Accepted Manuscript Evaluation of the oxidative stability of chia oil-loaded microparticles by thermal, spectroscopic and chemometric methods Alexandre Guimarães-Inácio, Cristhian Rafael Lopes Francisco, Valquíria Maeda Rojas, Roberta Souza Leone, Patrícia Valderrama, Evandro Bona, Fernanda Vitória Leimann, Ailey Aparecia Coelho Tanamati, Odinei Hess Gonçalves PII: S0023-6438(17)30706-5 DOI: 10.1016/j.lwt.2017.09.031 Reference: YFSTL 6546 To appear in: LWT - Food Science and Technology Received Date: 11 April 2017 Revised Date: 15 September 2017 Accepted Date: 18 September 2017 Please cite this article as: Guimarães-Inácio, A., Francisco, C.R.L., Rojas, Valquí.Maeda., Leone, R.S., Valderrama, Patrí., Bona, E., Leimann, Fernanda.Vitó., Tanamati, A.A.C., Gonçalves, O.H., Evaluation of the oxidative stability of chia oil-loaded microparticles by thermal, spectroscopic and chemometric methods, LWT - Food Science and Technology (2017), doi: 10.1016/j.lwt.2017.09.031. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. 1 ACCEPTED MANUSCRIPT 1 EVALUATION OF THE OXIDATIVE STABILITY OF CHIA OIL-LOADED 2 MICROPARTICLES BY THERMAL, SPECTROSCOPIC AND CHEMOMETRIC METHODS GUIMARÃES-INÁCIO, ALEXANDREa; FRANCISCO, CRISTHIAN RAFAEL LOPESb; ROJAS, 4 VALQUÍRIA MAEDAa; LEONE, ROBERTA SOUZAb; VALDERRAMA, PATRÍCIAa; BONA, 5 EVANDROa; LEIMANN, FERNANDA VITÓRIAa; TANAMATI, AILEY APARECIA COELHOb; 6 GONÇALVES, ODINEI HESSa* RI PT 3 SC 7 8 a 9 Via Rosalina Maria dos Santos, 1233, CEP 87301-899, POBox 271, Campo Mourão - PR – 10 Brasil. Phone/Fax: +55 (44) 3518-1400 M AN U Federal University of Technology - Paraná, Post-graduation Program of Food Technology 11 b Federal University of Technology - Paraná, Food Department TE D 12 13 Via Rosalina Maria dos Santos, 1233, CEP 87301-899, POBox 271, Campo Mourão - PR – 14 Brasil. Phone/Fax: +55 (44) 3518-1400 EP 16 *corresponding author: firstname.lastname@example.org AC C 15 2 ACCEPTED MANUSCRIPT ABSTRACT 17 18 Chia oil has been widely studied due to its benefits to human health, being source of 20 important omega-3 and omega-6. Encapsulation has been studied in order to protect the oil 21 from thermal degradation. In this work, oil from chia seeds was microencapsulated in 22 carnauba wax and thermal stability was evaluated not only by thermal methods but also 23 Spectroscopy and chemometric based analyses. High encapsulation efficiency of both 24 omega-3 and -6 (up to 97%) was verified. Particles presented micrometric sizes, spherical 25 shape and no fissures. Differential Scanning Calorimetry was applied in isothermal and non- 26 isothermal modes and kinetic parameters were determined using the Ozawa-Flynn-Wall 27 (OFW) method. An effective protection of the chia oil was verified by an increase in the 28 oxidative stability in both isothermal (activation energy increased from 73.5 ± 1.5 to 91.8 ± 29 1.6 kJ.mol-1) and non-isothermal (from 87.4 ± 2.1 to 97.3 ± 2.9 kJ.mol-1) tests. Increase in the 30 onset oxidation temperature and in the oxidation induction time were also detected. Chia oil 31 and oil-loaded microparticles were subjected to accelerated stability test. Degradation was 32 evaluated by the UV-Vis spectroscopy coupled to chemometry and also by the extinction 33 coefficients, corroborating the increase in the oxidative stability. 36 SC M AN U TE D EP 35 Keywords: DSC; kinetic parameters; OFW methodology; MCR-ALS; chemometry. AC C 34 RI PT 19 3 ACCEPTED MANUSCRIPT 37 38 1. Introduction Chia seed oil is a rich source of polyunsaturated fatty acids (PUFAs), containing a 40 high proportion of α-linolenic acid (approximately 60%) when compared to other known 41 vegetable sources (Sandoval-Oliveros and Paredes-López, 2013). This fatty acid is part of 42 the omega 3 group and its consumption is fundamental to growth and development of human 43 body (Simopoulos, 2002). RI PT 39 Despite showing a favorable nutritional profile for heath, the high percentage of 45 PUFAs in chia oil results in low oxidative stability (Ixtaina, Nolasco, & Tomás, 2012; Santos, 46 Santos, Souza, Prasad, & Santos, 2002). Therefore, protection against lipid