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

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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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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]

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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

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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

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promoted by iron encapsulation in the aqueous phase, even at 25 °C. However, iron chelation

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reduced the oxidation rate. Emulsions based on triglycerides rich in polyunsaturated fatty

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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

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fractions of high molecular weight components reduced the oxidation rate when iron was

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present.

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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).

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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

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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

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storage conditions. In many food products, oil is present either in the dispersed phase of oil-

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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

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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

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the seminal papers allowed significant knowledge advances, there are still many questions

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regarding lipid oxidation in W/O emulsified systems. For instance, the impact of the dispersed

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aqueous phase on the rate and mechanisms of PUFA oxidation are controversial. Some

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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.%)

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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

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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.

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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

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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-

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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

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90

propagation step are the primary oxidation products, related to the early oxidation state.

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During the formation of hydroperoxides from unsaturated fatty acids, conjugated dienes are

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formed due to the rearrangement of the double bonds. The concentration of conjugated dienes

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can be followed easily by spectrophotometry due to the absorption of the diene chromophore

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around 233-235 nm (Corongiu and Milia, 1983; Shahidi and Zhong, 2005). Both the peroxide

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value and the diene concentration reveal the formation of primary oxidation products and are

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highly correlated (Shahidi & Zhong, 2005). However, compared with the peroxide index,

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spectrophotometric measurement of diene concentration is simpler, requires a smaller sample

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volume, and consumes less time and solvents. Thus, in this work, lipid oxidation was

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followed by measuring the concentration of conjugated diene hydroperoxides using a

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spectrophotometer.

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2. Experimental methods

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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

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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

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gas, split ratio of 50). The GC system consisted of a gas chromatograph (GC 2010 plus,

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Shimadzu, Kyoto, Japan) equipped with a flame ionization detector maintained at 280 °C. The

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injector was set at 250 °C. The column temperature was increased from 150 °C to 200 °C at

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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

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v2.4 integration system (Shimadzu). Fatty acids from Sigma–Aldrich (Saint Louis, MO,

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USA) and natural extracts of known composition were used as standards for column

120

calibration.

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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

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G1329A) of 20 µL loop size, and an analyzing software, Agilent ChemStation. An analytical

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pre-packed column (250×2 mm; 5 µm) YMC-Pack SIL (YMC Co, LTD, Kyoto, Japan) was

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used with isopropanol in hexane (0.5:99.5, v/v) as the mobile phase. The system was operated

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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

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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).

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Polyglycerol polyricinoleate (PGPR) and distilled mono-glycerides (DMG) were purchased

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from Palsgraad (Juelsminde, Denmark). Ferrous sulfate and Iotect, an iodine indicator, were

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from VWR Chemicals (Leuven, Belgium). Ferric chloride was purchased from Merck

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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

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Sigma Aldrich. Glacial acetic acid was purchased from Xilab (Bruges, France). Potassium

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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

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Jersey, USA). Solvents were of analytical grade. Hexane and isopropanol with HPLC-grade

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were purchased from Sigma Aldrich.

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2.2 Emulsion preparation and structural characterization

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W/O emulsions were prepared at room temperature using a homogenizer (RZR 2102 control

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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

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was required to inhibit destabilization phenomena such as Oswald ripening (Kabalnov, 2001)

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and coalescence (Aronson & Petko, 1993). In some cases, a second electrolyte based on iron

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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

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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

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prepared at least in triplicate.

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Direct visualization of the water droplets just after preparation and during the oxidative

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experiments was carried out using an optical microscope (Leica DM2500P microscope

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equipped with an oil immersion ×100 objective, Zeiss, Germany) and a digital camera for

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capturing images. The images were processed by a Leica IM50 software to estimate the

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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

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light-scattering, using a Coulter LS 230 apparatus. To avoid multiple scattering, W/O

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emulsion samples were diluted with a dodecane solution containing 0.5 wt.% sorbitan

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monooleate (Span 80) surfactant to ensure emulsion stability in the measuring cell. The

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measuring cell was filled with the dodecane solution, and a small volume of the sample was

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introduced under stirring.

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2.3 W/O emulsion oxidation experiments and lipid oxidation measurements

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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

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in Table 1. Lipid oxidation was followed in bulk oils or W/O emulsions with time (72 hours).

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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

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excess during the experiments. Lipid oxidation was followed as production of diene

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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

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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

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a final lipid concentration that ensured an absorbance measurement in the spectrophotometer

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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),

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since this parameter is proportional to the diene concentration according to the Beer-

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Lambert’s law. In order to take into account the dilution factor imposed by the measuring

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conditions, the OD measured at 233 nm was normalized by the mass of lipids (mg) per unit

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volume of solution (mL). Three measurements were performed for each formulation.

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2.4. Statistical analysis

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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).

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3. Results and discussion

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In this work, different edible oils were used to evaluate the effect of the unsaturated fatty acid

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content on the oxidability of W/O emulsions. All the oils complied with Codex Alimentarius

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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

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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

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based on plant oils, namely PGPR. This surfactant has been demonstrated to be highly

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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

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content and an acceptable kinetic stability of the W/O emulsions.

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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

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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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considered insufficient to modify the oxidation status of the oils. Ambrosone, Cinelli, Mosca

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& Ceglie (2006) also observed that the peroxide value of crude olive oil did not change after

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45 minutes of stirring. The incorporation of a 0.1 M NaCl aqueous phase to form the W/O

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emulsions did not increase the initial concentration of conjugated dienes. This result shows

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that water has no noticeable impact on the oxidation rate as long as it does not contain pro-

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oxidant ionic species.

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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

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increase in the conjugated diene concentration was observed in all cases, but more

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pronounced in processed systems than in unprocessed ones. It is likely that the oxygen

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incorporated during the stirring step promoted oxidation. The values were similar for stirred

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oils and for emulsions, again confirming that water droplets did not have any noticeable

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influence (see supplementary information n°2). The conjugated diene concentration increased

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in all cases (see supplementary information n°2). For crude oils, the following hierarchy in the

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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

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tocopherol content (Table 1). Interestingly, there was a positive correlation between the

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oxidation (normalized OD) and the average number of double bonds (R = 0.99) and the

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linolenic acid content (R = 0.97). This pattern of correlations was also reported for soybean

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germplasms and other unsaturated oils (Kamal-Eldin, 2006). This may be because n-3 PUFA

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have the highest methylene bridge index, i.e. the mean number of bisallylic methylene

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positions, which make them the most susceptible to oxidation (Shahidi & Zhong, 2010). The

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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

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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

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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

Influence of formulation on the oxidative stability of water-in-oil emulsions.

The oxidation of water-in-oil (W/O) emulsions was investigated, emphasizing the impact of compositional parameters. The emulsions had approximately th...
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