abtahi et al., 2014). To the best of our knowledge, there is
71
no previous
the
72
antioxidant properties of wheat flour and produced bread. Therefore, for the
73
first time, the current study was aimed to investigate the effects of iron
74
enrichment on change trend of the antioxidant compounds and total antioxidant
75
capacity (TAC) of wheat flour during storage and bread baking.
76
investigation
of
regarding
iron
the
into
effects
the
of
flour
iron
can
reduce
enrichment
on
SC
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with
2.1.
Chemical agents
77
M AN U
2. Materials and methods
78 79
Iron-enriched/unenriched refined and whole wheat flour were obtained from
80
Sina flour factory (Hamadan, Iran).
81
TE D
Folin–
82
Ciocalteu's phenol reagents, ascorbic acid, and gallic acid were purchased
83
from Sigma Aldrich (St. Louis, MO, USA). Sodium hydroxide, choleric acid,
84
(TROLOX),
chloride,
methanol,
2,2-Diphenyl-1-picrylhydrazyl
ethyl
acetate,
and
other
(DPPH),
chemicals
all
in
85
analytical grade were purchased from Merck Company (Darmstadt, Germany).
86
The chemicals were of analytical grade.
87
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aluminum
acid
EP
2-carboxylic
The 6-hydroxy-2,5,7,8-tetramethylchroman-
2.2.
Preparation of the flour samples
88
The whole and refined wheat flour samples were prepared from Alvand wheat
89
cultivar by using hammer and roller mill, respectively. Extraction rate for whole
90
and refined flour was 100 and 72%, respectively. In order to enrich the
91
4
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92
were added by using a micro feeder machine. Flour samples were then stored
93
at room temperature for 60 days.
94
2.3.
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samples, iron (30 mg/kg) as ferrous sulfate (FeSO4) and folic acid (1.5 mg/kg)
Bread-making
95 96
Lavash bread (a typical bread in Iran). First, water (about 600 to 900 mL,
97
SC
Bread were prepared according to the conventional method used for baking
98
fresh flour and then mixed for 2.5 min with dough mixer (Mashhad Baking
99
Industries
was
100
fermented at 30 °C for 90 min in an incubator with 85% relative humidity. The
101
fermented dough was divided into ball-shaped pieces (150 g each) and then
102
left at room temperature for another 10 min to final fermentation or final proof.
103
Mashhad,
Iran)
to
obtain
a
firm
dough.
The
dough
TE D
Co,
M AN U
depending on the type of flour) and 0.5% dried yeast were added to 1000 g of
104
thick) and baked in a portable rotating oven (Mashhad Baking Industries Co,
105
Mashhad, Iran) at 300 °C for about 2 min. The samples were cooled at room
106
EP
The ball-shaped samples were then flattened into elliptical sheets (1.7 mm
107
°C in a refrigerator.
108
The concentrations of antioxidants and TAC of the dough (immediately after
109
fermentation) and bread (one day after preparation) were determined.
110
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temperature for 10 min, packaged in polyethylene bags, and then stored at 4-6
2.4.
Iron determination
111
5
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112
electric furnace (Shimaz Co. Tehran, Iran). One mL of chloride acid was added
113
to the ash, and the volume made up to 50 ml with adding of deionized water.
114
The Iron content of samples were analysed (mg/kg in dried weight) using
115
atomic absorption spectrometry (Thermo Fisher Scientific, USA) according to a
116
previously recommended method (Heshmati et al., 2017).
117
SC
2.5.
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The flour samples (2 g) were transferred to ash forms at 500 °C by using an
Extraction of free phenolic acid compounds
118 119
reported method (Lu et al., 2014) with some modifications. About 2 g of the
120
wheat flour sample, dough or bread were weighted and mixed with 60 mL of a
121
methanol-acetone-water solution (7:7:6; v/v/v) for 5 min. The solution was then
122
centrifuged at 5000 rpm for 10 min. The supernatant was removed and the pH
123
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The isolation of phenolic compounds was performed according to a previously
was adjusted to 2 with further addition of HCl (4 N). The bottom phase was re-
124
extracted twice
The
125
supernatants were collected, combined and concentrated until about 30 mL
126
the
methanol-acetone-water
(7:7:6;
v/v/v)
solution.
EP
with
127
concentrated supernatants with diethyl ether-ethyl acetate solution (1: 1, v/v).
128
AC C
under a vacuum condition. The phenolic acids were then extracted from the
2.6.
Extraction of the bonded phenolic acid compounds
129
After extraction the free phenolic compounds, the remaining insoluble residue
130
was
was
131
centrifuged at 5000 rpm for 10 min. The upper layer was transferred into a new
132
extracted
with
NaOH
(one
molar)
6
for
3
h.
Then,
the
mixture
ACCEPTED MANUSCRIPT
tube and acidified to pH 2 with HCl (4 molar). The bonded phenolic acids were
133
extracted with diethyl ether-ethyl acetate solution (1:1, v/v).
134
Phenolic acid determination
135
RI PT
2.7.
136
carbonate were added into 0.2 mL of the above-mentioned extracted solution.
137
The solution was mixed for 5 min and left at room temperature for 30 min. The
138 139
(Shimadzu Corporation, Kyoto, Japan) at 765 nm. A calibration curve was
140
plotted using different concentrations (50 to 1000 µg/mL) of a standard ferulic
141
acid
the
142
concentrations of phenolic acid expressed as µg of the equivalent ferulic acid
143
per gram of dried sample (Abdel-Aal and Rabalski, 2013; Abozed et al., 2014).
144
solution.
2.8.
was
The
measured
calibration
using
a
UV/VIS
M AN U
spectrophotometer
equation
was
used
to
determine
Flavonoid determination
flavonoid
concentration
was
determined
145 using
a
spectrophotometric
146
method (Adom and Liu, 2002) with slight modifications. About 0.5 mL of the
147
extracted solution, 2 mL of distilled water and 0.15 mL of 5% sodium nitrate
148
AC C
EP
The
absorbance
TE D
solution
SC
An aliquot of Folin-Ciocalteu’s phenol reagent (1 mL) and 0.8 M sodium
were added into a polyethylene tube. After 5 min, 0.15 mL of 10% aluminium
149
chloride was added. Five min later, 1 mL of 1 M sodium hydroxide was added.
150
The solution was thoroughly mixed and kept at room temperature for 15 min.
151
Before analysis, a calibration curve was drawn using different concentrations
152
(50 to 1000 µg/L) of a standard quercetin solution. The absorbance of the
153
sample solution was measured at 510 nm and compared to the quercetin
154
7
ACCEPTED MANUSCRIPT
standards. The flavonoid concentration was expressed as µg of the equivalent
155
quercetin per gram of the dried sample.
156
Antioxidant capacity assays
157
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2.9.
2.9.1. DPPH method
158 159
a previous study (Yu et al., 2013). A DPPH methanol solution (3.9 mL, 60
160
µmol/L)
The
161
absorbance was immediately measured at 515 nm. Then, the solution was left
162
in the dark for 60 min before a second absorbance reading was taken. TAC
163
was reported as the µmole equivalent of Trolox per gram of the dried sample.
164
added
into
a
0.1
mL
extract
and
thoroughly
mixed.
