Whey PeptideâIron Complexes Increase the Oxidative Stability of Oil

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Whey peptide-iron complexes increase the oxidative stability of oil-in-water emulsions in comparison to iron salts Maria Elisa Caetano-Silva, Lilian Regina Barros Mariutti, Neura Bragagnolo, Maria Teresa Bertoldo Pacheco, and Flavia Maria Netto J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.7b04873 • Publication Date (Web): 04 Feb 2018 Downloaded from http://pubs.acs.org on February 5, 2018

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Journal of Agricultural and Food Chemistry

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Whey peptide-iron complexes increase the oxidative stability of oil-in-water

2

emulsions in comparison to iron salts

3 4 5

Maria Elisa Caetano-Silvaa, Lilian Regina Barros Mariuttia, Neura Bragagnolob, Maria

6

Teresa Bertoldo-Pachecoc, Flavia Maria Nettoa

7 8

a

9

Campinas, UNICAMP, 13083-862 Campinas, SP, Brazil.

Department of Food and Nutrition, Faculty of Food Engineering, University of

10

b

11

UNICAMP, 13083-862 Campinas, SP, Brazil.

12

c

13

Campinas, SP, Brazil.

Department of Food Science, Faculty of Food Engineering, University of Campinas,

Center of Food Science and Quality, Institute of Food Technology, ITAL, 13070-178

*Corresponding author: M. E. Caetano-Silva. e-mail [email protected] ACS Paragon Plus Environment


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ABSTRACT

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Food fortification with iron may favor lipid oxidation both in food matrices and the

16

human body. This study aimed at evaluating the effect of peptide-iron complexation on

17

lipid oxidation catalyzed by iron, using oil-in-water (O/W) emulsions as a model

18

system. The extent of lipid oxidation of emulsions containing iron salts (FeSO4 or

19

FeCl2) or iron complexes (peptide-iron complexes or ferrous bisglycinate) was

20

evaluated during 7 days, measured as primary (peroxide value) and secondary products

21

(TBARS and volatile compounds). Both salts catalyzed lipid oxidation, leading to

22

peroxide values 2.6- to 4.6-fold higher than the values found for the peptide-iron

23

complexes. The addition of the peptide-iron complexes resulted in the formation of

24

lower amounts of secondary volatiles of lipid oxidation (up to 78-fold) than those of

25

iron salts, possibly due to the antioxidant activity of the peptides and their capacity to

26

keep iron apart from the lipid phase, since the iron atom is coordinated and takes part of

27

a stable structure. The peptide-iron complexes showed potential to reduce the

28

undesirable sensory changes in food products, and decrease the side effects related to

29

free iron and the lipid damage of cell membranes in the organism, due to the lower

30

reactivity of iron in the complexed form.

31 32 33

Keywords: Food fortification, HS-SPME-GC-MS, iron complexes, volatile lipid

34

oxidation products (VLOPs), whey protein hydrolysate.

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ACS Paragon Plus Environment

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1. Introduction

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Food fortification with iron is a strategy employed in many countries to decrease

38

the anemia prevalence, one of the main nutritional problems in the world 1. For this

39

practice to be effective, it is necessary to avoid sensory changes and assure iron

40

bioavailability. However, the most bioavailable forms of iron are generally more

41

reactive 2, thus, food fortification with this mineral may result in changes in physical

42

and sensory properties of foods 3. Among the compounds that can be used for this

43

purpose, ferrous sulfate has been the most used, despite its side effects, such as

44

heartburn, abdominal pain, nausea and diarrhea 4. The use of this salt has also been

45

related to the formation of hydroxyl radicals, which can start the peroxidation of

46

membrane lipids, inactivate enzymes and cause damage to DNA 5, 6.

47

In iron-fortified food products, lipid oxidation has a crucial role in the quality

48

and shelf life due to the deleterious effects on polyunsaturated fatty acids and other

49

oxidizable substrates

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emulsified lipids, leads to rancidity, with the development of undesirable flavors 9. Iron

51

can also catalyze lipid oxidation of cell membranes

52

reactive oxygen species (ROS). These ROS participate in tissue injuries and have been

53

related to cardio and neurological diseases

54

related to gastric mucosa damage 13 and may intensify inflammatory disturbs 14, 15.

7, 8

. The production of free radicals, catalyzed by iron ions in

10

, resulting in the formation of

11, 12

. In addition, free iron has also been

55

Iron may lead to the formation of lipid radicals by the Fenton reaction, in which

56

the metal yield to hydroxyl radicals (•OH) from the radical anion superoxide (O2•−) and

57

hydrogen peroxide (H2O2) 16, which can then abstract hydrogen atoms from unsaturated

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fatty acids 9. These unsaturated fatty acids can form alkyl and peroxyl radicals as a

59

consequence of free radical chain reactions in the presence of oxygen

17

. Lipid

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hydroperoxides (LOOH), the primary compounds of lipid oxidation, are formed by the

61

reaction of these radicals and give rise to low molecular weight secondary products,

62

such as aldehydes, ketones, and alcohols

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products of lipid oxidation should be monitored to follow the oxidative process.

