Effect of α-tocopherol on oxidative stability of oil during spray drying and storage of dried emulsions

    Effect of α-tocopherol on oxidative stability of oil during spray drying and storage of dried emulsions Maria del Rayo Hernandez Sanchez, Marie-Elisabeth Cuvelier, Christelle Turchiuli PII: DOI: Reference:

S0963-9969(16)30181-8 doi: 10.1016/j.foodres.2016.04.035 FRIN 6267

To appear in:

Food Research International

Received date: Revised date: Accepted date:

4 December 2015 25 April 2016 25 April 2016

Please cite this article as: del Rayo Hernandez Sanchez, M., Cuvelier, M.-E. & Turchiuli, C., Effect of α-tocopherol on oxidative stability of oil during spray drying and storage of dried emulsions, Food Research International (2016), doi: 10.1016/j.foodres.2016.04.035

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ACCEPTED MANUSCRIPT Effect of α-tocopherol on oxidative stability of oil during spray drying and storage of dried emulsions

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Maria del Rayo Hernandez Sanchez1*, Marie-Elisabeth Cuvelier1, Christelle Turchiuli1,2

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1- UMR Ingénierie Procédés Aliments, AgroParisTech, Inra, Université Paris-Saclay, 91300

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Massy, France

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2- Université Paris-Sud, IUT Orsay, Plateau de Moulon, F- 91400 Orsay, France

E-mail:

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[email protected] – Tel. +33 (0) 1.69.93.51.30 [email protected] – Tel. +33 (0) 1.69.93.50.03

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*Corresponding author:

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[email protected] – Tel. +33 (0) 1.69.93.50.71

Maria del Rayo Hernandez Sanchez AgroParisTech

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1 avenue des Olympiades 91300 Massy

ABSTRACT Lipophilic compounds such as polyunsaturated fatty acids (PUFAs) and antioxidants can be encapsulated by spray drying in order to protect and prolong their functionalities and get new handling properties. The aim of this work was to study the effect of both spray drying stage and storage (60°C – 50% RH) on the oxidation of lipophilic compounds encapsulated in spray dried oil-in-water emulsions (10 % w/w oil in dry matter) using maltodextrin as matrix and Tween®20 as emulsifier. Emulsions were prepared with oil containing or not -tocopherol (482 ppm in oil) in order to also demonstrate the influence of antioxidant. Results showed that there is a beginning of oxidation during spray drying, evidenced by a slight increase of markers of rancidity, i.e. conjugated dienes and volatile organic compounds. During storage, the oxidative degradation of PUFAs and -tocopherol started quickly under the conditions of aging. This was shown to be due to the negative effect of the process and to the porosity of the solid matrix to oxygen, associated with the hollow structure of the particles.

ACCEPTED MANUSCRIPT An inhibitory action of maltodextrin on a-tocopherol was also hypothesized, but it has to be confirmed.

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Key words

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Encapsulation, spray drying, oxidation, accelerated oxidation, volatile organic compounds, conjugated dienes, α-tocopherol

1. Introduction

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Lipophilic compounds such as polyunsaturated fatty acids (PUFAs), flavors, aroma, vitamins and antioxidants are known for their positive effects on both organoleptic properties of foods (e.g. improving taste, appearance, flavour and texture) and human health (Arab-Tehrany et al., 2012; Chen, McClements, & Decker, 2013; Taneja & Singh, 2012). But, lipophilic compounds rich in large unsaturated hydrocarbon chains, such as PUFAs, are susceptible to lipid oxidation resulting in the deterioration and degradation of their functional properties. When PUFAs are in contact with oxidation initiators such as metals, light or heat, hydrogen atoms are removed from the polyunsaturated fatty chain producing alkyl radicals. These newly formed free radicals rapidly react with molecular oxygen producing peroxyl radicals, which keep high reactivity and generate hydroperoxides, which are primary oxidation products (Chaiyasit, 2007). Hydroperoxides have no smell and taste and are relatively stable at room temperature but can be decomposed into alkoxyl radicals by transition metal cations or at high temperatures. The alkoxyl radicals contribute to the propagation of oxidation and endure several scission reactions leading to the formation of secondary oxidation products such as aldehydes, ketones, alcohols, alkanes, esters, furans and lactones, which are responsible for undesirable odors and off-flavors associated with rancidity of lipids (Frankel, 1998; McClements, 1999 ; Min & Boff, 2002 ; Choe & Min, 2006 ; Chaiyasit, 2007 ; Kolakowska & Sikorski, 2011). In some cases, lipophilic compounds also play a key role in the preservation and conservation of other oxidizable lipophilic compounds. Especially, some antioxidants are lipophilic compounds capable of delaying or interrupting oxidation of products rich in large unsaturated hydrocarbon chains, such as PUFAs. Among lipophilic antioxidants, tocopherol is an effective antioxidant that can be either endogenously present in vegetable oils or added as an additive (Ko et al., 2010; Xu et al., 2013; Karmowski et al., 2015; Kim et al., 2015). Besides the use of antioxidants, encapsulation in powder enhances the protection and prolongation of the functional properties of lipophilic compounds by adding a physical protection. It is also a way to modify handling properties when going from the liquid to the powdered form. This encapsulation of lipophilic compounds involves two main steps: a) the dispersion of lipophilic compounds in oil-in-water emulsions (Jayasinghe et al., 2013; Wang et al., 2016; Di Mattia et al., 2009) and b) the spray drying of these emulsions in order to replace the liquid barrier formed during emulsification by another stronger solid barrier (encapsulation matrix), providing protection against degradation and volatile losses. The physical barrier or encapsulation matrix is mainly formed by the wall materials entrapping lipophilic compounds within (Ahn et al., 2012; Damerau et al., 2014; Hernandez-Sanchez et al, 2015; Quintanilla-Carvajal., 2014; Carneiro et al., 2012; Turchiuli et al., 2013, 2014;

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Munoz-Ibanez et al., 2015). During the encapsulation of lipophilic compounds by spray drying, emulsions are pumped, atomized and later dried in contact with a hot air current resulting in a fine dried powder (20-100 m) with low water activity and modified handling properties (Fuchs et al., 2006; Gharsallaoui et al., 2007; Tonon et al., 2011; Kaushik et al., 2014).

