Accepted Manuscript Influence of formulation on the oxidative stability of water-in-oil emulsions Wafa Dridi, Wafa Essafi, Mohamed Gargouri, Fernando Leal-Calderon, Maud Cansell PII: DOI: Reference:
S0308-8146(16)30145-5 http://dx.doi.org/10.1016/j.foodchem.2016.01.145 FOCH 18719
To appear in:
Food Chemistry
Received Date: Revised Date: Accepted Date:
8 July 2015 5 January 2016 30 January 2016
Please cite this article as: Dridi, W., Essafi, W., Gargouri, M., Leal-Calderon, F., Cansell, M., Influence of formulation on the oxidative stability of water-in-oil emulsions, Food Chemistry (2016), doi: http://dx.doi.org/ 10.1016/j.foodchem.2016.01.145
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Influence of formulation on the oxidative stability of water-in-oil emulsions
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Wafa Dridi1,2,3,4,5, Wafa Essafi1, Mohamed Gargouri5, Fernando Leal-Calderon2,3,4, Maud
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Cansell2,3,4
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Physico-Chimique, Pôle Technologique de Sidi Thabet, 2020 Sidi Thabet, Tunisie
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Univ. Bordeaux, CBMN, UMR 5248, 33600 Pessac, France
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3
CNRS, CBMN, UMR 5248, 33600 Pessac, France
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Bordeaux INP, CBMN, UMR 5248, 33600 Pessac, France
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INSAT, Université de Carthage, Centre Urbain Nord, 1080 Tunis Cedex, Tunisie
Laboratoire Matériaux, Traitement et Analyse, Institut National de Recherche et d’Analyse
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Running title: Oxidation of water-in-oil emulsions
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* Corresponding authors: Maud Cansell,
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Université Bordeaux, Laboratoire CBMN, UMR 5248, Bordeaux INP, Allée Geoffroy Saint
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Hilaire, 33600 Pessac, France.
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Tel: +33 5 40 00 66 95
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Fax: +33 5 56 37 03 36
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e-mail:
[email protected] 21 22
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Abstract
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The oxidation of water-in-oil (W/O) emulsions was investigated, emphasizing the impact of
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compositional parameters. The emulsions had approximately the same average droplet size
27
and did not show any physical destabilization throughout the study. In the absence of pro-
28
oxidant ions in the aqueous phase, lipid oxidation of the W/O emulsions was moderate at 60
29
°C and was in the same range as that measured for the neat oils. Oxidation was significantly
30
promoted by iron encapsulation in the aqueous phase, even at 25 °C. However, iron chelation
31
reduced the oxidation rate. Emulsions based on triglycerides rich in polyunsaturated fatty
32
acids were more prone to oxidation, whether the aqueous phase encapsulated iron or not. The
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emulsions were stabilized by high- and low-molecular weight surfactants. Increased relative
34
fractions of high molecular weight components reduced the oxidation rate when iron was
35
present.
36 37 38
Key words: lipid oxidation, water-in-oil emulsions, emulsion formulation, conjugated dienes.
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1. Introduction
41 42
Many studies have focused on the health benefits of polyunsaturated fatty acids (PUFA) from
43
the n-3 series (Simopoulous, 1991; Sharma, 2013) leading to PUFA enrichment in various
44
foods and nutraceuticals (Jacobsen, Nielsen, Horn & Sørensen, 2013; Stratulat et al., 2015).