64

18

. Therefore, both primary and secondary

Proteins dispersed in the continuous phase of oil-in-water (O/W) emulsions can 10, 19, 20

65

inhibit lipid oxidation

, but peptides have higher antioxidant potential and may

66

enhance the protection against lipid oxidation in an emulsion system

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oxidation lowering effect can be attributed to radical scavenging or complexation with

68

pro-oxidant metals naturally present within the system 19, 20. Some authors have reported

69

the effect of iron complexation by proteins on the decrease of the extent of lipid

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oxidation in milk or emulsion model systems containing iron 23-26.

21, 22

. Lipid

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Although peptides have been considered as good ligands to coordinate iron ions

72

and form stable complexes 27-32, the capacity of peptide-iron complexes to decrease lipid

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oxidation through coordination of the iron ions has not been studied. In previous

74

studies, we demonstrated that peptides from whey protein hydrolysate form stable

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complexes with iron, by bidentate coordinate covalent bonds

76

during in vitro gastrointestinal digestion and lead to an increase in iron uptake by Caco-

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2 cells 32. In the present study, we evaluated the primary and secondary products of lipid

78

oxidation in O/W emulsions containing iron in various forms: iron salts (FeSO4 and

79

FeCl2) and whey peptide-iron complexes. We aimed at evaluating the effect of peptide-

80

iron complexation on lipid oxidation catalyzed by iron, using O/W emulsions as a

81

model system.

31

, which protect iron

82 83

2. Material and Methods

84

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2.1.

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Material

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Whey protein isolate (WPI) PROVON® was obtained from Glanbia Nutritionals

87

(Kilkenny, Ireland) (90.4 ± 1.1 g protein/100 g, determined by the micro-Kjeldahl

88

method

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232-468-9, P1750), Tween 20 (Polyethylene glycol sorbitan monolaurate, P9416) and

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ferrous sulfate (FeSO4.7H2O) were purchased from Sigma-Aldrich® (St. Louis, MO,

91

USA). Ferrous chloride (FeCl2.4H2O) was purchased from J.T. Baker (Phillipsburg, NJ,

92

USA). The patented complex Ferrochel® (ferrous bisglycinate) was kindly donated by

93

Albion Laboratories (Clearfield, Utah, USA). The canola (Brassica campestris L.) oil

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used for emulsion preparation was purchased in a local market. This oil is constituted by

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13 to 81% of monounsaturated fatty acids and 16 to 39% of polyunsaturated fatty acids

96

with 11 to 23% (w/w) of linoleic acid (n-6) and 5 to 13% (w/w) of α-linolenic acid (n-3)

97

34

98

octen-3-ol, 1-heptanol were used in HS-SPME-GC-MS analysis to identify and quantify

99

the volatile compounds and were purchased from Sigma Aldrich® (St. Louis, MO,

100

33

– conversion factor: 6.38). Pancreatin (4xUSP, from porcine pancreas, EC

. Standards of pentanal, hexanal, 1-penten-3-ol, 1-pentanol, 1-hexanol, nonanal, 1-

USA), with purity varying from 95 to 99%.

101 102

2.2.

Synthesis of peptide-iron complexes

The peptide-iron complexes were synthesized according to previous experiments

103 104

31, 32

105

(enzyme:substrate ratio E/S 4% w/w) at pH 8.0 and 40 °C for 180 min and the enzyme

106

was deactivated by heating (85 °C/15 min), according to previously defined conditions.

107

The hydrolysate was ultrafiltered using a cut-off 5 kDa membrane, and the filtrate

108

fraction (F), molecular mass < 5 kDa, was freeze-dried and used as a ligand. The

109

complexes were synthesized at pH 7.0, using 4% (w/v) protein and 0.1% (w/v) iron

. Briefly, WPI (10% protein solution; w/v) was hydrolyzed with pancreatin

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from FeCl2 or FeSO4. After 60 min stirring (25 ± 2 °C), the solutions were centrifuged

111

(5000 g/20 min) and the supernatant, containing the complexes, were freeze-dried. The

112

complexes synthesized with the filtrate and FeCl2 (F-Fe C) or filtrate and FeSO4 (F-Fe

113

S) were stored frozen (-18 °C) until further analysis. The iron content of F-Fe C and F-

114

Fe S, determined as described below (section 2.3), was 18.2 ± 0.3 and 17.2 ± 0.8 µg

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Fe/mg complex, respectively, while iron content of ferrous bisglycinate (Bis-Fe) was

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193.3± 3.1 µg Fe/mg complex. The protein content of F-Fe C and F-Fe S, determined

117

by the micro-Kjeldahl method 33, was 709 ± 90 and 738 ± 80 µg protein/mg complex,

118

respectively.

119 120

2.3.

Iron analysis

121

The iron content of the freeze-dried peptide-iron complexes (F-Fe C and F-Fe

122

S) and Bis-Fe was assessed by atomic absorption spectrophotometry (AAS), using a

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Perkin-Elmer Analyst 300 spectrometer (USA) equipped with a deuterium lamp

124

background corrector. The procedures were carried out according to Boen, et al.

125

Briefly, samples were digested with concentrated nitric acid and hydrogen peroxide (2:1

126

v/v) at 110 °C for 2 h. After transferring the samples to volumetric flasks and

127

completing the volume with ultrapure water, the iron content was measured using a

128

hollow cathode lamp for iron (248.3 nm). A standard curve ranging from 0.2 to 2.6 mg

129

Fe/L was built. The experiments were carried out in triplicate.

35

.

130 131 132 133

2.4.

Oil-in-water emulsion preparation

The O/W emulsion was prepared with canola (Brassica campestris L.) oil due to its high content of polyunsaturated fatty acids 34.

