Effect of iron-enrichment on the antioxidant properties of wheat flour and bread

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,...

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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

11

EP

TE D

M AN U

*Corresponding author:

12 13 14 15 16

AC C

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

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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

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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.

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

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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.

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

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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

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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

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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

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

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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

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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

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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

15

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

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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.