45
However, because these PUFA are extremely sensitive to oxidation (Frankel, 1991; Schaich,
46
2005; Shahidi & Zhong, 2010), precautions have to be taken in the formulation, processing
47
and handling of such products to avoid oxidation. Lipid oxidation may generate off-flavors,
48
rancid odors (Frankel, 1991; Fritsh, 1994; Jacobsen, 1999) and molecules that can be harmful
49
to health (Halliwell & Gutteridge, 1995; Kubow, 1992). Several factors are known to promote
50
lipid oxidation such as oxygen, light, heat, transition metal ions (Chen, McClements &
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Decker, 2011; Shahidi & Zhong, 2010; Schaich, 2005). Manufacturers can control or at least
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delay lipid oxidation through the addition of anti-oxidants in food formulations (Frankel,
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2012; Fritsh, 1994; Shahidi and Zhong, 2010), removal of oxygen (nitrogen atmosphere or
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vacuum) during processing, adequate packaging for food conditioning, and appropriate
55
storage conditions. In many food products, oil is present either in the dispersed phase of oil-
56
in-water (O/W) emulsions, as in salad dressings, or in the continuous phase of water-in-oil
57
(W/O) emulsions such as low fat spreads, butter, and margarines. Depending on the emulsion
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formulation, oil dispersion may either promote lipid oxidation (Chen et al., 2011; Mei, Decker
59
& McClements, 1998) or protect PUFA against oxidation (Kobayashi, Yoshida & Miyashita,
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2003; Miyashita, Nara & Ota, 1993). The abundant literature about lipid oxidation in
61
emulsions deals mainly with O/W systems (Berton-Carabin, Genot, Gaillard, Guibert &
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Ropers, 2013; Charoen, Jangchud, Jangchud, Harnsilawat, Decker & McClements, 2012;
63
Chen et al., 2011; Waraho, Cardenia, Decker & McClements, 2010), but only a few papers
64
address lipid oxidation in W/O emulsions (Calligaris, Manzocco & Nicoli, 2007). Although
3
65
the seminal papers allowed significant knowledge advances, there are still many questions
66
regarding lipid oxidation in W/O emulsified systems. For instance, the impact of the dispersed
67
aqueous phase on the rate and mechanisms of PUFA oxidation are controversial. Some
68
studies showed that the oxidation rate in W/O emulsions was similar to that of bulk oils
69
(Fritsch, 1994). In contrast, other studies revealed that even a small amount of water (3 wt.%)
70
dispersed in crude olive oil slowed down the oxidation process (Ambrosone, Angelico,
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Cinelli, Di Lorenzo, & Ceglie, 2002). The presence of hydrophilic emulsifiers in the aqueous
72
droplets (Yi, Zhu, McClements & Decker, 2014) or of anti-oxidants in the lipid phase (Mosca,
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Diantom, Lopez, Ambrosone & Ceglie, 2013) were pointed out as having a strong influence
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on lipid oxidation. However, much remains to be learned about the effect of the oil-soluble
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surfactant nature and concentration, the nature of the oil phase and the ion content of the
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aqueous droplets. Further studies are thus required to better understand and control lipid
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oxidation in W/O emulsions.
78
In this context, we have investigated the oxidative behavior of five plant oils, namely olive,
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rapeseed (crude and refined), camelina and linseed in bulk state and in W/O emulsions with
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different formulations in terms of oil-soluble surfactant content and water phase composition.
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The emulsions were stabilized with a mixture of polymer (polyricinoleate of polyglycerol)
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and low-molecular weight surfactant (distilled monoglycerides). The average droplet size of
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all emulsions was consistent under the initial conditions and throughout the experiments,
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allowing direct comparison of the different systems. Lipid auto-oxidation is classically
85
described as a three-step process: initiation, propagation, and termination (Frankel, 1991;
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Schaich, 2005; Shahidi & Zhong, 2010). In the initiation stage, hydrogen is abstracted from
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an olefinic compound to yield a free radical. This can combine with oxygen to form a peroxy-
88
free radical, which can in turn abstract hydrogen from another unsaturated molecule to yield a
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peroxide and a new free radical, promoting propagation. The hydroperoxides formed in the
4
90
propagation step are the primary oxidation products, related to the early oxidation state.
91
During the formation of hydroperoxides from unsaturated fatty acids, conjugated dienes are
92
formed due to the rearrangement of the double bonds. The concentration of conjugated dienes
93
can be followed easily by spectrophotometry due to the absorption of the diene chromophore
94
around 233-235 nm (Corongiu and Milia, 1983; Shahidi and Zhong, 2005). Both the peroxide
95
value and the diene concentration reveal the formation of primary oxidation products and are
96
highly correlated (Shahidi & Zhong, 2005). However, compared with the peroxide index,
97
spectrophotometric measurement of diene concentration is simpler, requires a smaller sample
98
volume, and consumes less time and solvents. Thus, in this work, lipid oxidation was
99
followed by measuring the concentration of conjugated diene hydroperoxides using a
100
spectrophotometer.