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The emulsion was formulated with 30% canola oil, 1% Tween 20 and ultrapure

135

water, at room temperature (25 ±2 °C). The preparation was carried out in two steps:

136

pre-emulsification in a rotor–stator homogenizer (Ultra Turrax IKA T18 Basic) (14.000

137

rpm/4 min), creating a coarse emulsion, followed by a two-stage high-pressure valve

138

homogenizer (30 MPa/5 MPa) (Panda 2K NS1001L, Niro Soavi, Parma, Itália) 36. The

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size of the droplets, expressed as the volume-surface mean diameter (D3.2) and

140

measured by laser diffraction using a Mastersizer 2000 (Malvern Instruments,

141

Worcester, UK), was 1.31 ± 0.02 µm. Immediately after the preparation, the pH of the

142

emulsion was adjusted to 2.0 to allow proper solubility of complexes and iron salts.

143

Seven emulsions were prepared with 1 mmol. L-1 Fe by the addition of: 1)

144

ferrous chloride (FeCl2); 2) ferrous sulfate (FeSO4); 3) peptide-iron complex F-Fe C; 4)

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peptide-iron complex F-Fe S; 5) filtrate and FeCl2 (F + C); 6) filtrate and FeSO4 (F +

146

S); and 7) ferrous bisglycinate (Bis-Fe). Two emulsions with no iron addition were

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prepared: 1) emulsion with filtrate addition (F) and 2) emulsion with no other

148

component addition (control). The emulsions were stored in the dark at 30 oC in a BOD

149

(Biological Oxygen Demand) Incubator (TE-390, Tecnal, Piracicaba, SP, Brazil). The

150

primary and secondary products of lipid oxidation were measured every day during 7

151

days 10. Day 0 values correspond to the control emulsion immediately after preparation.

152 153

2.5.

Primary products of lipid oxidation: peroxide value (PV)

154

The primary products of lipid oxidation were quantified by the peroxide value

155

(PV) assay, using a protocol adapted from Rebellato, et al. 37. Briefly, 10 g of emulsion

156

were destabilized by chloroform addition (1:2 w/v) and 0.5 g sodium sulfate. After 2

157

min of vigorous shaking, the destabilized emulsion was centrifuged (3000 g/5 min, 4

158

°C), the aqueous phase was removed by aspiration and the bottom layer passed through

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a common filter paper. The analyses were carried out using the lipid fraction (micelle of

160

oil and chloroform). This procedure was carried out in triplicate for each sample, in

161

three independent trials. After addition of acetic acid (3:2 v/v) and 0.5 mL KI saturated

162

solution to the lipid fraction, the following procedure was repeated three times: 20 s of

163

vigorous shaking and 10 s of rest in the dark. The PV was determined by titration with

164

sodium thiosulphate using starch (1% w/v solution) as indicator as described by

165

American Oil Chemists´ Society, Cd 8-53 38. The PV was given by Equation 1: / =

1000

166

Where: PV = peroxide value (meqv /kg); M = molarity of sodium thiosulphate solution

167

(N); V = volume of sodium thiosulphate solution (mL); m = sample mass (g)

168 169

2.6.

Secondary products of lipid oxidation

170

The formation of secondary products of lipid oxidation was evaluated by two

171

different methods: determination of thiobarbituric acid reactive substances (TBARS)

172

and identification and quantification of volatile compounds by headspace solid-phase

173

microextraction - gas chromatography coupled with mass spectrometry (HS-SPME-GC-

174

MS).

175 176

2.6.1. TBARS

177

TBARS test indirectly quantifies the formation of malonaldehyde (MDA), a

178

dialdehyde formed during the oxidation of polyunsaturated fatty acids and largely used

179

as a bioindicator 39. The reaction between 2-thiobarbituric acid and MDA leads to a red

180

compound, measured spectrophotometrically (532 nm). TBARS values were

181

determined according to Di Mattia, et al. 40 with modifications. Briefly, emulsions were

182

diluted with ultrapure water (1:5 to 1:100) and aliquots of 2.0 mL were transferred to 8


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test tubes. Four milliliters of TBA reagent (15% w/v trichloroacetic acid and 0.375%

184

w/v thiobarbituric acid in 0.25 mol/L HCl) were added to the tests tubes, incubated in a

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boiling water bath for 15 min, then cooled in an ice bath for 10 min and centrifuged

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(6800 g/15min). The absorbance of supernatants was measured at 532 nm using an

187

Agilent 8453 spectrophotometer (Agilent Technologies, Waldbronn, Germany).

188

Each analysis was accomplished in four replicates of two independent trials. The

189

concentrations of TBARS were determined using a standard curve prepared with

190

1,1,3,3-tetraethoxypropane (TEP), which is hydrolyzed under the experimental

191

conditions and leads to MDA formation. The curve ranged from 0.17 to 5.5 mg. L-1

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TEP, corresponding to 0.055 to 1.801 mg. L-1 MDA.

193 194 195

2.6.2. Volatile lipid oxidation products (VLOPs) by HS-SPME-GC-MS The volatile lipid oxidation products (VLOPs) produced during the 7 days of

196

emulsion storage were

identified and quantified by headspace solid-phase

197

microextraction (HS-SPME) gas chromatography coupled with a mass spectrometer

198

detector (GC-MS) in a GCMS-QP2010 Ultra spectrometer (Shimadzu, Kyoto, Japan).