oxidation is a 47 crucial factor when aiming for chia oil quality. Oil microencapsulation is an alternative that 48 has been studied to protect vegetable oils presenting high amounts of unsaturated fatty 49 acids. Encapsulation of PUFAs-rich oils is reported in the literatures (González, Martínez, 50 Paredes, León, & Ribotta, 2016; Ixtaina, Julio, Wagner, Nolasco, & Tomás, 2015; Timilsena, 51 Adhikari, Barrow, & Adhikari, 2016) demonstrating that the encapsulation efficiency is highly 52 dependent on the encapsulant as well as the encapsulation technique used. The production 53 of solid lipid particles has been reviewed in the literature demonstrating the advantages of 54 such kind of system mainly due to the compatibility of the lipid encapsulant and the oil 55 (Müller, Radtke, & Wissing, 2002; Tamjidi, Shahedi, Varshosaz, & Nasirpour, 2013). Oil- 56 loaded microparticles may be readily obtained by the hot homogenization technique in which 57 the oily phase is melted and dispersed in an aqueous medium. Solid lipid powders are 58 obtained after total water evaporation (Milanovic, Levic, Manojlovic, Nedovic, & Bugarski, 59 2011). In this context, carnauba wax is a promising encapsulant material since it is accepted 60 in food formulations and is classified as Generally Accepted as Safe (GRAS). Moreover, it 61 presents high melting temperature meaning that it may not melt during most food processing 62 conditions. AC C EP TE D M AN U SC 44 63 Since oxidation is an exothermic reaction, thermal analyses have been used to follow 64 the reaction course through continuous monitoring the thermal effects of lipid oxidation. 4 ACCEPTED MANUSCRIPT Vecchio Ciprioti and coworkers (2017) reported that Differential Scanning Calorimetry (DSC) 66 has been used to evaluated thermal oxidation of oils for over 60 years and DSC experiments 67 may be carried out in dynamic (linear increase in temperature) or in isothermal (constant 68 temperature) conditions (Ostrowska-Ligeza et al., 2010). Moreover, the correlation between 69 classical testes like Rancimat and DSC was demonstrated showing that DSC is reliable, fast 70 and less expensive technique to evaluate oxidative stability of vegetable oils (Noello, 71 Carvalho, Silva, & Hubinger, 2016; Pardauil et al., 2011; Tan, Man, Selamat, & Yusoff, 2002; 72 Vecchio Ciprioti et al., 2017). A wide array of edible oils were evaluated by DSC such as 73 olive oil (Qi et al., 2016), blackberry and raspberry oil (Micić et al., 2015), toogga (Balanites 74 aegyptiaca) oil (Gardette & Baba, 2013), buriti, rubber tree and passion fruit oil (Pardauil et 75 al., 2011). M AN U SC RI PT 65 Integral isoconversional methods are well stablished methodologies to perform kinetic 77 analysis of thermal data which may be obtained isothermally or non-isothermally (Vyazovkin 78 et al., 2011). It is worth noting that the kinetic parameters found must be associated to 79 processes and not to compounds. As described by Ostrowska-Ligeza and coworkers (2010), 80 the correct use of the OFW method in DSC experiments relies on the comparison of systems 81 at the same degree of conversion (α), defined by Equation 1, where ∆Hτ is the heat evolved 82 at an specific time τ and ∆Htotal is the heat evolved during the process. It may be assumed 83 that the degree of conversion at the beginning of the oxidation process is low but constant. AC C EP TE D 76 = 84 85 ΔH ΔH (1) The approach proposed by Ozawa, Flynn and Wall (OFW) (Flynn & Wall, 1966; 86 Ozawa, 1970; Vyazovkin et al., 2011) is one of the most used when evaluating the stability of 87 edible oils (Grompone, Irigaray, Bruno. Rodríguez, & Sammán, 2013; Litwinienko & 88 Kasprzycka-Guttman, 1998; Micić et al., 2015; Musialik & Litwinienko, 2007; Ostrowska- 89 Ligeza et al., 2010; Qi et al., 2016). Integral isoconversional methods are powerful tools to 90 determine the relationship between the activation energy and the degree of conversion 5 ACCEPTED MANUSCRIPT throughout the reaction. However, in the case of the stability of edible oils, researchers agree 92 that the evaluation of a single activation energy at