M AN U
was
SC
The antioxidant capacity was determined using DPPH method as described in
TE D
2.9.2. FRAP (Ferric Reducing Antioxidant Power) method
165 166
previous work (Selimović et al., 2014). The FRAP reagent was prepared by
167
mixing acetate buffer (25 mL, 0.1 M at pH 3.6), TPTZ (2,4,6-Tri(2-pyridyl)-s-
168
triazine) (2.5 mL, 10 mM) and ferric chloride (2.5 mL, 20 mM). The solution was
169
then incubated at 30 °C for 10 min. An aliquot of 200 µL was taken and
170
incubated at 37° C for an additional 10 min. The solution’s absorbance was
171
measured at 593 nm. The TAC was determined as the µmole equivalent of
172
Fe2+ per gram of dried sample, using an equation obtained from a calibration
173
curve prepared with different concentrations (50 to 1000 µmol/L) of FeSO4
174
7H2O (Benzie and Devaki, 2017).
175
AC C
EP
The TAC was determined using the FRAP method that was described in
8
ACCEPTED MANUSCRIPT
176
Data were analysed using version 17.0 of SPSS software (SPSS Inc., Chicago,
177
IL). Phenolic acid and flavonoid contents and TAC are reported as mean ±
178
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2.10. Statistical analysis
179
preparation and bread baking were determined. Their contents were separately
180
compared among unenriched and iron-enriched whole/refined flour, dough, and
181
SC
standard deviation and their reduction percentage during flour storage, dough
182
conducted to determine differences among the samples. A p-value of less than
183
0.05
was
considered
statistically
M AN U
bread samples. One-way analysis of variance (ANOVA) and Tukey's test were
significant.
All
chemical
tests
and
bread
preparation were carried out in triplicate. 3. Results
185 186
Iron content
187
TE D
3.1.
184
188
iron level in the iron-enriched whole flour (70.62 ± 2.98 mg/kg) was significantly
189
EP
The concentration of iron in various flour samples were shown in Table 1. The
higher than other samples (P<0.05). Change
trend
AC C
3.2.
in
190 wheat
phenolic
acid
concentration
during
flour storage and bread making
191 192
The phenolic acid content in different types of wheat flour as well as their
193
change trend during flour storage, dough preparation, and bread baking were
194
demonstrated in Table 2. During flour storage, phenolic acid content was
195
decreased which was not statistically significant. However, Iron enrichment no
196
9
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197
phenolic acid in whole wheat and refined flour was noted throughout storage
198
for 60 days at room temperature. Similar trends were observed in unenriched
199
and iron-enriched refined flour samples. The total, free, and bound phenolic
200
acid contents in unenriched whole flour sample was 3.44, 1.71, and 1.85 folds
201
higher than that in unenriched refined flour, respectively. Also, the levels of
202
total, free, and bound phenolic acids in iron-enriched whole flour samples were
203
3.67, 1.67, and 1.81 folds higher than corresponded values for iron-enriched
204
refined samples, respectively.
205
M AN U
SC
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significant changes in the decreasing trend of the total, free, and bound
206
change notably, significant reduction in the levels of bound and total phenolic
207
acid was observed after the completion of the baking process. No significant
208
difference was noted in the phenolic acid reduction percentages of unenriched
209
and iron-enriched samples during baking.
210
Change trend in wheat flavonoid content during flour storage
EP
3.3.
TE D
However, during the dough preparation, the phenolic acid content did not
and bread-making
211
AC C
212
Among flour investigated samples, the highest and lowest of total flavonoid
213
contents were found in unenriched fresh whole flour (1073.14 ± 16.17 µg/g)
214
and iron-enriched fresh refined flour (450.13 ± 14.43 µg/g) ,respectively. As
215
shown in Table 3, no significant change in the flavonoid content in unenriched
216
and non-enriched whole or refined flour during the storage time and dough
217
preparation was observed. In this regard, the baking did not influence the
218
10
ACCEPTED MANUSCRIPT
219
content significantly (p<0.05).
220
During baking, the reported reduction in the total flavonoid concentration was
221
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levels of free flavonoids although it decreased the bonded and total flavonoid
slightly higher for iron-enrich bread than unenriched samples (11.02 vs. 5.47%
222
for whole samples and 11.21 vs. 6.61 for refined samples).
223
Change
trend
in
antioxidant
capacity
of
wheat
flour
and
SC
3.4.
M AN U
bread with iron-enrichment
224 225
The highest TAC (4.68 µmole Trolox/g) was assigned for unenriched whole
226
fresh flour The TAC (as assessed by the DPPH and FRAP methods) of the
227
unenriched whole and refined flours
higher than
228
corresponded values for iron-enriched whole and refined fresh flours (Table 4).
229
Moreover,
was
230
significantly decreased. However, the TAC no significant change in TAC was
231
noted after the dough preparation.
232
storage
of
iron-enriched
flour
and
was
baking,
TAC
EP
TE D
during
and bread samples
233
the FRAP method) were 3.19 and 2.83 fold higher than those of the iron-
234
AC C
TAC of the iron-enriched and unenriched whole bread (as assessed by
enriched and unenriched refined bread, respectively. Higher reduction of TAC
235
was
iron-enriched
236
samples. During baking, the reductions in TAC was recorded as 8.40 and
237
18.28% in the unenriched and iron-enriched whole bread samples, and 18.46
238
and
239
noted
25.10%
in
the
in
the
refined
bread
unenriched
samples,
and
respectively.
especially
iron-enriched
in
refined
the
bread
samples,
240 11
ACCEPTED MANUSCRIPT
241 4. Discussion
242
Due to their specific chemical structures, polyphenols and flavonoids could
243
offer
Brumaghim,
244
2009). In the present study, the levels of phenolic acid and flavonoid in the flour
245
samples were higher than those reported in previous investigations (Menga et
246
al., 2010; Ragaee et al., 2006; Yu et al., 2004; Yu et al., 2013). For instance,
247
according to Ragaee et al. (2006), the amounts of phenolic acid in hard and
248
soft wheat were 562 and 501 µg equivalent of gallic acid/kg, respectively. In
249
another study, the concentration of phenolic acid in prepared wheat flour from
250
the Akron, Trego, and Platte varieties were measured as 212 to 113, 190 to
251
137, and 200 to 306 µg/g, respectively (Yu et al., 2004). These differences in
252
phenolic acid and flavonoid contents of wheat flour can be correlated with
253
as
powerful
antioxidants
(Perron
and
RI PT
properties
TE D
M AN U
SC
reducing
various parameters, such as the degree of flour extraction and environmental
254
and genetic factors (Yu et al., 2013).
255
been
reported
affect
256
phenolic acid yield in a higher rate while compared with genetic factors (Menga
257
EP
has
that
environmental
factors
also
could
AC C
It
et al., 2010). In the present study, the total amount of phenolic acid in iron-
258
enriched and unenriched whole flour was noted as 3.67 and 3.44 folds higher
259
than iron-enriched and unenriched refined flour, respectively. A 10 folds higher
260
concentration of phenolic acid in whole flour than refined flour also was
261
reported previously (Lu et al., 2015).
262
12
ACCEPTED MANUSCRIPT
our
results,
iron
enrichment
did
not
cause
significant
263
changes in the reduction trend of phenolic acid and flavonoid contents of whole
264
or refined wheat flour during 60 days of storage at room temperature. In other
265
words, no significant differences between the levels of
phenolic acid and
266
flavonoid in the iron-enriched and unenriched flour samples was noted. In
267
contrast, TAC of the iron-enriched samples was lower than the unenriched
268
ones. These findings showed that iron enrichment resulted in a significant
269
increment
polyphenols
270
depending on their structure and medium pH, chelate iron (including Fe3+) and
271
copper ions. Interactions between iron, flavonoids, and polyphenols in iron-
272
enriched
pro-oxidant
273
properties and could reduce TAC (Alvarez-Jubete et al., 2010; Mira et al.,
274
2002).