199

Sample preparation, i.e, pre-incubation conditions and adsorption conditions, were

200

carried according to Waraho, et al.

201

conditions were described by Souza and Bragagnolo 42.

41

, while chromatographic separation and MS

202

Briefly, 1.0 g of emulsion was weighed in a glass headspace vial with magnetic

203

screw cap (SU860103, Sigma-Aldrich®, St. Louis, MO, USA). The emulsion was pre-

204

incubated at 55 °C/13 min in an autosampler (AOC-5000, Shimadzu, Kyoto, Japan)

205

heating block before fiber (50/30 µm DVB/ Carboxen/PDMS, Supelco) exposure in the

206

headspace for 1 min. Then, the fiber was transferred to the GC injector port (250 °C/3

207

min), which was operated in splitless mode. The separation of the volatile compounds

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was achieved on a capillary column RTX-Wax (30 m × 0.25 mm inner diameter, 0.20

209

µm thick stationary phase, polyethylene glycol, Restek, Bellefonte, PA, USA). The

210

column temperature started at 30 °C/5 min, increased up to 115 °C at 10 °C/1 min, with

211

hold time of 1.0 min. Then, it increased up to 220 °C at 30 °C/1 min rate, held at this

212

temperature for 12.0 min.

213

The carrier gas was helium, in linear flow control mode (column flow: 1.22

214

mL/min). Ion source was used in electron ionization (EI) mode at 70 eV and 250 °C.

215

The scan mode (m/z 35−350) of the quadrupole mass/charge analyzer was used to

216

identify the compounds, and the solvent cut-off was 3.0 min. The analyses were carried

217

out in duplicate of two independent trials. The spectra were processed using the selected

218

ion monitoring (SIM) mode. The target ion (base peak) used for quantification and the

219

reference ions used to confirm the compound identity are shown in Table 1.

220

The identification of the volatile compounds in the mass spectra of samples was

221

done by comparison with the mass spectra of the analytical standards obtained under the

222

same experimental conditions and by consulting the mass spectra library (Wiley9). The

223

VLOPs were quantified using analytical curves obtained with the respective standards

224

added to a fresh emulsion. The concentration (ng/g) of each identified compound at Day

225

1 (D1) and Day 7 (D7) was determined, as well as the difference between them (∆ =D7-

226

D1) (Table 2). Limits of detection (LOD) and quantitation (LOQ) (Table 2) were

227

determined using the parameters from the analytical curves

228

concentration (ng/g) vs. time (days) was used to evaluate the hexanal formation during

229

the 7 days of storage.

43

. The plot of the

230 231

2.7.

Statistical analysis

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The results were expressed as mean ± standard deviation. The statistical analysis

233

was performed using the statistical package GraphPad Prism 6 (GraphPad Software

234

Inc., La Jolla, USA) by one-way analysis of variance (ANOVA), followed by Tukey’s

235

test. Values of p < 0.05 were considered significant. The Pearson correlation coefficient

236

(r) was calculated for all samples between the variables PV and TBARS and TBARS

237

and hexanal values. The closer the value of r is to 1, the stronger the correlation between

238

the two variables 44.

239 240

3. Results

241 242

3.1.

Primary products of lipid oxidation

243

Figure 1 A and B shows the PV of emulsions containing iron from iron salts or

244

complexes during 7 days of storage at 30 °C. The control emulsion reached 10.2

245

meqv/kg after 7 days. The PV increased with the addition of 1 mmol. L-1 iron to the

246

emulsion, especially in those with iron salts addition. This fact evidences the pro-

247

oxidant effect of iron, which catalyzed the initiation of lipid oxidation, yielding a large

248

amount of hydroperoxides.

249

The addition of peptide-iron complexes to the emulsions increased the PV in

250

less extent than iron salts. From Day 1 to Day 7, the PV of the emulsions with peptide-

251

iron complexes (F-Fe S and F-Fe C) increased around 3 to 5-fold, while the values of

252

the emulsions with filtrate along with iron salts (F + S and F + C) increased

253

approximately 9 to 12-fold. The addition of F + S or F + C led to lower PV than the

254

ones containing only iron salt, especially in the first 2 days, when the PV of F + S and F

255

+ C were similar to F-Fe S and F-Fe C. In the emulsion F, PV was around 80% lower

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than the control at Day 7, which reinforces the antioxidant character of this fraction

257

containing only peptides and no iron ions.

258

From Day 5 on, the emulsion FeCl2 presented a decrease in PV (Figure 1 A)

259

possibly because the active phase of hydroperoxide decomposition had begun. This

260

phase is marked by an exponential increase in the formation of secondary oxidation

261

products resulting from hydroperoxide decomposition, and the rate of hydroperoxide

262

degradation is greater than the rate of their formation 8. The formation of hydroperoxide

263

in all emulsions, except for emulsion FeCl2, was greater than its decomposition, since

264

the PV values increased until Day 7.

265

The iron source influenced the PV developing in emulsions. Comparing the

266

complexes synthesized with both iron sources, F-Fe S (Figure 1 B) led to a PV

267

approximately 30% lower than F-Fe C (Figure 1 A) at Day 7. The lipid oxidation in the

268

presence of iron added as FeCl2 differed from FeSO4 with or without peptide addition.

269 270

3.2.