the start of the oil degradation is sufficient 93 to compare different samples that were subjected to the same experimental conditions (same 94 heating rates, oxidizing atmosphere and so on). This may be also valid when comparing pure 95 and encapsulated oils in order to determine the actual gains in stability caused by 96 encapsulation. RI PT 91 Besides thermal analyses, spectroscopy techniques have been used to evaluate the 98 degradation of edible oils such as the use of UV-Vis (Gonçalves, Março, & Valderrama, 99 2014; Poyato, Ansorena, Navarro-Blasco, & Astiasarán, 2014; Valderrama, Março, Locquet, 100 Ammari, & Rutledge, 2011; W. Zhang et al., 2015) and FTIR (Mahboubifar, Hemmateenejad, 101 Javidnia, & Yousefinejad, 2017) spectroscopy coupled to chemometric methods. The 102 degradation process leads to the formation of oxidized compounds with intense absorption at 103 232 and 270 nm (conjugated dienes and trienes) which enables efficient monitoring (AOCS, 104 2009). M AN U SC 97 DSC technique is well established when it comes to vegetable oils and oxidative 106 stability evaluation. However, few studies use this technique to determine the actual gains in 107 stability caused by encapsulation. The microparticles stability during storage also needs to 108 be evaluated by conventional or chemometric methods. The objective of this work was to 109 evaluate the oxidative stability of chia oil encapsulated in carnauba wax microparticles. For 110 the stability tests, Differential Scanning Calorimetry analyses in isothermal and non- 111 isothermal modes were carried out, comparing encapsulated and in natura (before 112 encapsulation) oil. The accelerated stability test of the microparticles were also carried out 113 and the samples were subjected to classical extinction coefficient determination and also to 114 chemometric procedures. AC C EP TE D 105 115 116 117 118 2. Materials e methods 6 ACCEPTED MANUSCRIPT 2.1 Materials 120 Chia oil (Veris do Brasil Ltda) was stored at -10°C and protected from light until use. 121 Distilled water was use as continuous medium in the microparticles production. Carnauba 122 wax (Sigma-Aldrich, analytical grade) and sodium caseinate (Sigma-Aldrich, analytical 123 grade) were used as wall material and stabilizer, respectively. Neutral ether-alcohol solution 124 (2:1 v/v); phenolphthalein (Dinâmica, analytical grade) solution (1%); sodium hydroxide 125 (Dinâmica, analytical grade) solution 0.01 mol.L-1; methyl tricosanoate (23:0 Me, Sigma- 126 Aldrich, 127 esterifying reagent, methanolic sodium hydroxide solution 0.5 mol.L-1 and saturated sodium 128 chlorate solution were used in the characterization analyzes. standard); 130 131 chromatographic grade); 133 2.2 Methods TE D 132 135 (Dinâmica, M AN U 129 134 isooctane SC chromatographic RI PT 119 2.2.1 Microparticles production 137 The aqueous phase was prepared dissolving sodium caseinate (0.0550 g) in water 138 (50 g) and heating up to 368.15 K under gentle stirring. Separately, a jacketed borosilicate 139 flask was connected to a thermostatic bath at 368.15 K and, then, carnauba wax (3.350 g) 140 was added to be melted. After, chia oil (1.650 g) was added to the flask under gentle 141 agitation for 1 minute. Then, the aqueous phase was added under stirring (16,000 rpm, 5 142 minutes) using a high efficiency disperser (Ultraturrax IKA, T25). At the end of this step, the 143 obtained dispersion was poured in a recipient in ice bath for quick quenching and 144 microparticles solidification. Lastly, the dispersed microparticles were freeze-dried without AC C EP 136 7 ACCEPTED MANUSCRIPT 145 separation and stored at 263.15 K protected from light. The same proceeding also was 146 carried out to microparticles without chia oil (blank microparticles). 