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Since the delay or restriction of lipid oxidation is a quality parameter of lipophilic compounds-enriched food products, it is important to study the evolution of oxidation indicators upon processing and storage of these products. The use of accelerated oxidation tests allows estimating the oxidative stability when a product is exposed at highly aggressive conditions such as temperature and humidity (Gomez-Alonso et al., 2004; Mancebo-Campos et al., 2008; Vandamme et al., 2015). Although accelerated oxidation conditions generally do not correlate with shelf life conditions commonly used in industry, these tests are a useful tool to evaluate both the antioxidant capacity and encapsulation efficiency.

2.1. Materials

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2. Material and Methods

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The aim of this work was to study the effect of spray drying stage and storage on the oxidation of lipophilic compounds in oil-in-water emulsions (10 % w/w oil in dry matter constituted of maltodextrin) and to demonstrate the influence of -tocopherol (482 ppm in oil) on the oxidative stability of these compounds. Stripped sunflower oil was used as a source of PUFAs and for accelerated oxidation, powder samples were stored at 60°C under 50 % relative humidity. The oxidative stability was evaluated by following the concentration of primary oxidation products such as conjugated dienes (CD), the formation of secondary oxidation products such as total volatile organic compounds (VOCs) and the loss of αtocopherol (α-toc).

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Commercial sunflower oil (0.05% w/w α-tocopherol, 11% w/w saturated palmitic and stearic acids, 29% w/w mono-unsaturated oleic acid and 60% w/w poly-unsaturated linoleic acid) was purchased from a local store (Cora, FR). Before use, it was stripped from its natural tocopherols (See section 2.2) and later enriched with α-tocopherol (-toc) (Sigma-Aldrich, GE). The emulsions were stabilized by adding polyoxyethylene (20) sorbitan monolaurate (Tween® 20) (Sigma-Aldrich, FR) as surfactant and Maltodextrin DE 12 (MD) (Glucidex®, Roquette, FR) was used as wall material for the encapsulation process. Hexane (quality CHROMASOLV®) and isopropanol (quality CHROMASOLV® Plus) were purchased from used Sigma-Aldrich (FR). Tetrahydrofuran (THF), n-heptane and isooctane were purchased from Carlo Erba Reagents (FR).

2.2. Method for stripping sunflower oil The aim of the stripping process was to remove endogenous tocopherols present in commercial sunflower oil. The stripping method is based on the affinity chromatography principle and it is solvent-free. For the purpose of this work, an adaption of the method described by Yoshida et al. (1992) was made: 600 g of aluminum oxide were previously activated at 200ºC for at least 3 h before use and then cooled in a dessicator (high temperatures may promote oil oxidation during stripping process). Then 700 g of commercial

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sunflower oil were stripped by passage over aluminum oxide (200 g) placed in a Büchner funnel and vacuum was applied (initial vacuum pressure 75 mbar, reduced progressively to 30 mbar). This process was repeated twice and the aluminum oxide was changed at each new passage. The removal of tocopherols was later confirmed by HPLC analysis (See section 2.9.3). The photo-oxidation of stripped sunflower oil during storage was delayed by blowing a stream of nitrogen in the collection vessel that was wrapped in an aluminum foil and stored at 4ºC.

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2.3. Preparation of initial liquid emulsions

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Oil-in-water emulsions containing oil (4 g/100 g), maltodextrin (MD) (36 g/100 g) and Tween® 20 (0.14 g /100 g i.e. 3.5 g /100 g of oil) were prepared by following the protocol described previously by Hernandez-Sanchez et al. (2015) for the preparation of stable emulsions. Briefly, the protocol consisted in first, an emulsification of oil in water containing Tween® 20 in order to obtain a first emulsion with the required oil size (e.g. about 2 m) and second, a dilution of this emulsions with an aqueous phase containing the wall material. Two types of emulsions were prepared: i) a control emulsion (without antioxidant) and ii) an emulsion enriched with 482 ppm of α-tocopherol added in the oil phase prior homogenization.

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2.4. Oil droplet size distribution

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The oil droplet size distribution of liquid emulsions was measured by laser light diffraction (Mastersizer 2000, Malvern, FR) in wet mode (Hydro 2000) after dispersion in purified water. The refractive index values used for the dispersing water and for the oil droplets were 1.33 and 1.475 respectively (Hernandez-Sanchez et al. ,2015) 2.5. Preparation of spray dried emulsions

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Liquid emulsions were spray dried in a Niro Minor pilot scale spray dryer (Niro, DK). It is a one step co-current spray dryer with an evaporative capacity comprised between 1 and 4 kg.h1 . For spraying, a rotary wheel was used with a rotation speed of 25,000 rpm (5.5 bar compressed air). The emulsions flow rate was 25 g.min−1. Drying air was taken from the ambient by a fan (43 Hz) with a flow rate of 110 kg.h−1 (Hernandez et al., 2015). The inlet air temperature was fixed at 200 ºC and its outlet temperature varied from 94 to 110 ºC for the different trials. Two series of powders were produced: P0 - control powder without antioxidant; and P1 – with oil enriched with α-tocopherol. 2.6. Particle size distribution of dry emulsions The particle size distribution of powders was measured by laser light diffraction (Mastersizer 2000, Malvern, FR) in dry mode (Scirocco 2000) (Hernandez-Sanchez et al. ,2015). 2.7 Reconstitution of emulsion Liquid emulsions were reconstituted by adding 4 g of dry emulsions in 6 g of pure water, resulting in emulsions having the same dry matter content as the initial liquid emulsions (40 g /100 g emulsion)