101 102
2. Experimental methods
103 104
2.1 Materials
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Five different edible oils were used: refined rapeseed oil (Fleur de Colza, Lesieur, France),
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crude rapeseed oil (Vigean, Clion-sur-Indre, France), crude linseed oil (Bioplanète, Bram,
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France), crude camelina oil (Bioplanète, Bram, France), and crude olive oil (Elbarka, Ksar
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Said, Tunisia). The fatty acid compositions and the tocopherol contents of the different oils
109
are presented in Table 1. To determine their fatty acid composition, the oils were
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transmethylated in the presence of boron trifluoride-methanol complex (Morrison & Smith,
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1964). The resulting fatty acid methyl esters were identified by gas chromatography (GC)
112
using a BPX 70 capillary column (60-m long, 0.25-µm film, 0.25-mm i.d., SGE, N2 as carrier
113
gas, split ratio of 50). The GC system consisted of a gas chromatograph (GC 2010 plus,
114
Shimadzu, Kyoto, Japan) equipped with a flame ionization detector maintained at 280 °C. The
5
115
injector was set at 250 °C. The column temperature was increased from 150 °C to 200 °C at
116
1.3 °C.min-1, held for 20 min at 200 °C, increased again from 200 °C to 235 °C at 10°C.min-1
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and finally held for 23 min at 235 °C. Data were collected and integrated by a GC solution
118
v2.4 integration system (Shimadzu). Fatty acids from Sigma–Aldrich (Saint Louis, MO,
119
USA) and natural extracts of known composition were used as standards for column
120
calibration.
121
Tocopherol composition of the oils was determined using high performance liquid
122
chromatography (HPLC). Samples (2 g oil /25 mL hexane) were analyzed with an HPLC
123
system consisting of an Agilent L1200 liquid chromatographic system (Santa Clara, CA,
124
USA) equipped with a G1311A Quat Pump solvent delivery system, an injector (model
125
G1329A) of 20 µL loop size, and an analyzing software, Agilent ChemStation. An analytical
126
pre-packed column (250×2 mm; 5 µm) YMC-Pack SIL (YMC Co, LTD, Kyoto, Japan) was
127
used with isopropanol in hexane (0.5:99.5, v/v) as the mobile phase. The system was operated
128
isocratically at a flow rate of 0.3 mL.min-1. The fluorescence detector (Agilent 1100 Series)
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was set at 290 nm excitation wavelength and 330 nm emission wavelength. Quantification
130
was based on an external standard method. Mixed tocopherol standards in hexane solution
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(0.1 mg.mL-1), were prepared from standard compounds: α-, β-, γ-, δ-tocopherol (Sigma
132
Aldrich).
133
Polyglycerol polyricinoleate (PGPR) and distilled mono-glycerides (DMG) were purchased
134
from Palsgraad (Juelsminde, Denmark). Ferrous sulfate and Iotect, an iodine indicator, were
135
from VWR Chemicals (Leuven, Belgium). Ferric chloride was purchased from Merck
136
Millipore (Darmstadt, Germany). Sodium chloride, ferrous chloride, iron gluconate, iron
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lactate, boron trifluoride-methanol solution and sorbitan monooleate (Span 80) were from
138
Sigma Aldrich. Glacial acetic acid was purchased from Xilab (Bruges, France). Potassium
139
iodide was purchased from Analar Normapur (Leuven, Belgium). Sodium thiosulfate was
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from Scharlau (Barcelona, Spain). Dodecane was purchased from Acros Organics (New
141
Jersey, USA). Solvents were of analytical grade. Hexane and isopropanol with HPLC-grade
142
were purchased from Sigma Aldrich.
143 144
2.2 Emulsion preparation and structural characterization
145
W/O emulsions were prepared at room temperature using a homogenizer (RZR 2102 control
146
Z Heidolph, Schwabach, Germany) with a stainless steel propeller rotating between 700 and
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1000 rpm, according to the emulsion formulation. In all systems, sodium chloride at 0.1 M
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was dissolved in water as a background electrolyte. The presence of salt within the droplets
149
was required to inhibit destabilization phenomena such as Oswald ripening (Kabalnov, 2001)
150
and coalescence (Aronson & Petko, 1993). In some cases, a second electrolyte based on iron
151
was incorporated to probe the influence of a pro-oxidant species in the aqueous phase. The oil
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phase was obtained by dissolving various mixtures of PGPR and DMG in plant oil at 25 °C.
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The aqueous phase was gradually incorporated into the oil phase, under stirring up to a
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fraction of 80 g per 100 g. This large droplet fraction was adopted to take advantage of the
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high viscosity of the material, allowing the implementation of large viscous stresses even at
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relatively low shear rates (Mabille et al., 2000). The fine W/O emulsions obtained were
157
diluted with plant oil to set the final drop fraction at 40 wt.%. The supplementary information
158
n°1 presents the formulations of the different W/O emulsions studied. Each formulation was
159
prepared at least in triplicate.