147 2.2.2 Chia oil and microparticles characterization 149 Physicochemical parameters (acidity, peroxide and humidity index) of the chia oil 150 were determined according to the methodology described by the Adolfo Lutz Institute in 151 triplicate (Instituto Adolfo Lutz, 2008). SC RI PT 148 Fatty acids quantification was performed by Gas Chromatography (GC) using methyl 153 tricosanoate (23:0, Sigma-Aldrich) as internal standard according to Hartman and Lago 154 methodology (Milinsk, Matsushita, Visentainer, Oliveira, & Souza, 2008). Fatty acid methyl esters 155 (FAMEs) were separated, identified and quantified using chromatograph standards (Sigma- 156 Aldrich, F.A.M.E. Mix C14-C22) (Joseph & Ackman, 1992). Transesterifications were performed 157 in triplicate. TE D M AN U 152 Encapsulation efficiency was measured for omega 3 and omega 6 by GC. An aliquot 159 of the dispersion containing the microparticles were washed with absolute ethanol and 160 filtered (3 µm average porosity) to remove the oil on the particles surface (non-encapsulated 161 fraction). Then, the washed microparticles were dissolved in cyclohexane at 323.15 K then 162 quenched (283.15 K) to precipitate the carnauba wax. Sample was then centrifugated 163 (14,500 rpm, 15 min) and the supernatant was carefully separated, esterified and analyzed 164 by GC. This procedure was also applied to the microparticles without being washed to 165 determine the total amount of fatty acids present in the sample. Encapsulation efficiency 166 (EE%) was determined according to Equation 2 considering the total concentration of fatty 167 acids present in the microparticles ([FA]total) and the concentration of each fatty acid in the 168 washed microparticles ([FA]encapsulated). AC C EP 158 8 ACCEPTED MANUSCRIPT % = 100 [ ] [ (2) ] 169 For the determination of peak melting temperature of the microparticles, Differential 171 Scanning Calorimetry (DSC, Perkin Elmer 4000) was performed at 20 K.min-1 (Hayati, 172 Aminah, & Mamot, 2000) from 273.15 K to 713.15 K under nitrogen atmosphere (50 mL.min- 173 1 174 (Perkin Elmer). RI PT 170 SC ) in closed aluminum sample holders. DSC data were evaluated using software Pyris 11 Size and morphology of the microparticles were analyzed by Scanning Electron 176 Microscopy (Carl Zeiss - EVO MA15) operating at 15 kV with a secondary electron detector. 177 Particles diameter were measured with an image analyzer (SizeMeter software). Medium 178 infrared spectra of chia oil and the microparticles were obtained in a spectrophotometer 179 equipped with the Attenuated Total Reflectance accessory (FTIR-ATR, PerkinElmer Frontier 180 Spectrum 100). Oil samples were directly analyzed while microparticles were dissolved in 181 cyclohexane moments before reading to expose the encapsulated oil. X-ray diffraction trial 182 (DRX, Bruker model D8 Advance) were performed with Cu-Kα radiation, generated at 40 KV 183 to 35 mA, varying from 3° to 60° (2θ) at 5.9°.min-1. EP TE D M AN U 175 Thermogravimetric analyses were performed with chia oil and with the oil-loaded 185 microparticles. Samples (approximately 4 mg) were conditioned in aluminum sample holders 186 in a thermobalance (Shimadzu, TGA-50) and submitted to heating from 303.15 K to 923.15 K 187 at 10 K.min-1 using synthetic air atmosphere (mixture 79% N2, 21% O2, 150 mL.min-1). AC C 184 188 2.2.3 Thermal stability evaluation 189 Samples (5 - 10 mg) of chia oil or microparticles were submitted to Differential 190 Scanning Calorimetry (DSC, Perkin Elmer model 4000) in isothermal and non-isothermal 191 modes. Heating rates of non-isothermal experiments were 1, 5, 10 and 20 K.min-1 in a 192 temperature range of 273.15 K to 713.15 K. Isothermal tests were performed at 383.15 K, 9 ACCEPTED MANUSCRIPT 393.15 K, 403.15 K and 413.15 K during 220 minutes. In both tests, open aluminum sample 194 holders and synthetic air (mixture 79% N2, 21% O2) at 100 mL.min-1 were used. Calorimeter 195 calibration was performed for each analysis condition with zinc and indium standards (429.75 196 K and 692.68 K, respectively). RI PT 193 For the non-isothermal tests, calculation of the activation energy (Ea) involved in the 198 lipid oxidation reaction was carried out with the data obtained during the non-isothermal tests 199 by the method proposed by Ozawa, Flynn and Wall (OFW) (Flynn & Wall, 1966; Ozawa, 200 1970; Qi et al., 2016). The onset oxidation temperature was found by extrapolating the 