ACCEPTED MANUSCRIPT 2.8 Scanning Electron Microscopy (SEM)

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2.9. Conditions of accelerated thermo-oxidation

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A Quanta 200 model Scanning Electron Microscopy (FEI, EE.UU) was used to study the microstructure of the powders (dry emulsions). The samples were mounted on aluminum stubs with double-side sticky carbon tape, and subsequently subjected to a gold sputter coating process. The coated samples were analyzed under high vacuum (< 6.10-4 Pa) at accelerating voltage of 5kV. The micrographs shown in this paper were taken by the instrument’s software installed on the PC connected to the SEM.

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After production, the powder was stored for accelerated thermo-oxidation as follows: 150 g of each powder (P0 - control without antioxidant; and P1 - enriched with α-tocopherol) were equally distributed in 3 open vessels (50 g powder/vessel). The vessels were randomly placed into a climatic test chamber VCN 100 (VÖTSCH Industrietechnik®, FR) at 60 °C and 50 % RH during 150 days. The samples were manually homogenized and randomly changed of place every week and before sampling. Regular samplings were done at different times. For practical reasons, analysis of samples may need to be delayed. In this case samples were stored under reduced temperature (e.g. -20°C) for few days and it was checked that no evolution of the sample regarding oxidation occurred during freezing storage for both powders (results not shown).

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2.10 Measurement of lipid oxidation

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In this study three markers of lipid oxidation were evaluated: conjugated dienes (CD), total volatile organic compounds (VOCs) and α-tocopherol content. The given values correspond to the mean between three values obtained for three samples of the same powder picked up in three different vessels.

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2.10.1. Oil-organic phase extraction for the measurement of conjugated dienes and αtocopherol) concentration The measurement of conjugated dienes (standard ISO 3656:2011) and α-tocopherol (NF ISO 9936:2006) in oil droplets contained in dried emulsions classically requires the extraction of oil from the powder sample by organic solvent and evaporation, for example according to the method described by Kim et al. (2005). In this study, the method was modified to allow measuring conjugated dienes and -tocopherol directly in the organic phase of extraction without solvent evaporation. For that purpose, 2 mL of ultra-pure water at 50 °C were added to 0.5 g of powder and agitation was maintained until the emulsion was reconstituted. Then, 50 mL of hexane/isopropanol (3:1, v/v) were added to the reconstituted emulsion. The mixture was agitated for 15 min and centrifuged at 1000×g for another 15 min. Centrifugation allowed to recover around 47 mL of organic phase, where 1 mL was used to measure conjugated dienes and 46 mL for the measurement of α-tocopherol concentration. The analysis was repeated three times for each sample. In the case of initial emulsions, 2.5 g of emulsion were used to perform the extraction. 2.10.2 Measurement of conjugated dienes

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Abs 234nm Wg

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SA 

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The conjugated dienes can be detected by spectrophotometry UV at 234 nm. For that purpose, 1 mL of the oil-organic phase were diluted 1:6 adding 5 mL of hexane/iso-propanol (3:1, v/v) and vortexed for 60 s. Then the sample absorbance (Abs 234 nm) was measured using a UVvisible spectrophotometer (Specord 210 Plus, Serlabo Technologies, FR). Finally, the specific absorbance (SA) representing the absorbance of conjugated dienes at 234 nm and indicative of the primary oxidation products content in the oil was calculated according to Equation 1:

Where Abs234nm is the absorbance of the sample measured at 234 nm and Wg is the oil mass (g) in 100 mL of the solvent solution analyzed.

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In order to validate this method, it was necessary to compare it with the one described in standard ISO 3656:2011. The specific absorbance measured for oxidized stripped sunflower oil following the present method and the standard ISO 3656:2011 was 24 ±1.7 and 22.2 ± 1.1, respectively confirming the validity of the method used. 2.10.3 Measurement of α-tocopherol concentration

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1 g of enriched oil was weighed in a 10 mL volumetric flask and n-heptane HPLC grade was added to complete the volume. The flask was then vortexed for 60 s and placed in an ultrasonic bath for 30 s. The solution was filtered through a nylon syringe filter with 0.45 μm pore size (Uptidisc, Interchim, FR) and put into vials. 20 µL of the sample were injected and analyzed with an HPLC system (Waters®, Milford, MA, USA) equipped with a pump (Waters® 2695) coupled with a UV-Visible diode array detector (DAD) at 298 nm (Waters® 1996). The stationary phase consisted in a bonded silica column 100 Diol (Lichrosphere, length 250 mm, internal diameter 4 mm, particle size 5 μm), thermostated at 25 °C and equipped with a precolumn (13 mm) with similar characteristics. The mobile phase was a mixture of n-heptane/tetrahydrofuran (96.15/3.85, v/v) eluted isocratically at a flow rate of 1 mL/min with a runtime of 12 min. The identification and quantification of -toc were conducted in both detectors. The HPLC system controlling and data acquisition was carried out using the Empower software 2® (Waters Corporation, Milford, MA, USA). To measure the -toc in the oil phase of initial liquid emulsions and powder samples, 46 mL of the oil-organic phase (cf. 2.10.1) were evaporated in a rotary rotavapor RE 120 (Buchi, CH). 5 mL of n-heptane were added to the extracted oil (around 0.042 g ± 0.011) and submitted to vortex and ultrasonic agitation before analysis as described above. Different solutions of -toc in n-heptane were used to make a calibration curve between 0.05 and 100 mg/100 L. The results obtained were expressed by peak area measured at 298 nm (y) as a function of concentration of α-toc in n-heptane (x) according to y= 8059.61  x with R2 = 1. Since the results given by the calibration curve were expressed as mg α-toc / L n-heptane, it was necessary to take into account the quantity of extracted oil added to n-heptane (0.042 g/ 5 mL = 8.4 x 10-3 kg/L) in order to express the concentration of α-toc (mg) in kg of oil (ppm). 2.10.4 Identification of total volatile organic compounds by Headspace solid-phase microextraction (SPME) and gas chromatography-mass spectrometry (GC-MS).