Secondary products of lipid oxidation

271 272

3.2.1. TBARS

273

Formation of secondary products of lipid oxidation, measured as TBARS, was

274

observed from Day 1 in all iron added samples (Figure 2), indicating the fast breakdown

275

of hydroperoxides to other products. At Day 7 the control showed TBARS values lower

276

than the samples with iron addition at Day 1. The TBARS final values (Day 7) for the

277

control emulsion were 38 to 40-fold lower than those with iron salts and 17 to 21- and

278

28 to 29-fold lower than those with peptide-iron complexes or iron salts along with

279

peptides, respectively. By the other hand, the control emulsion reached value around

280

2.7-fold higher than the emulsion F after 7 days.

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The TBARS increasing during storage showed the same profile observed for

282

peroxide formation. TBARS values showed high correlation with PV results (r > 0.96)

283

for all iron added samples, except for FeCl2. The lower correlation for this sample (r =

284

0.85) is due to the decrease of PV from the Day 5 on.

285

Although the TBARS assay gives the indirect measurement of malonaldehyde 45

286

(MDA)

, it is worth mentioning that TBA can also react with substances other than

287

MDA, such as amino acids, sugars, bile pigments and other lipid oxidation products

288

(Knight et al., 1988; Gutteridge, 1982). Thus, it is a nonspecific test, and the generated

289

artifacts may overestimate TBARS results 46.

290 291

3.2.2. Volatile lipid oxidation products (VLOPs) by HS-SPME-GC-MS

292

The aldehydes hexanal, pentanal, and nonanal, and the alcohols 1-pentanol, 1-

293

penten-3-ol, 1-octen-3-ol, 1-hexanol, and 1-heptanol were identified and quantified at

294

the first and the last days of storage. Table 2 shows the concentration (ng/g) of the

295

volatile compounds in the emulsions at Day 1 (D1) and Day 7 (D7). The color scale

296

(from green to red) shows the increase in the concentration of each compound. The

297

amount of VLOPs formed in the emulsions containing iron in free and complexed form

298

was different (see color scale in Table 2). The addition of peptide-iron complexes led to

299

the formation of all VLOPs in lower amounts than of iron salts, resulting in values up to

300

233-fold lower. At D7, the main secondary products in the iron containing emulsions

301

were hexanal, followed by nonanal, pentanal, 1-pentanol, and 1-penten-3-ol for iron

302

salts, whey peptides along with iron salts and Bis-Fe. For peptide-iron complexes, the

303

main compounds, besides hexanal, were 1-penten-3-ol and 1-hexanol.

304

The identified compounds were found in the emulsions from the D1, and their

305

concentration increased throughout the storage, except the alcohol 1-heptanol, which

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306

was not detected in any sample at the first day. The volatile compounds pentanal and 1-

307

heptanol were not detected in the control and F emulsions neither in the first nor the

308

last day of storage. Concerning the different iron sources, FeSO4 and FeCl2 led to

309

different levels of lipid oxidation products, but the pathway of lipid oxidation was not

310

influenced, since the compounds found in all samples did not differ.

311

After 7 days, the amount of hexanal was 1.5 to 6.7-fold higher than the sum of

312

all the other volatile compounds identified for all the iron containing emulsions. Figure

313

3 shows the total ion chromatograms (TIC) obtained by HS-SPME-GC-MS and the

314

great difference of intensity between hexanal and the other compounds is clear in the

315

chromatogram of emulsions with FeCl2, F + C, and F-Fe C. Despite the lack of

316

specificity of TBARS assay, hexanal values showed good correlation with TBARS (r >

317

0.85) for emulsions with iron addition, except with peptide-iron complexes (r=0.44-

318

0.72).

319

Hexanal, the major saturated aldehyde originated from the breakdown of n-6

320

fatty acid peroxides 47, was identified and quantified in the emulsions during the 7 days

321

of storage (Figure 4).

322

From the first day of storage, emulsions with iron salts and Bis-Fe showed

323

higher hexanal contents than the emulsions with F + C (Figure 4a) and F + S (Figure

324

4b). Among the iron added samples, the emulsions with F-Fe C and F-Fe S showed the

325

lowest levels, suggesting that the complexes hinder the lipid oxidation catalyzed by

326

iron. This fact can be explained either by the lower formation of primary products or by

327

the capacity of peptides to scavenge the secondary products.

328 329

4. Discussion

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330

Iron, a transition metal, exhibits strong pro-oxidant effect in various food

331

systems. It is capable of initiating the oxidative process, by generating reactive oxygen

332

species (ROS), including the hydroxyl (•OH) and superoxide anion (O2•−) radicals.

333

These ROS, via a series of chain reactions, can react with unsaturated fatty acids to

334

produce hydroperoxides and promote oxidative damage at different levels

335

knowledge, this is the first study which addresses the protective effect of peptide-iron

336

complexes on lipid oxidation catalyzed by iron. Oil in water emulsions prepared with

337

canola oil, rich in polyunsaturated fatty acids, were used as model system. The

338

complexes F-Fe C and F-Fe S evaluated in the present work showed the best in vitro

339

iron bioavailability results in a previous work

340

iron bioavailability, the current results provide evidence that peptide-iron complexation

341

has potential to minimize the changes caused by the iron induced oxidation when added

342

to fortify or supplement food products, extending their shelf life by preserving the

343

sensory and quality characteristics.