201 baseline and the tangent line (leading edge) of each thermogram (Ostrowska-Ligeza et al., 202 2010). The onset temperature was used because the onset of the reaction occurs at about 203 the same extent of conversion which is in turn close to zero (Micić et al., 2015). Activation 204 energy was calculated using a set of onset oxidation temperature obtained at different 205 heating rates (β) using Equation 3 and 4. M AN U SC 197 207 208 209 !" (3) (4) # $% AC C = −2,19, EP !" = # $% + ' TE D 206 where β is the heating rate (K.min-1), T is the onset oxidation temperature (Ton in K), 210 “a” and “b” are the slope and intercept of Equation 3, respectively and R is the universal gas 211 constant (8.314 J.mol-1K-1). 212 For the isothermal DSC condition, the isoconversional method was used to determine 213 the activation energy using Equation 5 (Grompone et al., 2013; Micić et al., 2015). The 214 oxidation induction time (Ot) was found in the DSC curves as the intersection of the 10 ACCEPTED MANUSCRIPT 215 extrapolated baseline and the tangent line (leading edge) of the exothermic peak. The 216 activation energy was then calculated as the slope of the curve found by the least squares 217 regression of the ln(1/Ot) and T-1. 1 = ln / − ,# RI PT ln 218 (5) For both isothermal and non-isothermal modes, oxidation induction time and onset 220 oxidation temperature, respectively, were measured in duplicate and the averages for each 221 condition were used in the calculations. In all experiments, the maximum deviation found for 222 the parameters were less than 1.1% in the non-isothermal mode and 2.3% in the isothermal 223 mode, which is in accordance with the literature (Ostrowska-Ligeza et al., 2010). Linear 224 regression analyses for fitting of kinetics data (p-values and lack of fit) were carried out using 225 STATISTICA 7.0 (StatSoft Inc., Tulsa, OK, USA). M AN U SC 219 227 TE D 226 2.2.4 Accelerated stability test 229 Microparticles samples and the chia oil were conditioned in an open recipient and 230 evaluated by the Schaal oven test (González et al., 2016; Poyato et al., 2013). Ultraviolet- 231 visible spectra were acquired from 200 to 550 nm at 353.15 K and analyzed by Multivariate 232 Curve Resolution Alternating Least-Squares (MCR-ALS) as described by Gonçalves et al. 233 (2014), using software Matlab R2007b. Spectra were assigned to their respective 234 compounds according to Valderrama et al. (2011) in respect to degradation products and 235 also to tocopherols. Similarly, UV-Vis spectra were acquired at 353.15 K and the specific 236 extinction coefficients were calculated at 232 and 270 nm (American Oil Chemists Society, 237 2009; Instituto Adolfo Lutz, 2008). In both experiments, aliquots were removed at time AC C EP 228 11 ACCEPTED MANUSCRIPT 238 intervals defined in preliminary experiments, and dissolved in cyclohexane moments before 239 analysis in triplicate. 241 3. Results and Discussion 242 243 3.1 Chia oil and microparticles characterization 246 Table 1 presents the physicochemical parameters determined for the chia oil before encapsulation (in natura oil). M AN U 245 247 (Table 1) 248 TE D 249 250 SC 244 RI PT 240 Ixtaina et al. (2011) found acidity index from 0.07 to 0.20 goleic acid.100goil -1 when studying the properties of chia oil. In similar work, Segura-Campos et al. (2014) also found 252 small variations when comparing their results with other studies and suggested that this 253 difference may be influenced by cultivation or the oil extraction method. However, in both 254 works and also in the present study values found are within the established standards by 255 Codex Alimentarius (Codex Alimentarius Commission, 1999). 