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For the determination of total volatile organic compounds (VOCs), 0.5 g of powder were weighed in 20 ml crimp-sealed glass vials and 0.25 g of water were added in order to facilitate the release of volatiles. Using a GERTEL Multipurpose Sampler (MPS) (Mülmheim an der Rur, GE), the sample vials were transported to the heated agitator. Once there, the samples were agitated at 3000 rpm and incubated at 40ºC for 30 min to establish thermodynamic equilibrium between the headspace and the sample itself. Then, a 50/30-µm SPME fiber assembly Divinylbenzene/Carboxen/Polydimethylsiloxane (DVB/CAR/PDMS) (Supelco Inc, Bellafonte, PA, EE.UU) was inserted through the septum of the sample vial and left for 30 min to allow the absorption of volatiles onto the coating of the SPME fiber. The fiber equilibrated with the headspace volatiles was then inserted into the injector of the gas chromatograph (GC) Combipal Agilent 6090 (Agilent Technologies®, Palo Alto, CA) coupled to a mass spectrometer (MS) Agilent 5975 (Agilent Technologies®, Palo Alto, CA). The absorbed compounds were thermally desorbed and introduced in the GC in splitless mode using helium as a carrier gas (16 cm.s-1). For the separation of volatiles, a DB-5ms capillary column (30m x 0.32-mm inner diameter x 0.5 µm, J&W Scientific, Folsom, Calif., USA) was used. The oven temperature program was set as follows: 40 ºC for 40 min, 60ºC to 160°C by 2ºC/min, then 160ºC for 4 min and finally, 160° to 240 ºC by 15 ºC/min. The mass spectrometer was operated in electron ionization mode. The temperature of the ion source was 230ºC. The ionization energy of MS was 70 eV and a full-scan ranged from 33 to 330 amu.

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The data acquisition was performed with the software Enhanced Chemstation (MSD Chemstation D.02.00275, Agilent Technologies, Palo Alto, CA) and the identification of the volatile organic compounds was carried out by comparing the mass spectrum with those from Wiley7 and Nist 05 Library data. Results were expressed as the total area of all the volatile compounds obtained in full scan mode. 2.11 Surface oil extraction

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The surface oil extraction was carried out by following an adaptation of the method described by Kim et al., (2005) for the quantification of non encapsulated oil. 10g of powder (P0) were weighed on a filter paper (No. 4, Whatman, Maidstone, Kent, UK), and then washed with 4×50ml of hexane. The recovered organic phase was evaporated with a rotavapor and later removed by blowing a stream of nitrogen until constant weight. The surface oil was expressed as g surface oil/ g powder.

2.12 Oxygen permeability test A qualitative test to estimate oxygen permeability of powders was carried out following an adapted non-invasive optical method described by Cuvelier et al. (2016). This method allowed measuring the oxygen partial pressure in a sealed test tube containing the powder sample. For this purpose, we used a fiber optic oxygen transmitter (Fibox 3 LCD trace, PreSens, DE) with a 2 mm optical fiber and an oxygen sensor spot which was glued inside the glass tube. The tip of the fiber was directed on the sensor spot through the wall and the oxygen partial pressure was measured from the fluorescence emission resulting of the quenching of luminescence caused by collision between molecular oxygen and luminescent dye molecules in the excited state. Samples (powders) were put into the glass flask and then a stream of nitrogen was blown to remove the oxygen from the headspace around the powder. Then, the flask was closed and the oxygen partial pressure was measured until reaching

ACCEPTED MANUSCRIPT equilibrium (e.g. stabilisation). The increase of partial pressure measured upon time was assumed to correspond to the oxygen released from the powder sample.

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2.13 Influence of oil dispersion in solid matrix on the oxidative stability of agitated bulk oil and oil dispersed on maltodextrin particles

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In order to estimate the effect of encapsulation by spray drying on the oxidative stability of oil, it was necessary to evaluate the oxidative stability of non-encapsulated oil. For that purpose, two different tests were performed: first, 30 g of control and -toc enriched oil were put in Erlenmeyer flasks under constant agitation (550 rpm) with the aim of increasing the surface contact with oxygen. Secondly, control oil and enriched oil were dispersed on maltodextrin particles in order to simulate the same surface/volume (S/V) ratio as the one expected in dry emulsions (e.g. 2.104 cm-1). In each system, 3.6 g of oil (without and with 482 ppm of α-toc) were diluted in 300 mL of hexane and then added to 30 g of maltodextrin particles with a median diameter d50 = 16 µm. The solvent was then removed by blowing a stream of nitrogen. Samples were stored in accelerated thermo-oxidation conditions (cf. 2.9) and conjugated dienes and α-tocopherol concentration were periodically determined (cf. 2.10.1, 2.10.2 and 2.10.3).