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Direct visualization of the water droplets just after preparation and during the oxidative
161
experiments was carried out using an optical microscope (Leica DM2500P microscope
162
equipped with an oil immersion ×100 objective, Zeiss, Germany) and a digital camera for
163
capturing images. The images were processed by a Leica IM50 software to estimate the
164
average size of the drops. In parallel, the mean water droplet diameter (as evaluated by the
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volume weighted average diameter d4,3) and the size distribution were determined by static
166
light-scattering, using a Coulter LS 230 apparatus. To avoid multiple scattering, W/O
167
emulsion samples were diluted with a dodecane solution containing 0.5 wt.% sorbitan
168
monooleate (Span 80) surfactant to ensure emulsion stability in the measuring cell. The
169
measuring cell was filled with the dodecane solution, and a small volume of the sample was
170
introduced under stirring.
171 172
2.3 W/O emulsion oxidation experiments and lipid oxidation measurements
173
The initial lipid oxidation status of the neat oils was quantified using the peroxide value on
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the bulk systems according to the standardized method AFNOR T60-220. Results are reported
175
in Table 1. Lipid oxidation was followed in bulk oils or W/O emulsions with time (72 hours).
176
Experiments were performed at 25 or 60 °C. Samples of 8 g of oil or emulsion were stored in
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darkness, in closed glass flasks. The total flask capacity of 26 g ensured that oxygen was in
178
excess during the experiments. Lipid oxidation was followed as production of diene
179
hydroperoxides generated in the early stages of the lipid oxidation process (Schaich, 2005).
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They were directly detected at 233 nm, corresponding to the peak in the absorption band of
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the conjugated diene function (Corongiu & Milia, 1983; Shahidi and Zhong, 2005). Aliquots
182
of W/O emulsions were centrifuged to extract the oil phase (30 min/14000 rpm, minispin plus
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spinner, Brumath, France). Pre-weighed oil was dissolved in hexane (purity 96%, Scharlau) to
184
a final lipid concentration that ensured an absorbance measurement in the spectrophotometer
185
linear range. The lipid solution was immediately scanned from 300 nm to 200 nm for
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absorbance measurements using a Hitachi (U-2810) double beam spectrophotometer using a
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1-cm thick quartz cell. Hereafter, results will be expressed in terms of optical density (OD),
188
since this parameter is proportional to the diene concentration according to the Beer-
189
Lambert’s law. In order to take into account the dilution factor imposed by the measuring
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190
conditions, the OD measured at 233 nm was normalized by the mass of lipids (mg) per unit
191
volume of solution (mL). Three measurements were performed for each formulation.
192 193
2.4. Statistical analysis
194
Differences between two groups were tested for significance by using the ANOVA or
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Student’s t-tests. Data are expressed as means ± standard deviation (SD).
196 197
3. Results and discussion
198 199
In this work, different edible oils were used to evaluate the effect of the unsaturated fatty acid
200
content on the oxidability of W/O emulsions. All the oils complied with Codex Alimentarius
201
guidelines concerning their oxidative status, as measured by the peroxide value (maximum
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regulation limit of 15 meq.kg-1 for crude oils and 10 meq.kg-1 for refined oils) (Table 1). It has
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been shown that lipid oxidation in O/W emulsions is influenced by the average droplet size
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(Nakaya, Ushio, Matsukawa, Shimizu & Ohshima, 2005). In our case, to rule out any possible
205
impact of this parameter, emulsions with approximately the same average droplet size were
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fabricated. The droplet size distributions were measured just after preparation and during the
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oxidation experiments. Figure 1a shows a typical optical micrograph of a W/O emulsion just
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after preparation. Water droplets were homogeneously distributed in the oil phase with an
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average droplet size of 1 µm. This result was confirmed by particle sizing measurements
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based on light scattering (see supplementary information n°2). This size distribution was
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maintained after 72 h-storage at 25 °C (Figure 1b) and at 60 °C for all the W/O emulsions
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reported in this study (see an example in Figure 1c). The physical stability was ensured by the
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lipophilic surfactant mixture of PGPR and DMG.