275
et
also
al.
could
trend
of
decrease
(2017)
TAC.
TAC.
TE D
flour
reduction
Flavonoids
M AN U
the
investigated
the
Besides,
correlation
and
iron
between
had
the
levels
of
276
phenolic acid, flavonoids, and metals in the fungal species of Imleria Badia.
277
They cultured fungal samples in media contaminated with arsenic, cadmium,
278
mercury, and lead as well as in a non-contaminated media. They found no
279
AC C
EP
Gasecka
in
SC
RI PT
Considering
differences between the phenolic and flavonoids contents of the fungi grown in
280
the
They
281
also found that the antioxidant capacity of the fungi grown in the contaminated
282
media was lower than in the non-contaminated media. Therefore, heavy metals
283
conform to the complex with phenolic acids and may reduce the possibility of
284
electron donating ability of phenolic acid to free radicals (Gąsecka et al., 2017).
285
contaminated
environment
and
the
13
non-contaminated
environment.
ACCEPTED MANUSCRIPT
286
observed during bread-making. In contrast to our findings, Lu et al. (2014)
287
reported that the amount of phenolic acid did not decrease significantly during
288
bread-making, regardless of whether whole or refined flour was used. They
289
observed a slight non-significant increment in the amount of phenolic acid in
290
breads prepared from whole or refined flour during fermentation (Lu et al.,
291
2014). Abdel-Aal and Rabalski (2013) found that in contrast to bonded phenolic
292
acid, the amount of free phenolic acids increased during the baking of bread,
293
muffins, and cookies. The thermal process liberated the bonded phenolic acid
294
and converted it to free phenolic acid. The total phenolic acid content in the
295
final products was lower than the initial sample (Abdel-Aal and Rabalski, 2013).
296 acid,
297
including ferric acid, was increased during dough fermentation (Ktenioudaki et
298
al., 2015) While the level of phenolic acid did not significantly change or may
299
have
300
slightly
studies
also
showed
that
the
TE D
Previous
M AN U
SC
RI PT
In the present study, a significant reduction in the amount of phenolic acid was
increased et
decomposed
of
al.,
2015).
EP
(Ktenioudaki
during
acid
However,
due
some
compounds,
such
of
to
free
the
Maillard
researchers as
phenolic
rutin,
reaction
reported during
the
301
baking
302
AC C
phenolic
bread-baking
amount
(Vogrinčič et al., 2010).
303 304
5. Conclusions The
present
study
was
305 the
first
report
iron
306
enrichment on change trend of the wheat flour antioxidant compounds and
307
TAC during storage and bread baking. According to results, iron enrichment did
308
14
regarding
the
influence
of
ACCEPTED MANUSCRIPT
309
whole or refined wheat flour samples during 60 days of storage at room
310
temperature. However, the TAC of iron-enriched samples was lower than that
311
of unenriched samples. The dough preparation stage did not have a significant
312
effect on the levels of phenolic acid, flavonoid, or TAC. During baking, the
313
amount
capacity
314
decreased. In enriched samples, the reduction of the mentioned compound
315
was
and
316
in
317
TAC reduction in iron enrichment bread should be evaluated to provide strong,
318
convincing
319
higher.
nutritionists.
Our
acid,
findings
Furthermore,
arguments
phenolic
could
making
useful regarding
decisions
TE D
enrichment.
Acknowledgment:
be
information
for
compounds,
The
authors
and
wish
to
antioxidant
SC
phenolic
for
the
industry,
on
consumers,
mechanisms
M AN U
of
RI PT
not cause significant changes in the phenolic acid and flavonoids contents of
the
acknowledge
involved
continuation
of
iron
320 321 the
financial
support
322 323
The authors would also like to thank Amin Mousavi Khaneghah (PhD) who
324
EP
from Hamadan University of Medical Science (Project No: 935142388).
helps for the English proofreading as a native speaker.
325
AC C
326
Conflict of interest Authors has no conflict of interests.
327 328
References
329
Abdel-Aal, E.-S.M., Rabalski, I., 2013. Effect of baking on free and bound phenolic
330
acids in wholegrain bakery products. Journal of Cereal Science 57, 312-318.
331
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ACCEPTED MANUSCRIPT
332
contents and antioxidant activities of various solvent extracts from whole wheat and
333
bran. Annals of Agricultural Sciences 59, 63-67.
334
Abtahi, M., Neyestani, T.R., Pouraram, H., Siassi, F., Dorosty, A.R., Elmadfa, I.,
335
Doustmohammadian, A., 2014. Iron-fortified flour: can it induce lipid peroxidation?
336
International journal of food sciences and nutrition 65, 649-654.
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RI PT
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K.R.,
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387
Optimization
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the
viability
of
Lactobacillus
acidophilus
and
TE D
of
M AN U
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Perron, N.R., Brumaghim, J.L., 2009. A review of the antioxidant mechanisms of
391
polyphenol compounds related to iron binding. Cell Biochem Biophys 53, 75-100.
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Degradation of rutin and polyphenols during the preparation of tartary buckwheat
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different locations for their antioxidant properties. Food Chemistry 86, 11-16.
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Refined and Whole Wheat Flour and Bread. Antioxidants 2, 370.
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SC
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Rao, S., Santhakumar, A.B., Chinkwo, K.A., Wu, G., Johnson, S.K., Blanchard, C.L.,
Zhou, X., Hao, T., Zhou, Y., Tang, W., Xiao, Y., Meng, X., Fang, X., 2015.
412
Relationships
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413
buckwheat during germination. Journal of Food Science and Technology 52, 2458-
414
antioxidant
compounds
and
antioxidant
activities
of
EP
2463.
between
415 416
2010. Bread and durum wheat compared for antioxidants contents, and lipoxygenase
417
and peroxidase activities. International journal of food science & technology 45, 1360-
418
1367.
419
AC C
Žilić, S.a., Dodig, D., Šukalović, V.H.-T., Maksimović, M., Saratlić, G., Škrbić, B.,
420
19
ACCEPTED MANUSCRIPT
Table 1. Iron concentration in various wheat flour Flour type
Flour status
40.47±1.97Ba
Refined flour
15.02±1.54Bb
superscript
capital
letters
within
a
70.62±2.98Aa
45.08±2.45Ab
SC
Different
Whole flour
Iron-enriched
RI PT
unenriched
row
indicate
statistically
significant
M AN U
differences (p<0.05) among values. Different superscript small letters within a column
AC C
EP
TE D
indicate statistically significant differences (p< 0.05) among values.