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3. Results and Discussion

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3.1.Size distribution of liquid emulsions (initial and reconstituted) and dry emulsions

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Two series of initial oil-in-water emulsions composed by 40 g of dry matter/100 g (36 %w/w maltodextrin, 4 %w/w oil and 0.14 %w/w Tween® 20) were prepared either with stripped oil (control without antioxidant) or with oil containing 482 ppm of α-toc. The oil droplet size distribution was monomodal in each case with similar median diameters (d50) around 1.2 and 1.7 μm and a span value of 2.3 corresponding to relatively straight distributions (Fig.1a). Regarding the oil droplet size in reconstituted emulsions from spray dried powders (Fig. 1b), it can be seen that both the d50 and the span have increased (e.g. up to 2.8 and 3.9 µm for d50 and 11.7 and 15.6 for span) due to both a slight increase of the initial oil droplets size and the apparition of 100 m-droplets. During spray drying, initial liquid emulsions are subjected to mechanical and thermal stresses resulting, in some cases, in the modification of the oil droplet size (Munoz et al., 2015). Especially, when oil droplet breakup occurs during atomization, the quantity of non-encapsulated oil at the solid particle surface increases. And, depending on the surfactant content and mobility, some coalescence of oil droplets may occur when reconstituting the emulsion as it has been observed by Turchiuli et al. (2014) for spray dried emulsions prepared without emulsifying agent. In this study, initial emulsions were prepared lowering the quantity of Tween® 20 to avoid interactions between antioxidant and surfactant molecules in excess. This may explain the different oil droplet size distribution obtained for reconstituted emulsions and tends to indicate the presence of unencapsulated oil at the solid particle surface. Furthermore, the spray dried powders obtained here (e.g. P0 (without -toc) and P1 (482 ppm -toc in oil)) were very fine particles whose median size ranged from 15.3 to 19.4 μm with a span between 1.4 and 2.3 corresponding to straight particle size distributions (Fig. 1c). The smaller solid particles obtained had therefore diameters close to those of the larger oil droplets in the initial emulsion. This will correspond to oil droplets not well encapsulated in the smaller solid particles and may explain the presence of larger coalesced oil droplets in the reconstituted emulsions.

ACCEPTED MANUSCRIPT 3.2 Microstructure and surface characterization of dry emulsions

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The spray drying of initial liquid emulsions resulted in powders (P0 and P1) with both shrunk and spherical particles (Fig.2 Top row). In both cases, their surface was homogeneous and smooth (Fig. 2 Middle row) with some bumps, especially for P1, which can be related to the presence of non-encapsulated oil (surface oil). Regarding the inner structure of the particles (Fig.2 Bottom row), it can be seen that spherical particles were hollow with the holes corresponding to oil droplets distributed throughout the thickness of the particle crust, but not in the close vicinity of the surface. The production of hollow particles can be related to the spray drying process conditions and to the formulation of the initial emulsion. When exposed to high drying air temperature, the evaporation of water from the surface of the emulsion droplet is fast leading to the formation of a solid crust, and resulting in hollow particles due to the migration of water from core to surface during further drying.. The composition of the liquid emulsion has also an impact in the final shape of the particles. Especially, the interfacial properties of the components in the formulation were found to influence the dry particle morphology. It tends to be smoother when the elasticity of the components at the drying drop surface is lower and permits the retraction of the drop during drying (Nuzzo et al., 2015; Jafari et al., 2008). 3.3. Effect of spray drying process on the oxidative stability of oil in emulsions

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The effect of spray drying on the oxidative stability of oil was estimated by comparison of the oxidative status of oil in the initial liquid emulsion before spray drying with that of oil in the spray dried powder immediately after production. The three oxidation markers were therefore measured in both series of samples with and without antioxidant: conjugated dienes content (SA value) as primary oxidation products (Tab.1), total volatiles organic compounds (VOCs) content as secondary oxidation products (Fig.3), and α-toc residual content in the case of samples containing -toc enriched oil (Tab.1). Assuming that no oil oxidation occurs during the emulsification stage (Martinez et al., 2015), SA values in initial liquid emulsions (before spray drying) with and without -toc were expected to be similar (Tab. 1). The higher SA value obtained for the liquid emulsion containing -toc was therefore attributed to the absorbance of -toc at 234 nm, which is taken into account in the SA value measured here. This was checked on the SA value measured for stripped oil which was higher when -toc was added (results not shown). Anyway, the results in Tab.1 show that spray drying caused a slight increase of conjugated dienes content with an increase of the SA value of 2.1 and 3.4 for respectively P0 and P1 after spray drying. Considering the variation coefficient on SA measurements, this increase was similar for both samples (e.g. about 20%) (Tab. 1) and indicates that oil was subjected to a slight initial oxidation during spray drying. This was confirmed with the analysis of volatile organic compounds in both the head-space of the -toc enriched oil used for the preparation of the initial emulsion and the head-space of the corresponding dry emulsion P1 (Fig. 3). The GC-MS analysis revealed that no VOCs were present in enriched initial oil whereas some compounds have appeared in the dry emulsion. Among the VOCs detected, the aldehydes (hexanal and nonanal) and octanoic acid are good markers of lipids oxidation, particularly coming from linoleic acid. So, the apparition of these VOCs just after spray drying confirms the initiation of oxidation during the process. This result is consistent with those of Takeungwongtrakul et al. (2015) who detected VOCs in shrimp oil after encapsulation by spray drying (Tinlet air = 180  2ºC, Toutlet air = 90  2ºC) using mixed wall materials (e.g. whey protein concentrate, sodium caseinate, acacia gum, glucose syrup and maltodextrin). Actually, during spray drying process, emulsion drops are in contact with a hot air current (e.g. up to 200°C). Even if during the beginning of water

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removal, drying liquid drops remain at the air wet bulb temperature (e.g. about 40°C), their temperature may start increasing at the end of drying when the air current has reached a temperature close to its outlet temperature (e.g. 94 – 110°C). Dry particles may therefore be exposed for a very short time (seconds) to high temperatures and leave the chamber at about 50-60°C before recovery. This may enhance the development of some oxidation products at the initial stage such as oxidation precursors or free radicals (Reineccius, 2004). On the other hand, Serfert et al. (2009b) suggested that oxidation of encapsulated fish oil during spray drying is rather due to the oxygen content in the initial liquid emulsion than to the oxygen content in the drying gas. To end, during a study of the encapsulation of chia oil using sodium caseinate and lactose as wall materials, Ixtaina et al. (2015) stated that the oil droplet size of initial emulsion might have an impact on the amount of oxidation precursors that can be formed during spray drying. A smaller oil droplet size produces a higher contact surface with hot air, increasing the contact area between prooxidative transtition metal ion and lipid hydroperoxides present at the oil-water interface (Jacobsen et al., 2000; Kargar et al., 2011).

