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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
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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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1. Introduction
36 37
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
50
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
58
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
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reaction of these radicals and give rise to low molecular weight secondary products,
62
such as aldehydes, ketones, and alcohols
63
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
67
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
70
oxidation in milk or emulsion model systems containing iron 23-26.
21, 22
. Lipid
71
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
73
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
75
complexes with iron, by bidentate coordinate covalent bonds
76
during in vitro gastrointestinal digestion and lead to an increase in iron uptake by Caco-
77
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
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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
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(Kilkenny, Ireland) (90.4 ± 1.1 g protein/100 g, determined by the micro-Kjeldahl
88
method
89
232-468-9, P1750), Tween 20 (Polyethylene glycol sorbitan monolaurate, P9416) and
90
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,
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USA). The patented complex Ferrochel® (ferrous bisglycinate) was kindly donated by
93
Albion Laboratories (Clearfield, Utah, USA). The canola (Brassica campestris L.) oil
94
used for emulsion preparation was purchased in a local market. This oil is constituted by
95
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
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(5000 g/20 min) and the supernatant, containing the complexes, were freeze-dried. The
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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-
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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
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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
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S) and Bis-Fe was assessed by atomic absorption spectrophotometry (AAS), using a
123
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
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water, at room temperature (25 ±2 °C). The preparation was carried out in two steps:
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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
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measured by laser diffraction using a Mastersizer 2000 (Malvern Instruments,
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Worcester, UK), was 1.31 ± 0.02 µm. Immediately after the preparation, the pH of the
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emulsion was adjusted to 2.0 to allow proper solubility of complexes and iron salts.
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Seven emulsions were prepared with 1 mmol. L-1 Fe by the addition of: 1)
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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
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(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
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°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
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oil and chloroform). This procedure was carried out in triplicate for each sample, in
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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
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determined according to Di Mattia, et al. 40 with modifications. Briefly, emulsions were
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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
185
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
192
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.
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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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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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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
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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
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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-
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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
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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].
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450
References
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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.
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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
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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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