ACCEPTED MANUSCRIPT
Table 2. Phenolic acid content (µg equivalent of ferulic acid/g) in iron-enriched and unenriched wheat flour and its changes during the
RI PT
baking process of bread Sample type Type of Status
Whole
product Bound
Fresh Flour
242.15±15.12a
1312.14±25.10a
Stored
241.62±17.15a
1306.23±33.17a
Ironenriched
Bound
Total
1556.11±20.42a
72.03±5.32a
768.15±22.10a
841.15±11.15a
1547.85±35.62a
70.23±8.21a
762.15±20.14a
832.38±12.32a
M AN U
TE D
1345.23±20.19a
EP
Bread
262.23±12.15a
185.23±23.25a
1210.23±15.12b
AC C
Dough
Free
(0.53)
flour* unenriched
Total
SC
Free
Refined
1607.46±23.25a
(1.04) 73.45±2.56a
752.15±10.26a
(-3.30) 1395.46±28.96b
825.6±19.72a (1.85)
64.15±4.96a
623.12±9.26c
(13.19)
687.27±14.25c (16.76)
Fresh Flour
228.10±11.15a
1330.11±35.56a
1558.18±19.05a
64.52±2.87a
800.32±18.17a
864.52±10.32a
Stored
225.23±10.26a
1324.09±42.12a
1549.32±31.02a
61.24±5.34a
792.28±11.12a
853.52±16.38a
ACCEPTED MANUSCRIPT
Dough
(0.57)
245.23±10.26a
1330.23±29.42a
1575.46±35.24a (-1.11)
215.56±15.15a
1195.35±30.42b
1410.91±26.59b
SC
Bread
(1.27)
RI PT
flour*
* Flour samples were stored for 60 days in room temperature.
M AN U
(10.44)
62.24±3.80a
59.12±6.81a
779.41±21.39a
841.65±8.35a (2.65)
663.12±19.39b
722.24±23.12b (14.19)
Mean values of each column followed by different superscript letter significantly differ when subjected to Tukey’s
TE D
multiple range test (p<0.05).
AC C
EP
Values within parenthesis indicate reduction percentage in comparison with fresh flour.
ACCEPTED MANUSCRIPT
Table 3. Changes in flavonoid content (µg equivalent of quercetin/g) in unenriched and iron-enriched wheat flour and its changes
RI PT
during the baking process of bread Sample type
Type of
Whole
Status product
Total
Free
Bound
Fresh Flour
214.23±11.128a
858.87±29.65a
1073.14±16.17a
114.23±15.32a
324.32±17.87a
438.55±17.98a
Stored flour*
210.08±15.08a
852.21±29.65a
1062.96±32.16a
110.23±12.35a
319.12±15.36a
429.23±32.11a
SC
Bound
M AN U
unenriched
Free
Refined
(0.95)
203.12±26.32a
831.26±11.25a
785.12±22.32b
1066.58±12.25a
(2.13) 112.32±2.12a
295.09±9.65b
(0.95) 1008.24±14.25b
407.41±11.02a (7.10)
90.23±4.05a
290.21±12.63b
(5.47)
380.44±19.65b (6.61)
EP
Bread
235.32±12.23a
TE D
Dough
Total
enriched
Fresh Flour
239.32±27.17a
811.15±14.12a
1050.08±31.41a
109.01±5.17a
341.12±12.51a
450.13±14.43a
Stored flour*
233.02±30.12a
801.09±26.35a
1034.08±23.66a
105.01±9.12a
335.43±17.85a
440.01±25.19a
Dough
AC C
Iron-
251.45±15.87a
(1.52%) 772.31±23.21b
1023.76±15.63a (2.51)
(2.24) 113.25±5.22a
303.11±11.25b
416.36±29.65a (7.50)
ACCEPTED MANUSCRIPT
Bread
215.56±15.15a
695.35±30.42b
910.91±26.59b
89.65±10.21a
RI PT
(11.02%)
* Flour samples were stored for 60 days in room temperature.
280.02±20.12b
369.67±18.25b (11.21)
Mean values of each column followed by different superscript letter significantly differ when subjected to Tukey’s multiple range test
SC
(p<0.05).
AC C
EP
TE D
M AN U
Values within parenthesis indicate reduction percentage in comparison with fresh flour.
ACCEPTED MANUSCRIPT
Table 4. Antioxidant capacity in unenriched and iron-enriched wheat flour and its changes during
Refined
Whole
Refined
4.68±0.45a
1.89±0.32a
42.09±2.82a
16.98±1.09a
Stored flour*
4.61±0.62a
1.87±0.25a
41.23±3.45a
16.23±1.21a
SC
Fresh Flour
(1.50)
(1.06)
(2.04)
(4.42)
4.51±0.21a
1.8±.30a
40.95±3.56a
16.25±1.23a
(3.63)
(4.76)
(2.71)
(4.30)
3.71±0.19b
1.38±0.12b
37.51±3.02b
13.25±1.78b
(17.74)
(23.33)
(8.40)
(18.46)
Fresh Flour
4.63±0.16a
1.90±.0.55a
39.45±1.17a
15.86±1.87a
4.15±0.32b
1.56±.0.42b
35.12±2.15b
12.03±2.12b
(10.37)
(17.89)
(10.98)
(24.15)
4.1±0.15b
1.54±.21b
36.11±2.89b
12.35±2.06b
(11.45)
(18.95)
(8.47)
(22.13)
3.23±0.23c
0.95±.09c
29.51±1.81c
9.25±1.02c
(21.22)
(38.31)
(18.28)
(25.10)
Dough
TE D
Bread
Stored flour*
AC C
Iron-enriched
Fe2+/g)
of trolox/g) Whole
FRAP (µmole equivalent of
RI PT
DPPH (µmole equivalent
M AN U
unenriched
Applied method for antioxidant capacity assay
EP
product
Status (Iron-
Type of
unenriched/enriched)
the baking process of bread
Dough
Bread
* Flour samples were stored for 60 days in room temperature.
ACCEPTED MANUSCRIPT
Mean values of each column followed by different superscript letter significantly differ when subjected to Tukey’s multiple range test (p<0.05).
AC C
EP
TE D
M AN U
SC
RI PT
Values within parenthesis indicate reduction percentage in comparison with fresh flour
ACCEPTED MANUSCRIPT
highlights The change trend of antioxidant of wheat bread enriched with iron was assessed.
•
The iron had no effect on the change trend of phenolic acid and flavonoid levels.
•
Dough fermentation did not significantly alter the antioxidant attributes.
•
The iron enrichment raised the TAC reduction during flour storage and bread
RI PT
•
AC C
EP
TE D
M AN U
SC
baking.
S0733-5210(18)30891-9
DOI:
https://doi.org/10.1016/j.jcs.2019.03.010
Reference:
YJCRS 2735
To appear in:
Journal of Cereal Science
Received Date: 22 November 2018 Revised Date:
7 March 2019
Accepted Date: 12 March 2019
Please cite this article as: Ranjbar, A., Heshmati, A., Momtaz, J.K., Vahidinia, A., Effect of ironenrichment on the antioxidant properties of wheat flour and bread, Journal of Cereal Science (2019), doi: https://doi.org/10.1016/j.jcs.2019.03.010. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT
Effect of iron-enrichment on the antioxidant properties of wheat flour and bread
1 2
Akram Ranjbara, Ali Heshmatia*, Javad Karami Momtaza, , Aliasghar Vahidiniaa Nutrition Health Research Center, Hamadan University of Medical Sciences,
4
Hamadan, Iran
5
RI PT
a
3
SC
.