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One particular lipophilic emulsifier was predominant in the formulation of W/O emulsions
215
based on plant oils, namely PGPR. This surfactant has been demonstrated to be highly
216
effective for stabilizing fine W/O emulsions made with triglyceride oils (Benichou, Aserin &
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Garti, 2001). PGPR is an oligomeric lipophilic emulsifying agent obtained by partial
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esterification of fatty acids of castor oil with polyglycerol (Wilson, Van Schie & Howes,
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1998). Although it has gained the “Generally Recognized as Safe (GRAS)” status from the
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Food and Drug Administration (FDA), its total or partial removal from food products is
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sought after within the prospect of meeting clean label and consumer requirements. Within
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this context, we examined the potential to replace PGPR with a technically effective, cheap
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and legally acceptable alternative, i.e. distilled monoglycerides deriving from the partial
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hydrolysis of plant fats, classically used in margarine and bakery compounds. Unfortunately,
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DMG are much less efficient stabilizers than PGPR. With the oil used in this work, a 1:1
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wt.% mixture of PGPR and DMG was found to be the best compromise between a low PGPR
227
content and an acceptable kinetic stability of the W/O emulsions.
228 229
3.1 Influence of agitation and emulsification on the oil oxidation
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For the different types of oil used in this work, the conjugated diene concentration was
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measured in the bulk oil (UBO), in the bulk oil stirred for 15 min (SBO), and in the oil phase
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of the emulsions (EO). Emulsions were formulated using a mixer equipped with a stainless
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steel propeller to minimize the introduction of pro-oxidant metals. Because emulsification
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was not performed under nitrogen, oxygen could potentially be incorporated during the
235
process, thus promoting hydroperoxide formation. A stirring step was thus applied to the bulk
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oils in order to assess the impact of oxygen enrichment. Within experimental uncertainty, the
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conjugated diene concentrations were comparable for UBO, SBO and EO systems (see
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supplementary information n°2). Thus, the relatively short stirring time (15 minutes) was
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239
considered insufficient to modify the oxidation status of the oils. Ambrosone, Cinelli, Mosca
240
& Ceglie (2006) also observed that the peroxide value of crude olive oil did not change after
241
45 minutes of stirring. The incorporation of a 0.1 M NaCl aqueous phase to form the W/O
242
emulsions did not increase the initial concentration of conjugated dienes. This result shows
243
that water has no noticeable impact on the oxidation rate as long as it does not contain pro-
244
oxidant ionic species.
245 246
3.2 Influence of the oil nature in W/O emulsion on lipid oxidation
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The samples, UBO, SBO and EO, were stored at 60 °C for 72 h. Upon storage, a significant
248
increase in the conjugated diene concentration was observed in all cases, but more
249
pronounced in processed systems than in unprocessed ones. It is likely that the oxygen
250
incorporated during the stirring step promoted oxidation. The values were similar for stirred
251
oils and for emulsions, again confirming that water droplets did not have any noticeable
252
influence (see supplementary information n°2). The conjugated diene concentration increased
253
in all cases (see supplementary information n°2). For crude oils, the following hierarchy in the
254
oxidative stability was obtained: linseed oil < camelina oil < rapeseed oil < olive oil. Linseed
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oil, with the highest n-3 PUFA content (57 %), was the least stable oil despite its high
256
tocopherol content (Table 1). Interestingly, there was a positive correlation between the
257
oxidation (normalized OD) and the average number of double bonds (R = 0.99) and the
258
linolenic acid content (R = 0.97). This pattern of correlations was also reported for soybean
259
germplasms and other unsaturated oils (Kamal-Eldin, 2006). This may be because n-3 PUFA
260
have the highest methylene bridge index, i.e. the mean number of bisallylic methylene
261
positions, which make them the most susceptible to oxidation (Shahidi & Zhong, 2010). The
262
commercial sample of refined rapeseed oil used in this work was less prone to oxidation, as
263
revealed by the conjugated diene concentration that increased to a lesser extent than the crude
11
264
oil. This is consistent with the fact that, in the oil industry, the refining step is typically
265
applied to remove pro-oxidant species such as free fatty acids, polar and oxidized compounds
266
(Farhoosh, Einafshar & Sharayei, 2009).