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For powder P1, containing -toc, the antioxidant content in the oil phase of emulsions was measured before and after spray drying (Tab.1). Despite the theoretical content was 482 ppm, the content of -toc in initial liquid emulsion before spray drying was measured as 444 ± 9 ppm. This difference (e.g. 7.8%) may be explained by the yield of the extraction procedure before analysis and, we hypothesized that some -toc could be retained in Tween micelles in the aqueous phase of emulsion. However, when comparing the α-toc content in initial emulsion to the content in dry emulsion, it was observed that, taking into account the average standard deviation, antioxidant content was totally maintained, indicating that it was not degraded during the spray drying process. The increase of oxidation markers observed was therefore due solely to the oxidation of oil. This result is consistent with those obtained by Serfert et al. (2009a). They showed that autoxidation of fish oil encapsulated with a mixture of n-octenylsuccinate-derivatized starch and glucose syrup occurred during the process of encapsulation and oxidative stability cannot be achieved only by the addition of tocopherols alone.

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3.4. Effect of accelerated thermo-oxidation conditions (aging) on the oxidative stability of oil in dry emulsions Dry emulsions P0 and P1 were stored at 60 ºC and 50 % RH in order to study the oxidative stability of encapsulated oil and -toc under thermo-oxidation conditions (aging). Whilst spray drying encapsulation was expected to protect PUFAs from oxidation during aging, evolutions of SA value obtained (Fig. 4) demonstrate the absence of lag phase even during the very first days of aging with, for both samples, a fast increase of conjugated dienes content during the first days of storage. In P1, conjugated dienes content reached a maximum (SA about 60) after about 12 days of aging, followed by a notable decrease and then a stabilization (SA around 23) after about 60 days. For P0, the SA value increased similarly during the seven first days up to about 30 and then a slow down was observed until reaching a maximun of 43 at 30th day of aging. Beyond day 30, the evolution became similar again to the one of powder P1. Results in Fig. 4 are consistent with those presented by Wang et al. (2011) during the study of the oxidative stability of fish oil encapsulated with barley protein. They also observed the apparently absence of lag phase, with a sharp increase and then a reduction of peroxide values upon storage at 40ºC for 8 weeks. The authors attributed the absence of lag phase to the fact that there was some non-encapsulated oil at the particle surface oxidizing rapidly and causing the fast increase of peroxide content. Since these primary oxidation products oxidize to

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aldehydes and ketones (secondary oxidation products), when all the quantity of surface oil was oxidized, the peroxide content decreased indicating that the solid matrix actually ensured protection of the rest of the oil (encapsulated). The oxidative stability of powder P0, where non encapsulated or surface oil represented 15 % of the total oil (i.e. 0.015 ±0.001 g / g P0), was therefore compared with that of a powder sample where surface oil had been removed by solvent washing (hexane) (Fig.5). Results show that there is not a significant difference in the oxidation status between the two powders considering the presence or absence of surface oil, indicating that both surface and encapsulated oil fractions were oxidized similarly under accelerated thermo-oxidation conditions (aging). Our results therefore differ from the findings of Wang et al. (2011) on this point. Anyway, the fast development of oxidation markers observed in both P0 and P1 during the first 20 days of 60°C-50% RH aging and the apparent absence of lag phase can be attributed to three other causes: i) the initiation of oil oxidation during the spray drying process, which has been evidenced in section 3.3; ii) the oxygen permeability of the encapsulation matrix that would permits to oxygen to be in contact with encapsulated oil and iii) the high surface/volume ratio (S/V) due to the dispersion of oil into small droplets (e.g. inferior to 2 µm) to be encapsulated into about 20 µm particles.

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Regarding the oxygen permeability of the encapsulation matrix, a qualitative test of oxygen permeability in powders was carried out. Results showed that after 1 week, the partial pressure of oxygen increased by 7 %, indicating the presence of oxygen released by the powder. The detected oxygen could come either from the oil encapsulated in the matrix or from the air located in inner holes. Actually, as previously discussed (cf. 3.2), the powder samples contained some hollow particles (Fig. 2) with a thin solid wall and a large vacuole at the center possibly acting as an oxygen reservoir. During the first period of aging, oil droplets dispersed in the solid wall were therefore protected from surrounding air, but in contact with oxygen entrapped within the solid matrix. Oxidation thus arose until no more "inner oxygen" was available. This is in agreement with the results of Partanen et al. (2008) who observed that both encapsulated and bulk flaxseed oil displayed similar behavior regarding oxidation during 3 weeks of storage at 37°C and 0 – 91 % RH but then, the oxidation rate increased more rapidly in bulk oil. They attributed this behavior to the different oxygen availability at the oil surface in both samples. After three weeks, oxidation in the dry emulsion became limited by the concentration of oxygen whilst it was not for bulk oil in direct contact with ambient air. Finally, we also checked the influence of the dispersion of oil with a high surface/volume ratio in dry emulsions. Actually, whilst bulk oil oxidizes in continuous phase, the encapsulated oil oxidation is the result of individual oil droplets oxidizing at different reaction rates. Sarkar et al. (2016) observed that hydroperoxides in encapsulated oil were three-fold higher than those in bulk oil. Authors attributed this behavior to the exposure of oil to oxygen, light and higher temperature during every step involved in the process of encapsulation and also, to the huge surface area of oil droplets. Actually, the surface/volume ratio of oil plays a key role in the oxidation rate since the higher is the surface of exchange (S) with surrounding air related to the volume of oil (V), the higher is expected to be the concentration of the oxidation markers (Roman et al., 2013). In the present work, the S/V ratio of oil in dry emulsions was high, about 2.104 cm-1 (taking into account the oil droplet median diameter of reconstituted emulsions, which was assumed to be representative of the size of the oil droplets dispersed in the dry emulsion matrix). The oxidative stability of both control oil and -toc enriched oil when encapsulated in dry emulsions P0 and P1 respectively was therefore compared to the one of i) oil dispersed at the surface of maltodextrin particles of about 10µm