6 7 8 9
[email protected] (Ali Heshmati)
10
Tel: +98 8138381821 Fax: +98 8138381822
11
EP
TE D
M AN U
*Corresponding author:
12 13 14 15 16
AC C
17 18 19 20 21 22 23 1
ACCEPTED MANUSCRIPT
24
The enrichment of wheat flour with iron attracted notable attention in some
25
countries in order to prevent anaemia. For the first time, the current study was
26
aimed to investigate the effects of iron enrichment on change trend of the
27
antioxidant compounds and total antioxidant
wheat flour
28
the Folin–Ciocalteu and
29
aluminium chloride methods were used to measure the phenolic acid and
30
flavonoid concentrations, respectively. Also, the TAC was evaluated by using
31
the
no
32
significant change in the concentrations of flavonoid and phenolic acid in the
33
unenriched
TAC
34
reduction of the iron-enriched flour samples was higher than that of the iron-
35
unenriched samples. Moreover, no change in antioxidant attributes as a result
36
DPPH
and
methods.
iron-enriched
In
this
According
regard,
to
results,
flour
samples
was
during
observed.
storage,
However,
TE D
and
FRAP
baking.
of
SC
storage and bread
capacity (TAC)
M AN U
during
RI PT
Abstract
37
flavonoid, and TAC during bread baking, while in the iron-enriched samples,
38
the observed decrease was more pronounced probably due to further losses in
39
the
power
of
antioxidant
components
as
well
as
pro-oxidant
40
properties of iron. Obtained findings could be useful for making decisions on
41
the continuation of iron enrichment.
42
Keywords: wheat flour, bread, antioxidant capacity, phenolic acid, flavonoid
43
AC C
reducing
EP
of fermentation was noted. There was a decline in levels of phenolic acid,
1. Introduction Nowadays,
consumers
44 have
become
more
nutritional
45
values of the consumed foods. In this regard, they prefer to consume healthier
46
2
aware
regarding
the
ACCEPTED MANUSCRIPT
47
as important staple foods in many countries are considered as good sources
48
for energy and nutrients (Mousavi Khaneghah et al., 2018; Mousavi et al.,
49
2019; Zhou et al., 2015; Žilić et al., 2010). Because of the health benefits of
50
wholegrain cereals and antioxidant-rich cereal bread, the demands for such
51
products
Khaneghah,
52
Martins, et al., 2018; Rao et al., 2018; Zhou et al., 2015) While they have
53
captured attentions of both consumers and researchers due to their important
54
roles in the prevention of several diseases (Abdel-Aal and Rabalski, 2013; Rao
55
et al., 2018). In this context, their rich antioxidant contents can prevent the
56
accumulation
Brumaghim,
57
2009) consequently protect the body through a balance between pro-oxidants
58
and
The
59
production of ROS occurs when the levels of pro-oxidants and antioxidants is
60
imbalanced; a condition called oxidative stress (Qingming et al., 2010).
61
Cereals are foods
While among the,
62
phenolic compounds, especially phenolic acids, are the main types of available
63
antioxidants in cereals (Zhou et al., 2015). In several countries, especially in
64
low- and middle-income countries, wheat bread is the most consumed type of
65
cereal products (Khaneghah, Fakhri, Raeisi, et al., 2018). Although there is a
66
decline in the antioxidant content during processing and storage.
67
growing
(Khaneghah,
Fakhri
and
Sant'Ana,
2018;
reactive
and/or
dietary
species
(ROS)
antioxidants
(Perron
(Masisi
et
with considerable antioxidant content
and
al.,
2016).
AC C
EP
endogenous
oxygen
TE D
of
M AN U
SC
are
RI PT
foods to keep them away from the disease. Cereal products, particularly bread,
Due to the high prevalence of iron deficiency anaemia in most of under
68
devolving countries such as Iran, as a routine approach, wheat flour is enriched
69
3
ACCEPTED MANUSCRIPT
iron.
However,
incorporation
the
70
antioxidant capacity (Abtahi et al., 2014). To the best of our knowledge, there is
71
no previous
the
72
antioxidant properties of wheat flour and produced bread. Therefore, for the
73
first time, the current study was aimed to investigate the effects of iron
74
enrichment on change trend of the antioxidant compounds and total antioxidant
75
capacity (TAC) of wheat flour during storage and bread baking.
76
investigation
of
regarding
iron
the
into
effects
the
of
flour
iron
can
reduce
enrichment
on
SC
RI PT
with
2.1.
Chemical agents
77
M AN U
2. Materials and methods
78 79
Iron-enriched/unenriched refined and whole wheat flour were obtained from
80
Sina flour factory (Hamadan, Iran).
81
TE D
Folin–
82
Ciocalteu's phenol reagents, ascorbic acid, and gallic acid were purchased
83
from Sigma Aldrich (St. Louis, MO, USA). Sodium hydroxide, choleric acid,
84
(TROLOX),
chloride,
methanol,
2,2-Diphenyl-1-picrylhydrazyl
ethyl
acetate,
and
other
(DPPH),
chemicals
all
in
85
analytical grade were purchased from Merck Company (Darmstadt, Germany).
86
The chemicals were of analytical grade.
87
AC C
aluminum
acid
EP
2-carboxylic
The 6-hydroxy-2,5,7,8-tetramethylchroman-
2.2.
Preparation of the flour samples
88
The whole and refined wheat flour samples were prepared from Alvand wheat
89
cultivar by using hammer and roller mill, respectively. Extraction rate for whole
90
and refined flour was 100 and 72%, respectively. In order to enrich the
91
4
ACCEPTED MANUSCRIPT
92
were added by using a micro feeder machine. Flour samples were then stored
93
at room temperature for 60 days.
94
2.3.
RI PT
samples, iron (30 mg/kg) as ferrous sulfate (FeSO4) and folic acid (1.5 mg/kg)
Bread-making
95 96
Lavash bread (a typical bread in Iran). First, water (about 600 to 900 mL,
97
SC
Bread were prepared according to the conventional method used for baking
98
fresh flour and then mixed for 2.5 min with dough mixer (Mashhad Baking
99
Industries
was
100
fermented at 30 °C for 90 min in an incubator with 85% relative humidity. The
101
fermented dough was divided into ball-shaped pieces (150 g each) and then
102
left at room temperature for another 10 min to final fermentation or final proof.
103
Mashhad,
Iran)
to
obtain
a
firm
dough.
The
dough
TE D
Co,
M AN U
depending on the type of flour) and 0.5% dried yeast were added to 1000 g of
104
thick) and baked in a portable rotating oven (Mashhad Baking Industries Co,
105
Mashhad, Iran) at 300 °C for about 2 min. The samples were cooled at room
106
EP
The ball-shaped samples were then flattened into elliptical sheets (1.7 mm
107
°C in a refrigerator.
108
The concentrations of antioxidants and TAC of the dough (immediately after
109
fermentation) and bread (one day after preparation) were determined.
110
AC C
temperature for 10 min, packaged in polyethylene bags, and then stored at 4-6
2.4.
Iron determination
111
5
ACCEPTED MANUSCRIPT
112
electric furnace (Shimaz Co. Tehran, Iran). One mL of chloride acid was added
113
to the ash, and the volume made up to 50 ml with adding of deionized water.
114
The Iron content of samples were analysed (mg/kg in dried weight) using
115
atomic absorption spectrometry (Thermo Fisher Scientific, USA) according to a
116
previously recommended method (Heshmati et al., 2017).
117
SC
2.5.
RI PT
The flour samples (2 g) were transferred to ash forms at 500 °C by using an
Extraction of free phenolic acid compounds
118 119
reported method (Lu et al., 2014) with some modifications. About 2 g of the
120
wheat flour sample, dough or bread were weighted and mixed with 60 mL of a
121
methanol-acetone-water solution (7:7:6; v/v/v) for 5 min. The solution was then
122
centrifuged at 5000 rpm for 10 min. The supernatant was removed and the pH
123
TE D
M AN U
The isolation of phenolic compounds was performed according to a previously
was adjusted to 2 with further addition of HCl (4 N). The bottom phase was re-
124
extracted twice
The
125
supernatants were collected, combined and concentrated until about 30 mL
126
the
methanol-acetone-water
(7:7:6;
v/v/v)
solution.