25, 48

. To our

32

. Besides the previously demonstrated

344

The peptide-iron complexes seem to exert a crucial role in reducing the pro-

345

oxidant effect of iron and thus increasing the oxidative stability of emulsions since they

346

led to the lowest formation of primary and secondary oxidation products comparing to

347

iron salts and whey peptides along with iron salts (F + C and F + S). To explain such

348

differences, we propose two pathways, which can occur at the same time: the

349

antioxidant activity of the peptides and the previous iron complexation. First, the whey

350

peptides, known for their antioxidant activity 49-51, interacted and stabilized the reactive

351

species generated during emulsion storage. This property was proved by the lowering of

352

oxidation products formation observed when the peptides were included in the

353

emulsions (F), along with iron salt (F + C and F + S) and as peptide-iron complexes (F-

354

Fe C and F-Fe S). In the peptide-iron complexes, which are composed of numerous

15



Page 16 of 31

355

peptides besides those involved in iron coordination, part of the peptides does not

356

coordinate iron ions 31, therefore, they have the functional groups available to scavenge

357

reactive species.

358

The addition of Bis-Fe to the emulsion led to lipid oxidation levels similar to the

359

iron salts, despite the fact that the iron was in the complexed form, proving the great

360

importance of the whey peptides as antioxidants in the system. However, by comparison

361

of the results of the emulsions containing F + FeCl2 or F + FeSO4 and the emulsion

362

containing complexes, the impact of the previous formation of the peptide-iron complex

363

becomes evident. This stable structure may hinder the iron capacity to interact with the

364

lipid phase and to catalyze the lipid oxidation. Despite this protective effect, emulsions

365

containing peptide-iron complexes showed formation of lipid oxidation products in a

366

higher extent than the control and F emulsions. This fact suggests that part of the iron

367

ions, possibly those weakly bound, may act as pro-oxidants, albeit to a lower extent.

368

Besides, the peptides may have less functional groups available to scavenge the radicals

369

due to their participation in iron coordination.

370 371

Iron ions can promote the degradation of hydroperoxides by one of the following mechanisms 9, 52:

372

Fe2+ + LOOH

Fe3+ + LO• + OH-

373

Fe3+ + LOOH

Fe2+ + LOO• + H+

374

These lipid radicals can then abstract hydrogen atoms of unsaturated fatty acids,

375

and propagate the chain reaction 9. The degradation of hydroperoxides catalyzed by iron

376

ions can occur in the proximity of emulsion droplet interface

377

which constitutes around 70% of the emulsions, could facilitate the ions motion

378

their interactions in the oil:water interface. Cho, et al. 55 observed that iron chelators are

379

capable of increasing the transfer of iron from lipid droplets in O/W emulsions to the

53

. The aqueous phase, 54

and

16


Page 17 of 31


380

aqueous phase, thus decreasing its pro-oxidant activity, which is in accordance with our

381

results for the emulsions containing iron as peptide-iron complexes.

382

In an aqueous medium, iron salts are ionized and dissociate in iron ion and their 56

383

counterions. Osinchak, et al.

demonstrated that the chloride ion might have an

384

important effect on lipid oxidation, contributing to an oxidative effect, which suggests

385

that the counterions SO4-2 and Cl- may also play a role in the lipid oxidation process in

386

the system of the present study. This fact may have contributed to the different

387

oxidation levels observed for emulsions containing FeSO4 and FeCl2, although both

388

have the same iron concentration.

389

Alkoxyl (LO•) and peroxyl (LOO•) radicals, formed by hydroperoxides (LOOH)

390

degradation, lead to the formation of products such as aldehydes, ketones, acids, esters,

391

alcohols, and short-chain hydrocarbons

392

(control and F), the formation of fewer types of compounds and in much lower extent

393

than the iron containing emulsions (Table 2) was observed. Therefore, even more than

394

temperature and time, iron ions seem to be the main responsible for the formation of the

395

alcohols and aldehydes identified in the iron containing emulsions. Considering the low

396

levels of volatile compounds found in the emulsions with peptide-iron complexes, it

397

seems that iron remained mostly linked to peptides functional groups and out of reach

398

of the lipid interface, decreasing the rate of oxidation.

399

54, 57, 58

. In the emulsions without iron addition

Each volatile compound is formed from a different fatty acid. The degradation of 42

400

peroxides from n-3 fatty acids yields 1-penten-3-ol

, whereas the n-6 fatty acids

401

generate pentanal, hexanal, 1-pentanol, 1-hexanol, and 1-octen-3-ol

402

nonanal and the alcohol 1-heptanol are formed from the oxidation of n-9 fatty acids 58.

58

. The aldehyde

403

Although n-9 fatty acids constitute the majority of canola oil composition 34, the

404

volatile compounds formed in greater extent in the emulsions containing the peptide-

17



Page 18 of 31

405

iron complexes were 1-penten-3-ol from α-linolenic acid (n-3), and hexanal and 1-

406

hexanol originated from linoleic acid (n-6) (Table 2), due to the greater susceptibility of

407

these fatty acids to oxidation. The oxidation rate of oleic, linoleic and linolenic acid

408

generally follows the proportion 1:12:25, respectively, due to the number of double

409

bonds and the bond energy necessary for abstraction of hydrogen atom

410

containing free form of iron showed the greatest extent of volatile compounds

411

formation, including the ones originated from n-9 fatty acid.