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in order to ensure a similar S/V ration; and to the one of ii) oil laying in bulk under agitation to also ensure a high S/V ratio (Fig. 6). Considering bulk oil, whilst for control oil (no antioxidant) there was a rapid increase of specific absorbance within 2 days, followed by a decrease (Fig. 6a), for -toc enriched oil, the SA value increased very slowly until day 12 and then started increasing more rapidly. It is obvious here that the absence of lag phase for control oil was due to the lack of physical protection and to the high contact surface with oxygen under constant agitation. And, the different behavior observed for enriched oil was due to the antioxidant effect of -tocopherol delaying the beginning of PUFAs oxidation for about 12 days. However, when control oil was encapsulated in powder (P0), the SA values were lower than those of the agitated bulk oil. Despite there was an increase of SA values in P0 when exposed to the accelerated oxidation conditions, it seems that the encapsulation by spray drying provided a partial protection against oxidation when compared to agitated bulk oil. This protective effect of the encapsulation matrix can not be evidenced for P1, probably due to a strong influence of the encapsulation process on the oxidation stability when tocopherol was added. And, in both cases, results indicate a lower oxidative stability of oil encapsulated in powder (P0 and P1) compared to the one of oil dispersed on maltodextrin particles despite a similar S/V ratio. This seems to confirm the negative effect of the spray drying process on the oil oxidative stability during dry emulsion storage.

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To further investigate the behavior of enriched oil regarding oxidation, the evolution of the three oxidation markers during 5 months of aging was followed for powder P1 (Fig. 7). It can be seen that the VOCs presented the same behavior as conjugated dienes: no lag phase but a fast increase during the first 20 days then followed by a decrease and stabilization. It has already been observed that the abundance of VOCs correlates well with the hydroperoxides content, primary oxidation marker as conjugated dienes, indicating that the production of hydroperoxides enhances the development of VOCs (Takeungwongtrakul et al., 2015). These curves also show a rapid consumption of -tocopherol since we observed that after 10 days of aging, the antioxidant was completely gone. This may explain why we observed no difference between P0 and P1 regarding the evolution of conjugated dienes content (SA value) despite in the case of agitated α-toc-enriched bulk oil, results showed that the addition of α-tocopherol actually reduced the SA values in comparison to control oil (no α-tocopherol), demonstrating its antioxidant activity (Fig 7.b). The fast increase in oxidation products in P1 can now be related to the rapid consumption of αtocopherol. Velasco et al. (2009) studied the antioxidant activity of phenolic antioxidants in freeze-dried sunflower oil particles when stored at 40ºC using Cu (II) as an oxidation catalyst. They observed that α-tocopherol was completely exhausted in encapsulated oil after 12 days of storage whereas it took 56 days in non-encapsulated oil. The early loss of antioxidant was attributed to its direct interaction with Cu (II) to give the tocopheroxyl radical and Cu (I), being more favored in the encapsulated oil. In our work, we cannot exclude such induction of -tocopherol degradation by metal ions coming from water or wall material or processing, associated with aggressive conditions of aging (60°C and 50% RH). We therefore compared the consumption of α-tocopherol in sunflower oil either in bulk under constant agitation, or dispersed on maltodextrin particles, or encapsulated in dry emulsion P1 (Fig.8). Our results agree with those of Velasco et al. (2009): the consumption of α-tocopherol in agitated bulk oil was less aggressive than in P1 or when dispersed on MD particles. That can be also reflected in the values of SA correspondingly lower when the -tocopherol content was higher. These results could be also indicative of a possible prooxidant effect of α-tocopherol when in contact with MD. This would also explain why, in our study, it was not possible to evidence a difference in oxidation rate between surface and encapsulated oil as it has been observed in

ACCEPTED MANUSCRIPT other studies (Ahn et al., 2012; Velasco et al., 2003). Complementary experimental work is therefore necessary to evaluate the hypothesis concerning the role of metallic ions.

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To end, despite the protection brought by the encapsulation of -toc enriched oil in powder is still questionable regarding oxidative stability, we have checked that the quantity of extracted oil in powders did not change over aging time, which means the absence of polymerization in the encapsulated oil in contrary to what was observed for bulk oil (data not shown).