267 268
3.3 Influence of the water phase composition on lipid oxidation in W/O emulsions
269
Encapsulation of water-soluble pro-oxidants was performed in order to enhance lipid
270
oxidation. Iron was chosen because it is known to accelerate lipid oxidation by promoting the
271
decomposition of hydroperoxides (Schaich, 2005). Moreover, iron is one of the major pro-
272
oxidants found in foods (Cho, Alamed, McClements & Decker, 2003) and is widely used in
273
food fortification for consumers suffering from anaemia (Huma, Rehman, Anjum, Murtaza &
274
Sheikh, 2007). Figure 2 presents the evolution of the conjugated diene concentration with
275
time for different emulsified oils containing ferrous sulfate (Fe2+SO42-) in the water droplets,
276
stored at 25 °C. For each oil, after a 72 h-storage, lipid oxidation was greatly enhanced by
277
addition of ferrous sulfate compared with the control system devoid of iron at 60 °C: for
278
example, for linseed oil, the normalized OD was 0.87 ± 0.06 (mg of oil.mL-1)-1 and 1.78 ±
279
0.05 (mg of oil.mL-1)-1, in the absence and presence of ferrous sulfate, respectively. The
280
relationship between lipid oxidation and fatty acid composition was evaluated again and a
281
positive correlation determined between the normalized OD and the average number of
282
double bonds (R = 0.97), and the linolenic acid content (R = 0.98).
283
Figure 3 reveals the influence of the ferrous sulfate concentration in the aqueous drops of
284
W/O emulsions based on refined rapeseed oil: the higher the encapsulated iron concentration,
285
the faster the oxidation process. This result suggests that the surfactant layer does not impede
286
the contact of ferrous ions with lipid molecules at the interface. Charoen et al. (2012)
287
observed a similar trend in oil-in-water emulsions and they hypothesised that this was due to
12
288
either a partial solubilization of ferrous sulfate in the oil phase or to the easy access of the pro-
289
oxidant metal ions at the water/oil interface, in close proximity to the lipid substrate.
290
In order to understand better the impact of the interfacial composition, the PGPR to DMG
291
ratio in the oil phase was varied (Figure 4). The variation of the bulk concentration was
292
expected to modify the interfacial composition. In the following set of experiments, the DMG
293
concentration in the oil phase was set to 1.5 wt.% and 3 different PGPR concentrations,
294
namely 1.5, 3 and 5 wt.% were adopted. Increasing the PGPR fraction slowed down the lipid
295
oxidation process. It can be hypothesized that the polar moieties are larger in the oligomeric
296
PGPR chains than in DMG molecules. Consequently, the presence of PGPR at the interface
297
may contribute to build-up thick interfacial layers acting as a physical barrier that separates
298
the lipid substrates from iron in the aqueous phase. Within this scheme, increasing the PGPR-
299
to-DMG fraction in bulk, and thus at the interface, has the effect of preventing lipid oxidation,
300
as observed experimentally. It is worth noting that we did not notice any influence of the
301
amounts of PGPR on the conjugated diene concentrations for the W/O emulsions based on
302
refined rapeseed oil containing only NaCl in the aqueous phase, stored at 60 °C, for 72 h: the
303
normalized OD was 0.55 ± 0.03 (mg of oil.mL-1)-1 and 0.54 ± 0.02 (mg of oil.mL-1)-1, for 2
304
wt% and 5 wt% PGPR, respectively. These results suggest that lipid oxidation involves
305
interfacial and bulk contributions and that, in the absence of water-soluble pro-oxidant
306
species, the interfacial contribution becomes negligible.
307
Ferrous and ferric chloride solutions at the same concentrations (5.10-3 M) were used for the
308
fabrication of W/O emulsions based on refined rapeseed oil to examine the impact of the iron
309
valance. The ionic strength within the aqueous droplets was almost identical and mainly
310
determined by the background electrolyte (0.1 M NaCl). After 72 h at 25 °C, the normalized
311
OD was 1.10 ± 0.08 (mg of oil.mL-1)-1 for the emulsion containing ferrous chloride
312
( ݁ܨଶା 2 ) ି ݈ܥand 1.11 ± 0.02 (mg of oil.mL-1)-1 for the emulsion containing ferric chloride
13
313
( ݁ܨଷା 3) ି ݈ܥ. Thus, lipid oxidation was similar regardless of the charge of the iron ions
314
encapsulated in the water phase. A very distinct conclusion was drawn by Mei et al. (1998)
315
who studied O/W emulsions stabilized by sodium dodecyl sulfate (SDS). It was found that
316
Fe3+ had a stronger pro-oxidant activity than Fe2+, presumably because of its higher binding
317
capacity to the negatively charged surface covered by anionic dodecyl sulfate molecules.