59

. Emulsions

412

Although hydroperoxides are generally tasteless and odorless, the short-chain

413

volatile compounds have a great impact on the sensory quality of foods, including the

414

rancid off-flavor

415

impact on the sensory quality of foodstuffs in comparison to iron salts, ferrous

416

bisglycinate, and even to the concomitant addition of iron salts and peptides.

57, 60

. Thus, the addition of peptide-iron complex may lead to a lower

417

One of the major challenges at choosing a compound to fortify food with iron is

418

the relation bioavailability vs. reactivity, since, generally, the higher bioavailability of

419

the compound, the lower the stability 2, due to its reactivity. The complex F-Fe C could

420

be an exception to this statement. Comparing to compounds applied for food

421

fortification, such as ferrous sulfate and ferrous bisglycinate, the complex F-Fe C

422

showed lower reactivity and, according to previous results, around 70% higher in vitro

423

bioavailability

424

complexed iron is possibly transported across the brush border membrane through the

425

normal intestinal absorption route for peptides while remaining coordinated. Therefore,

426

this complex might not release the iron ions during gastrointestinal digestion,

427

preventing interactions with inhibitory dietary ligands and binding agents such as

428

phytate and oxalate. Moreover, an undissociated compound may be less likely to cause

429

gastrointestinal irritation in sensitive people 61.

32

. Previous in vitro bioavailability experiments showed that the

18


Page 19 of 31


430

In sum, whey peptide-iron complexes reduce the pro-oxidant effect of iron

431

comparing to the iron salt form in an emulsified model system. It is worth highlighting

432

that besides the capacity of whey peptides to neutralize the lipid radicals, the previous

433

formation of a peptide-iron complex can exert an indirect antioxidant capacity, since the

434

lipid oxidation of emulsions containing these complexes was quite lower than the

435

emulsions containing whey peptides along with iron salts. The formation of a ring

436

structure with the metal seems to protect iron from interacting with the lipid phase and

437

thus diminish the lipid oxidation. Therefore, these peptide-iron complexes can be

438

advantageous for food fortification, since they can potentially reduce the undesirable

439

sensory changes and hence be an alternative of less reactive iron. However, further

440

studies are necessary to evaluate if the behavior observed in this model system is also

441

observed in food products.

442 443

Acknowledgments

444

The authors thank the Laboratory of Process Engineering (Department of Food

445

Engineering) and the Food Chemistry Laboratory II (Department of Food Science) of

446

the Faculty of Food Engineering (University of Campinas) for providing assistance with

447

the emulsions production and the GC-MS analyses. This work was supported by

448

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) [grant number

449

2013/10356-7].

19



Page 20 of 31

450

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451 452 453 454 455 456 457 458 459 460 461 462 463 464 465 466 467 468 469 470

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10.1201/9781439822395.ch1. 61. Gerber, J. A review of mineral absorption with special consideration of chelation as a method to improve bioavailability of mineral supplements. (Feb 7th),

620

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Page 24 of 31

Figure captions

Figure 1. Peroxide value (PV) of canola oil O/W emulsions containing 1mM Fe in free and complexed forms. FeCl2 and FeSO4 - iron salts; F-Fe C and F-Fe S - peptide-iron complexes synthesized with filtrate and FeCl2 or FeSO4, respectively; F + C and F + S filtrate (fraction < 5 kDa) (2 mg protein/g emulsion) + FeCl2 or FeSO4, respectively; Bis-Fe - ferrous bisglycinate. Control emulsions without iron: control – with no other component addition; F - filtrate (fraction < 5 kDa) (2 mg protein/g emulsion). Samples stored at 30 oC. Figure 2. Thiobarbituric acid reactive substances (TBARS) of canola oil O/W emulsions containing 1mM Fe in free and complexed forms. FeCl2 and FeSO4: iron salts; F-Fe C and F-Fe S: peptide-iron complexes synthesized with filtrate and FeCl2 or FeSO4, respectively; F + C and F + S: filtrate (fraction < 5 kDa) (2 mg protein/g emulsion) + FeCl2 or FeSO4, respectively; Bis-Fe: ferrous bisglycinate. Control: emulsion with no other component addition; F: filtrate (fraction < 5 kDa) (2 mg protein/g emulsion), with no iron addition. Samples stored at 30 oC. Figure 3. Total Ion Chromatograms (TIC) (m/z 35−350) of volatile compounds obtained by HS−SPME−GC−MS, of canola oil O/W emulsions. The emulsions contained 1mM Fe from FeCl2, F-Fe C (peptide-iron complex synthesized with filtrate (fraction < 5 kDa) and FeCl2); and F + C (FeCl2 + filtrate fraction). The emulsions were stored for 7 days at 30 oC. Compounds are numbered according to Table 1. Figure 4. Hexanal content (ng/g) of canola oil O/W emulsions containing 1mM Fe in free and complexed forms. FeCl2 and FeSO4: iron salts; F-Fe C and F-Fe S: peptideiron complexes synthesized with filtrate and FeCl2 or FeSO4, respectively; F + C and F + S: filtrate (fraction < 5 kDa) (2 mg protein/g emulsion) + FeCl2 or FeSO4, respectively; Bis-Fe: ferrous bisglycinate. Control: emulsion with no sample addition; F: filtrate (fraction < 5 kDa) (2 mg protein/g emulsion), with no iron addition. Samples stored at 30 oC.