257 258 259 260 AC C 256 EP 251 In Table 2, composition of chia oil and chia oil loaded-microparticles is presented. Figure 1 shows the obtained chromatograms. (Table 2) (Figure 1) Omega 3 concentration found was 63.8% which is consistent with the data available in 12 ACCEPTED MANUSCRIPT the literature (González et al., 2016; Julio et al., 2015). The encapsulation efficiency found for 262 omega-3 and omega-6 were 96.4 ± 0.9 and 96.6 ± 1.0 %, respectively. Both results were 263 considered efficient and demonstrate the affinity between the chia seeds oil and the 264 encapsulating matrix chosen. RI PT 261 Proportions between polyunsaturated and saturated fatty acid (PUFA:SFA, Table 2) higher 266 than 0.45 and between omega 3 and 6 (n-6: n-3) from 1:1 to 2:1 are recommended for daily 267 human ingestion (Wood et al., 2004) meaning the oil have high nutritional quality. This is an 268 important data, since an imbalance in n-6: n-3 proportion, as in the modern western food 269 ingestion, are related with chronic diseases and metabolic disorders (Simopoulos, 2008). The 270 encapsulated oil presented characteristics similar to chia oil before encapsulation, meaning that 271 the microparticles may be used to protect the oil and to formulate products with the same high 272 nutritional values of chia oil since the wall material may be considered a food formulation 273 component. M AN U SC 265 Figure 2 (a) presents the image obtained by Scanning Electron Microscopy of the oil-loaded 275 microparticles, figure 2 (b) presents Infrared spectra and Figure 2 (c) the X-Ray diffractograms 276 also for the oil-loaded-microparticles and blank microparticles. EP 278 (Figure 2) Microparticles presented spherical morphology, continuous surface, without fractures AC C 277 TE D 274 279 and monomodal size distribution varying from 1.5 to 15 µm with average diameter of 3.71 ± 280 1.85 µm. This is important since porous and irregular structure may lead to oxygen diffusion 281 and oil migration which could accelerate the oxidation process. 282 DSC thermograms carried out under nitrogen flow (Figure 1S, Supplementary 283 Material) showed a decrease in the melting temperature of 4.2 K (from 357.37 K to 361.57 K) 284 and in the melting enthalpy of 47.68 J.g-1 (from 162.70 to 115.02 J.g-1) when comparing blank 285 and oil-loaded microparticles was observed. This may be related to the decrease in the 13 ACCEPTED MANUSCRIPT carnauba wax crystallinity caused by the partial solubilization of the wax in the oil which 287 suggests an effective encapsulation of the chia oil. Ribeiro et al. (2012) observed similar 288 behavior when producing lipid microparticles using different proportions of oleic acid and 289 stearic acid. Since oleic acid melts at 6 ºC, its presence decreased the melting temperature 290 and enthalpy of the stearic acid. RI PT 286 Infrared analyses of the oil showed bands corresponding to –CH, –CH2 and –CH3 292 groups from the fatty acids chains and other components present in the sample (Moros, 293 Roth, Garrigues, & Guardia, 2009). In Figure 2 (b) the region from 2800 to 3000 cm-1 294 presents few absorption bands that correspond to the C-H bonds such as asymmetric 295 stretching (CH2 at 2926 cm-1) and to symmetric stretching (CH2 at 2853 cm-1). The region 296 below 1600 cm-1 corresponds to a fingerprint region presenting a more complex profile of 297 absorption of various bands in 1450, 1256, 1040, 902 and 860 cm-1 presenting the 298 absorption bands of functional groups –C-H2, -C-H3, -C-O (ester), -HC=CH (cis) and =CH2 299 (Zhang et al., 2012). The characteristic bands of the chia oil were attenuated in the oil-loaded 300 microparticles indicating that the oil was located inside the microparticles. It is worth pointing 301 out that all spectra were normalized in order to allow comparison between samples. TE D M AN U SC 291 In Figure 2 (c) low intense peaks were found at 21.6° and 23.8° which correspond to 303 0.41 nm and 0.37 nm, respectively (Villalobos-Hernández & Müller-Goymann, 2006). They 304 are characteristic of β’, orthorhombic crystalline sub-cell arrangement of the carnauba wax 305 (Blake, Co, & Marangoni, 2014; Révérend, Fryer, Coles, & Bakalis, 2010). It was possible to 306 observe the appearance of an amorphous region from 15 to 20° in the oil-loaded 307 microparticles as well as a decrease in the peaks intensity. This may indicate that the 308 presence of the oil led to the disorganization of the crystalline structure of carnauba wax. AC C EP 302 309 310 311 3.2 Thermal