318
Unlike SDS, the surface-active species used in the present study were neutral, which may
319
explain why the oxidation was insensitive to the iron charge.
320
In Figure 5, we report the kinetic evolution of the normalized OD in W/O emulsion based on
321
refined rapeseed oil, encapsulating different iron ferrous salts at the same concentration (5.10 -
322
3
323
the iron charge remained constant (+ 2) and we thus explored the influence of the counter-ion
324
nature. The rate of lipid oxidation followed the order: ferrous chloride > ferrous sulfate >
325
ferrous lactate > ferrous gluconate. Such a hierarchy could reflect the ligand environment of
326
iron. It can be hypothesized that ferrous ions are efficient catalyzers for lipid oxidation as free
327
ions and that they become less efficient when strongly bound to their counter-ion. Indeed, the
328
slowest oxidation rates were obtained in the presence of counter-ions promoting iron
329
chelation: lactate and gluconate (Smith & Martell, 1975). Similarly, Hegenauer Saltman,
330
Ludwig, Ripley & Bajo (1979) compared simple ferrous and cupric salts to iron and copper
331
chelates in homogenized O/W milk emulsions. They found that chelated forms produced
332
significantly less lipid peroxidation at concentrations within the practical range of
333
fortification. In the same vein, Sugiarto, Ye, Taylor & Singh (2009) showed that the ability of
334
iron to catalyze lipid oxidation on milk emulsions was significantly reduced when iron was
335
bound to proteins compared to with when it was in its free form.
M), and in the presence of 0.1 M NaCl to fix the ionic strength. In this set of experiments,
336 337
4. Conclusion
14
338 339
We have studied the evolution of the conjugated diene concentration in plant oils with
340
different fatty acid compositions, and in the corresponding water-in-oil emulsions. In the
341
absence of water-soluble pro-oxidant metal cations, lipid oxidation preferentially occurred in
342
the bulk phase and was insensitive to the presence of water droplets. In this system, the main
343
parameter influencing lipid oxidation was the fatty acid composition of the triglycerides. As
344
expected, the encapsulation of pro-oxidant metal cations within the aqueous droplets
345
promoted lipid oxidation. The oxidation rate was then dependent on the interfacial
346
composition. The oxidation rate could also be tuned via the counter-ion nature of the metal
347
cation.
348
On the whole, this work has identified some key formulation parameters to control the
349
oxidation rate of water-in-oil emulsions. Our results might provide some guidance for the
350
design of functional foods based on emulsions, fortified by transition metal cations likely to
351
promote lipid oxidation (iron, magnesium, copper, etc.). It is within the reach of future work
352
to screen other parameters such as droplet size or fraction.
353 354
Acknowledgments The financial support of the Ministry for Higher Education and Research
355
of Tunisia is greatly acknowledged for its financial support through PhD research grant for
356
W.D. The authors are also grateful to the Aquitaine Regional Council for its support in
357
material investment. We thank also I. Mahwachi, for her technical support in HPLC
358
measurements.
359 360
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465 466
20
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Figure captions
468 469
Figure 1. Optical microscopy images of a W/O emulsion based on refined rapeseed oil
470
containing 2 wt.% PGPR and 2 wt.% DMG in the oil phase and 0.1 M NaCl in the water
471
phase: (a) just after preparation, (b) after 72 h-storage at 25 °C and (c) after 72 h-storage at 60
472
°C.
473 474
Figure 2. Evolution of the normalized optical density (OD) with time for different oils in W/O
475
emulsions: crude olive oil (), crude camelina oil (), crude linseed oil (), crude rapeseed
476
oil (), and refined rapeseed oil (). All emulsions were formulated with NaCl (0.1 M) and
477
FeSO4 (5.10-3 M) in the aqueous phase, and 1.5 wt.% PGPR and 1.5 wt.% DMG in the oil
478
phase. The storage temperature was 25 °C.
479 480
Figure 3. Evolution of the normalized optical density (OD) with time for refined rapeseed oil
481
in W/O emulsion and different FeSO4 concentrations in the aqueous phase: 0 M (), 2.10-3 M
482
(), and 5.10 -3 M (). The emulsions were formulated with 0.1 M NaCl in the aqueous
483
phase, and oil phases contained 1.5 wt% PGPR and 1.5 wt% DMG. The storage temperature
484
was 25 °C.