24 ACS Paragon Plus Environment

Page 25 of 31


Table 1. Target and reference ions (m/z) used to, respectively, quantify and identify the volatile lipid oxidation products (VLOPs) by headspace solid-phase microextraction gas chromatography coupled with a mass spectrometer detector (HS-SPME-GC-MS). Peak

Compound name

Target ion (m/z)

Reference ions (m/z)

1

Pentanal

44

58, 41

2

Hexanal

44

56, 41

3

1-penten-3-ol

57

41, 39

4

1-pentanol

42

55, 41

5

1-hexanol

56

43, 55

6

nonanal

57

41, 56

7

1-octen-3-ol

57

43, 72

8

1-heptanol

70

56, 41



Page 26 of 31

Table 2. Volatile lipid oxidation compounds (ng/g) determined by headspace solid-phase microextraction gas chromatography coupled with mass spectrometer detector (HS-SPME-GC-MS) in canola oil O/W emulsion containing 1mM Fe in free and complexed forms. Day 1 (D1)

Day 7 (D7)

[ ] (ng/g)

[ ] (ng/g)

VLOP Control FeCl2 FeSO4 3

FC

FS

Bis

F+C

F+S

F

Control

FeCl2

FeSO4

FC

FS

Bis

F+C

F+S

F

46.2

25.5 1204.5 838.8

292.6

nd

nd

6474.5

4282.3

83.1

58.0

5129.1

4750.7

1949.2

*

Pentanal

nd

566.6 1285.9

Hexanal

24.7

8241.1 1470.8 1018.6 482.0 1872.3 4573.0 2922.0 28.8

407.2

1-penten-3-ol

1.4

436.1

230.3

35.5

23.6

217.2

131.5

44.2

1.3

13.2

1596.3

1640.9

856.1

328.5

1721.8

1957.3

1734.1

4.2

1-pentanol

0.9

134.9

78.0

7.7

2.6

75.3

35.8

14.9

1.2

4.8

3019.5

3090.1

73.3

51.1

2202.7

2907.3

1048.6

1.8

1-hexanol

nd

16.7

10.2

1.9

0.9

8.6

5.7

3.2

nd

5.3

257.9

219.2

390.7

102.9

205.1

215.4

229.5

nd

nonanal

nd

687.0

487.9

nd

nd

409.3

340.4

254.7

nd

nd

8240.2

5540.0

179.3

98.2

5033.7

4788.8

1173.7

nd

1-octen-3-ol

2.0

447.0

237.0

10.2

7.5

217.9

88.2

17.8

1.9

6.7

2902.0

2758.9

123.3

62.2

2361.3

2755.9

1655.5

2.8

1-heptanol

nd

70.2

Nd

nd

nd

32.0

5.3

10.5

nd

nd

2068.8

890.3

nd

nd

1109.4

1211.8

367.4

nd

Bis

F+C

F+S

F

3924.6

3911.9

1656.6

*

136319.5 123345.7 3351.9 1578.6 108834.6 50087.2 26142.3 46.9

∆D7-D1 Peak

VLOP

RT2

[ ] (ng/g) FS

Control

FeCl2

FeSO4

FC

5907.9

2996.3

36.8

1

Pentanal

5.3

nd

2

Hexanal

7.9

382.5

1-penten-3-ol 9.7

11.8

1160.1

1410.6

820.6

304.9

1504.5

1825.8

1689.9

2.9

3

32.5

128078.4 121874.9 2333.3 1096.6 106962.3 45514.1 23220.2 18.1

4

1-pentanol

11.3

4.0

2884.5

3012.1

65.7

48.5

2127.4

2871.5

1033.7

0.6

5

1-hexanol

12.9

5.3

241.3

209.0

388.9

101.9

196.4

209.6

226.3

nd

6

nonanal

13.5

nd

7553.2

5052.1

179.3

98.2

4624.4

4448.4

919.0

nd

1-octen-3-ol 14.4

4.7

2455.0

2521.9

113.1

54.7

2143.3

2667.7

1637.7

0.9

nd

1998.6

890.3

nd

nd

1077.4

1206.5

356.9

nd

7 8

1-heptanol

14.5

1

The target ion for quantification was the base peak of each compound; 2 Retention time (min). 3not detected. Limit of Detection (LOD) and Limit of Quantitation (LOQ) (ng/g): Pentanal – 2.9 and 9.0; Hexanal – 7.8 and 23.8; 1-penten-3-ol – 0.3 and 1.0; 1-pentanol – 0.3 and 0.9; 1-hexanol – 0.2 and 0.7; nonanal – 26.2 and 79.3; 1-octen-3-ol – 0.6 and 1.8; 1-heptanol – 1.5 and 4.6. FeCl2 and FeSO4: iron salts; F-Fe C and F-Fe S: peptide-iron complexes synthesized with filtrate and FeCl2 or FeSO4, respectively; F + C and F + S: filtrate (fraction < 5 kDa) + FeCl2 or FeSO4, respectively; Bis-Fe: ferrous bisglycinate. Control: emulsion with no other component addition; F: filtrate (fraction < 5 kDa), with no iron addition. Samples stored at 30 oC for 7 days. The label colors were defined using color scale in conditional formatting of Excel. Green labels represent compounds formed in lower extent than red labels; yellow and orange labels represent intermediate values.

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Whey PeptideâIron Complexes Increase the Oxidative Stability of Oil

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