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Conclusions

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In the present paper we studied the effect of the addition of lipophilic antioxidant (tocopherol) on the oxidative stability of oil in oil-in-water emulsions both during spray drying and upon storage of the spray dried emulsions (60°C and 50% RH). The spray drying process showed to have an impact on two oxidation markers (conjugated dienes and VOCs) but not on the antioxidant concentration. It therefore results in the initiation of oil oxidation that can not be avoided by the presence of antioxidant (-tocopherol) in the oil phase. The spray drying conditions must therefore be chosen in order to lower thermal and mechanical constraints. Anyway, the encapsulation process provided a partial protection against oxidation in control oil (P0) but the oxidative stability was higher when oil was dispersed on MD particles, confirming that the beginning of oxidation arising during spray drying is detrimental to the dry emulsion oxidative stability. When powders (with and without -tocopherol) were subjected to accelerated thermooxidation (60 ºC and 50 % RH), both conjugated dienes and VOCs content rapidly increased with no lag phase. After about ten days, it was followed by a decrease and then stabilization after about fifty days of storage. In powder P1, containing antioxidant, the content of αtocopherol decreased rapidly and tended to zero within a short time, reducing the effect of protection of oil upon aging. We demonstrated that the rapid oxidation of encapsulated oil (thus the absence of lag phase) was mainly due to the oxygen permeability of the encapsulation matrix, especially when particles are hollow since the inner vacuole behaves as an oxygen reservoir. The control of the dry particle structure is therefore important for a good protection. In the case of oil containing antioxidant we highlighted a probable inhibitory action of maltodextrin on α-tocopherol that must be confirmed.

Acknowledgment

The authors would like to thank the Mexican National Council of Science and Technology (Consejo Nacional de Ciencia y Tecnologia, CONACYT), for financial resources provided to Maria del Rayo Hernandez Sanchez. Thanks also to Giana Almeida and Nathalie Ruscassier at Laboratoire de Génie des Procédés et Matériaux, at Ecole Centrale Paris for performing the SEM observations. The authors thank particularly Axelle Collinet, Katherine Quispe Puelles and Serife Kaymaz for their efficient technical help in this study.

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Fig. 2. Scanning electron microscope micrographs of spray dryed emulsions. Top row : General view of the powder particles. Middle row: Particle surface topography. Bottom row: Inner structure of particles (a) P0 (control) (b) P1 (α-toc).

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Fig. 3. Chromatographic profiles of some of volatile organic compounds detected in headspace of a) initial oil (with 482 α-toc) and b) powder P1. (Peak 1: Hexanal; Peak 2: Nonanal; Peak 3: Octanoic acid).

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Fig 4. Evolution of conjugated dienes content in the oil phase of spray dried emulsions P0 (control) and P1 (482 ppm α-toc in oil) from the evolution of the specific absorbance at 234 nm measured upon aging at 60 ºC and 50% RH.

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Fig. 5. Evolution of conjugated dienes content (expressed as SA value) in control powder (P0) and in control powder P0 after non-encapsulated surface oil removal by solvent washing (hexane).

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Fig. 6. Evolution of conjugated dienes in a) control oil and b) α-toc-enriched oil in dry emulsion (P0 or P1), dispersed on maltodextrin particles (MD) and without any treatment (agitated bulk oil) upon aging at 60°C and 50% RH.

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Fig.7. Oxidative stability of oil in dry emulsion P1 (482 ppm α-toc in oil) expressed as percentage of residual α-tocopherol, specific absorption at 234 nm (SA) and total volatiles organic compounds (VOCs) upon aging at 60 ºC and 50% RH.

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Fig.8. Evolution of α-tocopherol, and conjugated dienes (SA value) contents in agitated bulk oil, encapsulated oil in P1 and dispersed oil on MD particles during 15 days aging.

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60 40

SC R

Specific Absorbance at 234 nm

120

20 0 2

4

6

8

10

NU

0

12

14

Aging time (days) at 60 °C and 50 %HR

b) α-toc/ oil

D

80

TE

60 40 20 0

2

AC

0

CE P

Specific Absorbance at 234 nm

100

Figure 6

Agitated bulk α-toc /oil α-toc /oil in P1 α-toc /oil on MD

MA

120

4

6

8

10

Aging time (days) at 60 °C and 50 %HR

12

14

ACCEPTED MANUSCRIPT % residual α-toc SA at 234 nm VOCs (Peak area x 107)

160 140 120

T

100

IP

80

SC R

60 40 20 0

25

50

75

100

NU

0

MA

Aging time (days) at 60 °C and 50 %HR

AC

CE P

TE

D

Figure 7

125

150

ACCEPTED MANUSCRIPT α-toc – agitated bulk oil

90

80

60

α-toc – P1

60

α-toc – MD

IP

50 40 30 20 10

2

4

6

8

NU

0 0

Aging time (days) at 60 °C and 50 %HR

CE P

TE

D

MA

Figure 8

AC

50

SA – Agitated AS – agitated bulk bulkoiloil

T

70

SC R

% α- toc residual

70

SA AS –– P1 P1

80

10

SA AS –– MD MD

40 30 20 10 0

12

14

Specific Absorbance at 234 nm

100

ACCEPTED MANUSCRIPT Table 1. Specific absorbance (SA) and α-tocopherol content (ppm) measured in oil phase extracted from liquid emulsions before spray drying and from dry emulsions after spray drying for systems P0 (control without -toc) and P1 (enriched with 482 ppm α-toc).

Liquid emulsion

Dry emulsion

before spray after spray drying drying

8.2 ± 1,8

10.5 ± 2.1

13.9 ± 2,2

MA D TE

Dry emulsion

before spray drying

after spray drying

2,1

-

-

3,4

444 ± 9

433 ± 30

ΔSA

NU

6,1± 0,5

CE P

P1

0 482 ppm in oil

AC

P0

α-toc content in extracted oil (ppm) Liquid emulsion

IP

α-toc

SC R

System

T

Specific Absorbance (SA) in extracted oil

ACCEPTED MANUSCRIPT Highlights - Initiation of oil oxidation but no degradation of -tocopherol observed during spray drying.

T

- Addition of -tocopherol does not avoid PUFAs oxidation during spray drying and storage.

IP

- Encapsulation of PUFAs in maltodextrin particles provides partial protection during aging.

AC

CE P

TE

D

MA

NU

SC R

- Negative effect of oxygen permeability and hollow structure of particles on oil protection.