485 486
Figure 4. Evolution of the normalized optical density (OD) with time for refined rapeseed oil
487
in W/O emulsion stabilized by constant DMG concentration (1.5 wt.%) and different PGPR
488
concentrations: 1.5 wt.% (), 3 wt.% (), and 5 wt.% (). The emulsions were formulated
489
with 0.1 M NaCl and 5 10 -3 M FeSO4 in the aqueous phase. The storage temperature was 25
490
°C.
491
21
492
Figure 5. Evolution of the normalized optical density (OD) with time for refined rapeseed oil
493
in W/O emulsion and different pro-oxidant solutions in the aqueous phase: 5.10 -3 M ferrous
494
chloride (), 5.10-3 M ferrous sulfate (), 5.10 -3 M ferrous gluconate (), and 5.10-3 M
495
ferrous lactate (). All aqueous phase also contained 0.1 M NaCl. The emulsions were
496
formulated oil phases contained 1.5 wt.% PGPR and 1.5 wt.% DMG. The storage temperature
497
was 25 °C.
498 499 500 501
22
502
(a)
503 504
(b)
505 506
(c)
507 508 509 510
Fig. 1
511 512
23
513 514 515 516
OD (233nm)/(mg of oil.mL-1)
2.0
1.6
1.2
0.8
0.4
0.0 0
6 12 18 24 30 36 42 48 54 60 66 72 78
Time (hours) 517 518 519 520
Fig. 2
521 522
24
523 524 525
OD (233nm)/(mg of oil.mL-1)
1.6
1.2
0.8
0.4
0.0 0
6 12 18 24 30 36 42 48 54 60 66 72 78
Time (hours) 526 527 528 529 530
Fig. 3
531 532
25
533 534 535
OD(233nm)/(mg of oil.mL-1)
1.6
1.2
0.8
0.4
0.0 0
6 12 18 24 30 36 42 48 54 60 66 72 78
Time (hours)
536 537 538 539 540
Fig. 4
541 542
26
543 544
OD(233 nm)/(mg of oil.mL-1)
545
1.6
1.2
0.8
0.4
0.0 0
6 12 18 24 30 36 42 48 54 60 66 72 78
Time (hours) 546 547 548 549 550 551 552 553
Fig. 5
554
27
555
Table 1. Oil characteristics Refined
Virgin
Virgin
Virgin
Virgin
rapeseed oil
rapeseed oil
olive oil
camelina oil
linseed oil
16:0
4.4
4.5
15.6
5.8
5.3
16:1(n-7)
0.2
0.2
1.7
0.0
0.0
18:0
1.3
1.4
2.0
2.6
3.6
18:1(n-9)
58.9
59.9
59.5
13.5
17.6
18:1(n-7)
2.9
2.8
2.7
0.7
0.6
18:2(n-6)
19.9
19.5
16.1
20.5
15.0
18:3(n-3)
9.5
8.6
0.7
35.1
57.3
20:0
0.4
0.4
0.3
1.3
0.1
20:1(n-9)
1.2
1.0
0.2
13.6
0.1
20:2(n-6)
0.7
0.9
1.1
0.2
0.0
20:3(n-6)
0.0
0.0
0.0
2.0
0.0
21:0
0.0
0.0
0.0
1.5
0.0
22:1(n-9)
0.5
0.2
0.0
2.3
0.0
Othersa
0.1
0.4
0.0
0.8
0.3
Fatty acid (% wt)
Σ SFAb
6.2
6.7
18.0
11.5
9.1
b
Σ MUFA
63.7
64.3
64.1
30.6
18.4
Σ PUFAb
30.1
29.0
18.0
57.9
72.5
Σ n-6 PUFAb
20.6
20.4
17.2
22.8
15.2
Σ n-3 PUFAb
9.5
8.6
0.7
35.1
57.3
Tocopherol content (mg/100 g of oil)
α-tocopherol
9.9
7.4
14.9
1.3
0.5
β-tocopherol
3.3
2.8
0.5
0.5
4.3
γ-tocopherol
14.7
16.9
1.1
13.6
23.9
δ-tocopherol
0.8
0.8
0.1
1.1
0.8
28.7
27.9
16.1
16.5
29.5
3.8
3.0
2.8
2.5
Total tocopherols -1
Peroxide value (meq.kg of oil) 0.5
28
556
a
557
b
558
PUFA: sum of the polyunsaturated fatty acids of the n-3 series; Σ n-6 PUFA: sum of the
559
polyunsaturated fatty acids of the n-6 series.
Others represent fatty acids that contributed