EP
with
127
concentrated supernatants with diethyl ether-ethyl acetate solution (1: 1, v/v).
128
AC C
under a vacuum condition. The phenolic acids were then extracted from the
2.6.
Extraction of the bonded phenolic acid compounds
129
After extraction the free phenolic compounds, the remaining insoluble residue
130
was
was
131
centrifuged at 5000 rpm for 10 min. The upper layer was transferred into a new
132
extracted
with
NaOH
(one
molar)
6
for
3
h.
Then,
the
mixture
ACCEPTED MANUSCRIPT
tube and acidified to pH 2 with HCl (4 molar). The bonded phenolic acids were
133
extracted with diethyl ether-ethyl acetate solution (1:1, v/v).
134
Phenolic acid determination
135
RI PT
2.7.
136
carbonate were added into 0.2 mL of the above-mentioned extracted solution.
137
The solution was mixed for 5 min and left at room temperature for 30 min. The
138 139
(Shimadzu Corporation, Kyoto, Japan) at 765 nm. A calibration curve was
140
plotted using different concentrations (50 to 1000 µg/mL) of a standard ferulic
141
acid
the
142
concentrations of phenolic acid expressed as µg of the equivalent ferulic acid
143
per gram of dried sample (Abdel-Aal and Rabalski, 2013; Abozed et al., 2014).
144
solution.
2.8.
was
The
measured
calibration
using
a
UV/VIS
M AN U
spectrophotometer
equation
was
used
to
determine
Flavonoid determination
flavonoid
concentration
was
determined
145 using
a
spectrophotometric
146
method (Adom and Liu, 2002) with slight modifications. About 0.5 mL of the
147
extracted solution, 2 mL of distilled water and 0.15 mL of 5% sodium nitrate
148
AC C
EP
The
absorbance
TE D
solution
SC
An aliquot of Folin-Ciocalteu’s phenol reagent (1 mL) and 0.8 M sodium
were added into a polyethylene tube. After 5 min, 0.15 mL of 10% aluminium
149
chloride was added. Five min later, 1 mL of 1 M sodium hydroxide was added.
150
The solution was thoroughly mixed and kept at room temperature for 15 min.
151
Before analysis, a calibration curve was drawn using different concentrations
152
(50 to 1000 µg/L) of a standard quercetin solution. The absorbance of the
153
sample solution was measured at 510 nm and compared to the quercetin
154
7
ACCEPTED MANUSCRIPT
standards. The flavonoid concentration was expressed as µg of the equivalent
155
quercetin per gram of the dried sample.
156
Antioxidant capacity assays
157
RI PT
2.9.
2.9.1. DPPH method
158 159
a previous study (Yu et al., 2013). A DPPH methanol solution (3.9 mL, 60
160
µmol/L)
The
161
absorbance was immediately measured at 515 nm. Then, the solution was left
162
in the dark for 60 min before a second absorbance reading was taken. TAC
163
was reported as the µmole equivalent of Trolox per gram of the dried sample.
164
added
into
a
0.1
mL
extract
and
thoroughly
mixed.
M AN U
was
SC
The antioxidant capacity was determined using DPPH method as described in
TE D
2.9.2. FRAP (Ferric Reducing Antioxidant Power) method
165 166
previous work (Selimović et al., 2014). The FRAP reagent was prepared by
167
mixing acetate buffer (25 mL, 0.1 M at pH 3.6), TPTZ (2,4,6-Tri(2-pyridyl)-s-
168
triazine) (2.5 mL, 10 mM) and ferric chloride (2.5 mL, 20 mM). The solution was
169
then incubated at 30 °C for 10 min. An aliquot of 200 µL was taken and
170
incubated at 37° C for an additional 10 min. The solution’s absorbance was
171
measured at 593 nm. The TAC was determined as the µmole equivalent of
172
Fe2+ per gram of dried sample, using an equation obtained from a calibration
173
curve prepared with different concentrations (50 to 1000 µmol/L) of FeSO4
174
7H2O (Benzie and Devaki, 2017).
175
AC C
EP
The TAC was determined using the FRAP method that was described in
8
ACCEPTED MANUSCRIPT
176
Data were analysed using version 17.0 of SPSS software (SPSS Inc., Chicago,
177
IL). Phenolic acid and flavonoid contents and TAC are reported as mean ±
178
RI PT
2.10. Statistical analysis
179
preparation and bread baking were determined. Their contents were separately
180
compared among unenriched and iron-enriched whole/refined flour, dough, and
181
SC
standard deviation and their reduction percentage during flour storage, dough
182
conducted to determine differences among the samples. A p-value of less than
183
0.05
was
considered
statistically
M AN U
bread samples. One-way analysis of variance (ANOVA) and Tukey's test were
significant.
All
chemical
tests
and
bread
preparation were carried out in triplicate. 3. Results
185 186
Iron content
187
TE D
3.1.
184
188
iron level in the iron-enriched whole flour (70.62 ± 2.98 mg/kg) was significantly
189
EP
The concentration of iron in various flour samples were shown in Table 1. The
higher than other samples (P<0.05). Change
trend
AC C
3.2.
in
190 wheat
phenolic
acid
concentration
during
flour storage and bread making
191 192
The phenolic acid content in different types of wheat flour as well as their
193
change trend during flour storage, dough preparation, and bread baking were
194
demonstrated in Table 2. During flour storage, phenolic acid content was
195
decreased which was not statistically significant. However, Iron enrichment no
196
9
ACCEPTED MANUSCRIPT
197
phenolic acid in whole wheat and refined flour was noted throughout storage
198
for 60 days at room temperature. Similar trends were observed in unenriched
199
and iron-enriched refined flour samples. The total, free, and bound phenolic
200
acid contents in unenriched whole flour sample was 3.44, 1.71, and 1.85 folds
201
higher than that in unenriched refined flour, respectively. Also, the levels of
202
total, free, and bound phenolic acids in iron-enriched whole flour samples were
203
3.67, 1.67, and 1.81 folds higher than corresponded values for iron-enriched
204
refined samples, respectively.
205
M AN U
SC
RI PT
significant changes in the decreasing trend of the total, free, and bound
206
change notably, significant reduction in the levels of bound and total phenolic
207
acid was observed after the completion of the baking process. No significant
208
difference was noted in the phenolic acid reduction percentages of unenriched
209
and iron-enriched samples during baking.
210
Change trend in wheat flavonoid content during flour storage
EP
3.3.
TE D
However, during the dough preparation, the phenolic acid content did not
and bread-making
211
AC C
212
Among flour investigated samples, the highest and lowest of total flavonoid
213
contents were found in unenriched fresh whole flour (1073.14 ± 16.17 µg/g)
214
and iron-enriched fresh refined flour (450.13 ± 14.43 µg/g) ,respectively. As
215
shown in Table 3, no significant change in the flavonoid content in unenriched
216
and non-enriched whole or refined flour during the storage time and dough
217
preparation was observed. In this regard, the baking did not influence the
218
10
ACCEPTED MANUSCRIPT
219
content significantly (p<0.05).
220
During baking, the reported reduction in the total flavonoid concentration was
221
RI PT
levels of free flavonoids although it decreased the bonded and total flavonoid
slightly higher for iron-enrich bread than unenriched samples (11.02 vs. 5.47%
222
for whole samples and 11.21 vs. 6.61 for refined samples).
223
Change
trend
in
antioxidant
capacity
of
wheat
flour
and
SC
3.4.
M AN U
bread with iron-enrichment
224 225
The highest TAC (4.68 µmole Trolox/g) was assigned for unenriched whole
226
fresh flour The TAC (as assessed by the DPPH and FRAP methods) of the
227
unenriched whole and refined flours
higher than
228
corresponded values for iron-enriched whole and refined fresh flours (Table 4).
229
Moreover,
was
230
significantly decreased. However, the TAC no significant change in TAC was
231
noted after the dough preparation.
232
storage
of
iron-enriched
flour
and
was
baking,
TAC
EP
TE D
during
and bread samples
233
the FRAP method) were 3.19 and 2.83 fold higher than those of the iron-
234
AC C
TAC of the iron-enriched and unenriched whole bread (as assessed by
enriched and unenriched refined bread, respectively. Higher reduction of TAC
235
was
iron-enriched
236
samples. During baking, the reductions in TAC was recorded as 8.40 and
237
18.28% in the unenriched and iron-enriched whole bread samples, and 18.46
238
and
239
noted
25.10%
in
the
in
the
refined
bread
unenriched
samples,
and
respectively.
especially
iron-enriched
in
refined
the
bread
samples,
240 11
ACCEPTED MANUSCRIPT
241 4. Discussion
242
Due to their specific chemical structures, polyphenols and flavonoids could
243
offer
Brumaghim,
244
2009). In the present study, the levels of phenolic acid and flavonoid in the flour
245
samples were higher than those reported in previous investigations (Menga et
246
al., 2010; Ragaee et al., 2006; Yu et al., 2004; Yu et al., 2013). For instance,
247
according to Ragaee et al. (2006), the amounts of phenolic acid in hard and
248
soft wheat were 562 and 501 µg equivalent of gallic acid/kg, respectively. In
249
another study, the concentration of phenolic acid in prepared wheat flour from
250
the Akron, Trego, and Platte varieties were measured as 212 to 113, 190 to
251
137, and 200 to 306 µg/g, respectively (Yu et al., 2004). These differences in
252
phenolic acid and flavonoid contents of wheat flour can be correlated with
253
as
powerful
antioxidants
(Perron
and
RI PT
properties
TE D
M AN U
SC
reducing
various parameters, such as the degree of flour extraction and environmental
254
and genetic factors (Yu et al., 2013).
255
been
reported
affect
256
phenolic acid yield in a higher rate while compared with genetic factors (Menga
257
EP
has
that
environmental
factors
also
could
AC C
It
et al., 2010). In the present study, the total amount of phenolic acid in iron-
258
enriched and unenriched whole flour was noted as 3.67 and 3.44 folds higher
259
than iron-enriched and unenriched refined flour, respectively. A 10 folds higher
260
concentration of phenolic acid in whole flour than refined flour also was
261
reported previously (Lu et al., 2015).
262
12
ACCEPTED MANUSCRIPT
our
results,
iron
enrichment
did
not
cause
significant
263
changes in the reduction trend of phenolic acid and flavonoid contents of whole
264
or refined wheat flour during 60 days of storage at room temperature. In other
265
words, no significant differences between the levels of
phenolic acid and
266
flavonoid in the iron-enriched and unenriched flour samples was noted. In
267
contrast, TAC of the iron-enriched samples was lower than the unenriched
268
ones. These findings showed that iron enrichment resulted in a significant
269
increment
polyphenols
270
depending on their structure and medium pH, chelate iron (including Fe3+) and
271
copper ions. Interactions between iron, flavonoids, and polyphenols in iron-
272
enriched
pro-oxidant
273
properties and could reduce TAC (Alvarez-Jubete et al., 2010; Mira et al.,
274
2002).
275
et
also
al.
could
trend
of
decrease
(2017)
TAC.
TAC.
TE D
flour
reduction
Flavonoids
M AN U
the
investigated
the
Besides,
correlation
and
iron
between
had
the
levels
of
276
phenolic acid, flavonoids, and metals in the fungal species of Imleria Badia.
277
They cultured fungal samples in media contaminated with arsenic, cadmium,
278
mercury, and lead as well as in a non-contaminated media. They found no
279
AC C
EP
Gasecka
in
SC
RI PT
Considering
differences between the phenolic and flavonoids contents of the fungi grown in
280
the
They
281
also found that the antioxidant capacity of the fungi grown in the contaminated
282
media was lower than in the non-contaminated media. Therefore, heavy metals
283
conform to the complex with phenolic acids and may reduce the possibility of
284
electron donating ability of phenolic acid to free radicals (Gąsecka et al., 2017).
285
contaminated
environment
and
the
13
non-contaminated
environment.
ACCEPTED MANUSCRIPT
286
observed during bread-making. In contrast to our findings, Lu et al. (2014)
287
reported that the amount of phenolic acid did not decrease significantly during
288
bread-making, regardless of whether whole or refined flour was used. They
289
observed a slight non-significant increment in the amount of phenolic acid in
290
breads prepared from whole or refined flour during fermentation (Lu et al.,
291
2014). Abdel-Aal and Rabalski (2013) found that in contrast to bonded phenolic
292
acid, the amount of free phenolic acids increased during the baking of bread,
293
muffins, and cookies. The thermal process liberated the bonded phenolic acid
294
and converted it to free phenolic acid. The total phenolic acid content in the
295
final products was lower than the initial sample (Abdel-Aal and Rabalski, 2013).
296 acid,
297
including ferric acid, was increased during dough fermentation (Ktenioudaki et
298
al., 2015) While the level of phenolic acid did not significantly change or may
299
have
300
slightly
studies
also
showed
that
the
TE D
Previous
M AN U
SC
RI PT
In the present study, a significant reduction in the amount of phenolic acid was
increased et
decomposed
of
al.,
2015).
EP
(Ktenioudaki
during
acid
However,
due
some
compounds,
such
of
to
free
the
Maillard
researchers as
phenolic
rutin,
reaction
reported during
the
301
baking
302
AC C
phenolic
bread-baking
amount
(Vogrinčič et al., 2010).
303 304
5. Conclusions The
present
study
was
305 the
first
report
iron
306
enrichment on change trend of the wheat flour antioxidant compounds and
307
TAC during storage and bread baking. According to results, iron enrichment did
308
14
regarding
the
influence
of
ACCEPTED MANUSCRIPT
309
whole or refined wheat flour samples during 60 days of storage at room
310
temperature. However, the TAC of iron-enriched samples was lower than that
311
of unenriched samples. The dough preparation stage did not have a significant
312
effect on the levels of phenolic acid, flavonoid, or TAC. During baking, the
313
amount
capacity
314
decreased. In enriched samples, the reduction of the mentioned compound
315
was
and
316
in
317
TAC reduction in iron enrichment bread should be evaluated to provide strong,
318
convincing
319
higher.
nutritionists.
Our
acid,
findings
Furthermore,
arguments
phenolic
could
making
useful regarding
decisions
TE D
enrichment.
Acknowledgment:
be
information
for
compounds,
The
authors
and
wish
to
antioxidant
SC
phenolic
for
the
industry,
on
consumers,
mechanisms
M AN U
of
RI PT
not cause significant changes in the phenolic acid and flavonoids contents of
the
acknowledge
involved
continuation
of
iron
320 321 the
financial
support
322 323
The authors would also like to thank Amin Mousavi Khaneghah (PhD) who
324
EP
from Hamadan University of Medical Science (Project No: 935142388).
helps for the English proofreading as a native speaker.
325
AC C
326
Conflict of interest Authors has no conflict of interests.
327 328
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329
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420
19
ACCEPTED MANUSCRIPT
Table 1. Iron concentration in various wheat flour Flour type
Flour status
40.47±1.97Ba
Refined flour
15.02±1.54Bb
superscript
capital
letters
within
a
70.62±2.98Aa
45.08±2.45Ab
SC
Different
Whole flour
Iron-enriched
RI PT
unenriched
row
indicate
statistically
significant
M AN U
differences (p<0.05) among values. Different superscript small letters within a column
AC C
EP
TE D
indicate statistically significant differences (p< 0.05) among values.
ACCEPTED MANUSCRIPT
Table 2. Phenolic acid content (µg equivalent of ferulic acid/g) in iron-enriched and unenriched wheat flour and its changes during the
RI PT
baking process of bread Sample type Type of Status
Whole
product Bound
Fresh Flour
242.15±15.12a
1312.14±25.10a
Stored
241.62±17.15a
1306.23±33.17a
Ironenriched
Bound
Total
1556.11±20.42a
72.03±5.32a
768.15±22.10a
841.15±11.15a
1547.85±35.62a
70.23±8.21a
762.15±20.14a
832.38±12.32a
M AN U
TE D
1345.23±20.19a
EP
Bread
262.23±12.15a
185.23±23.25a
1210.23±15.12b
AC C
Dough
Free
(0.53)
flour* unenriched
Total
SC
Free
Refined
1607.46±23.25a
(1.04) 73.45±2.56a
752.15±10.26a
(-3.30) 1395.46±28.96b
825.6±19.72a (1.85)
64.15±4.96a
623.12±9.26c
(13.19)
687.27±14.25c (16.76)
Fresh Flour
228.10±11.15a
1330.11±35.56a
1558.18±19.05a
64.52±2.87a
800.32±18.17a
864.52±10.32a
Stored
225.23±10.26a
1324.09±42.12a
1549.32±31.02a
61.24±5.34a
792.28±11.12a
853.52±16.38a
ACCEPTED MANUSCRIPT
Dough
(0.57)
245.23±10.26a
1330.23±29.42a
1575.46±35.24a (-1.11)
215.56±15.15a
1195.35±30.42b
1410.91±26.59b
SC
Bread
(1.27)
RI PT
flour*
* Flour samples were stored for 60 days in room temperature.
M AN U
(10.44)
62.24±3.80a
59.12±6.81a
779.41±21.39a
841.65±8.35a (2.65)
663.12±19.39b
722.24±23.12b (14.19)
Mean values of each column followed by different superscript letter significantly differ when subjected to Tukey’s
TE D
multiple range test (p<0.05).
AC C
EP
Values within parenthesis indicate reduction percentage in comparison with fresh flour.
ACCEPTED MANUSCRIPT
Table 3. Changes in flavonoid content (µg equivalent of quercetin/g) in unenriched and iron-enriched wheat flour and its changes
RI PT
during the baking process of bread Sample type
Type of
Whole
Status product
Total
Free
Bound
Fresh Flour
214.23±11.128a
858.87±29.65a
1073.14±16.17a
114.23±15.32a
324.32±17.87a
438.55±17.98a
Stored flour*
210.08±15.08a
852.21±29.65a
1062.96±32.16a
110.23±12.35a
319.12±15.36a
429.23±32.11a
SC
Bound
M AN U
unenriched
Free
Refined
(0.95)
203.12±26.32a
831.26±11.25a
785.12±22.32b
1066.58±12.25a
(2.13) 112.32±2.12a
295.09±9.65b
(0.95) 1008.24±14.25b
407.41±11.02a (7.10)
90.23±4.05a
290.21±12.63b
(5.47)
380.44±19.65b (6.61)
EP
Bread
235.32±12.23a
TE D
Dough
Total
enriched
Fresh Flour
239.32±27.17a
811.15±14.12a
1050.08±31.41a
109.01±5.17a
341.12±12.51a
450.13±14.43a
Stored flour*
233.02±30.12a
801.09±26.35a
1034.08±23.66a
105.01±9.12a
335.43±17.85a
440.01±25.19a
Dough
AC C
Iron-
251.45±15.87a
(1.52%) 772.31±23.21b
1023.76±15.63a (2.51)
(2.24) 113.25±5.22a
303.11±11.25b
416.36±29.65a (7.50)
ACCEPTED MANUSCRIPT
Bread
215.56±15.15a
695.35±30.42b
910.91±26.59b
89.65±10.21a
RI PT
(11.02%)
* Flour samples were stored for 60 days in room temperature.
280.02±20.12b
369.67±18.25b (11.21)
Mean values of each column followed by different superscript letter significantly differ when subjected to Tukey’s multiple range test
SC
(p<0.05).
AC C
EP
TE D
M AN U
Values within parenthesis indicate reduction percentage in comparison with fresh flour.
ACCEPTED MANUSCRIPT
Table 4. Antioxidant capacity in unenriched and iron-enriched wheat flour and its changes during
Refined
Whole
Refined
4.68±0.45a
1.89±0.32a
42.09±2.82a
16.98±1.09a
Stored flour*
4.61±0.62a
1.87±0.25a
41.23±3.45a
16.23±1.21a
SC
Fresh Flour
(1.50)
(1.06)
(2.04)
(4.42)
4.51±0.21a
1.8±.30a
40.95±3.56a
16.25±1.23a
(3.63)
(4.76)
(2.71)
(4.30)
3.71±0.19b
1.38±0.12b
37.51±3.02b
13.25±1.78b
(17.74)
(23.33)
(8.40)
(18.46)
Fresh Flour
4.63±0.16a
1.90±.0.55a
39.45±1.17a
15.86±1.87a
4.15±0.32b
1.56±.0.42b
35.12±2.15b
12.03±2.12b
(10.37)
(17.89)
(10.98)
(24.15)
4.1±0.15b
1.54±.21b
36.11±2.89b
12.35±2.06b
(11.45)
(18.95)
(8.47)
(22.13)
3.23±0.23c
0.95±.09c
29.51±1.81c
9.25±1.02c
(21.22)
(38.31)
(18.28)
(25.10)
Dough
TE D
Bread
Stored flour*
AC C
Iron-enriched
Fe2+/g)
of trolox/g) Whole
FRAP (µmole equivalent of
RI PT
DPPH (µmole equivalent
M AN U
unenriched
Applied method for antioxidant capacity assay
EP
product
Status (Iron-
Type of
unenriched/enriched)
the baking process of bread
Dough
Bread
* Flour samples were stored for 60 days in room temperature.
ACCEPTED MANUSCRIPT
Mean values of each column followed by different superscript letter significantly differ when subjected to Tukey’s multiple range test (p<0.05).
AC C
EP
TE D
M AN U
SC
RI PT
Values within parenthesis indicate reduction percentage in comparison with fresh flour
ACCEPTED MANUSCRIPT
highlights The change trend of antioxidant of wheat bread enriched with iron was assessed.
•
The iron had no effect on the change trend of phenolic acid and flavonoid levels.
•
Dough fermentation did not significantly alter the antioxidant attributes.
•
The iron enrichment raised the TAC reduction during flour storage and bread
RI PT
•
AC C
EP
TE D
M AN U
SC
baking.