analysis 14 ACCEPTED MANUSCRIPT 312 3.2.1 Non-isothermal Differential Scanning Calorimetry In Figure 3 the thermograms obtained in the non-isothermal DSC tests are presented 314 (the adjusted curves are presented in the Supplementary Material, Figure 2S). Table 3 315 shows the activation energy and the adjusted coefficient of determination of each adjusted 316 curves found in the non-isothermal and isothermal Differential Scanning Calorimetry 317 experiments. RI PT 313 SC 318 M AN U 319 (Figure 3) 320 (Table 3) 321 322 For both samples (chia oil and oil-loaded microparticles) linear regression presented 324 satisfactory adjusted coefficient of determination (Table 3). Also, all linear models were 325 statistically significant (p<0.05) while lack of fit was statistically not significant (p>0.05) 326 meaning that the linear models may satisfactorily describe the experimental data. EP TE D 323 It is worth pointing out that chia oil (non encapsulated) is liquid under the conditions 328 used in all experiments meaning that the variations detected in the DSC runs could not be 329 caused by other thermal events like glass transition. Moreover, PUFAs rich oils like chia oil is 330 prone to degrade under heating under oxidizing atmospheres. The activation energy values 331 for the onset of the oxidation reaction of chia oil and for oil-loaded microparticles were of 87.4 332 ± 2.1 kJ.mol-1 and 97.3 ± 2.9 kJ.mol-1, respectively. Grompone et al (2013) found similar 333 value (97 kJ.mol-1) when analyzing the oxidation of chia oil. As stated by Santos at al. (2002), 334 the first step of thermal decomposition of edible oils is the most important one when 335 determining stability, as decomposition of unsaturated fatty acid starts in this step. Thermal AC C 327 15 ACCEPTED MANUSCRIPT 336 decomposition of fatty acids is considered a multistep process which is highly dependent on 337 fatty acids composition. When comparing the chia oil and the oil-loaded microparticles it was possible to 339 observe an increase in both oxidation temperatures and in the activation energy (increase of 340 9.89 kJ.mol-1, corresponding to 11.3% increase). This denotes a greater resistance of the oil 341 to oxidize, probably because the encapsulant acted as a barrier to oxygen while it was still 342 solid delaying the onset of the oxidation reaction. The encapsulant could still protect the oil 343 even after melting due to a dilution effect resulting in an efficient protection of the chia oil by 344 the carnauba wax. Grompone et al. (2013) carried out non-isothermal DSC experiments and 345 found different induction temperatures for the chia oil of those described in Table 3. This 346 variation may be associated with different analysis conditions, such as the flow gas in the 347 DSC or differences in oil composition and harvest conditions. Litwinienko and Kasprzycka- 348 Guttman (1998) also mentioned the influence of the atmosphere applied during DSC 349 analyses. 350 353 354 355 Isothermal Differential Scanning Calorimetry EP 352 3.2.2 In Figure 4 the thermograms obtained in the isothermal DSC tests are presented (the adjusted curves are presented in the Supplementary Material, Figure 3S). AC C 351 TE D M AN U SC RI PT 338 (Figure 4) 356 One may observe in Table 4 an increase of 77 minutes in the oxidation induction time 357 for the analysis at 393.15 K (120ºC). The experimental run of the microparticles at 383.15 K 358 did not present signs of oxidation during the whole analysis time (220 min) confirming the 359 protection given by the encapsulant. The activation energy values found was 73.5 ± 1.5 and 360 91.8 ± 1.6 kJ.mol-1 for the onset of the oxidation reaction of chia oil and for the oil-loaded 16 ACCEPTED MANUSCRIPT microparticles, respectively. As in the case of the non-isothermal DSC mode this 362 demonstrates that encapsulation led to an increased stability of the chia oil. Differences 363 between results from isothermal and non-isothermal setups were expected because the 364 mechanisms involved in the oil oxidation are influenced by the heating time and temperature 365 (Adhvaryu, Erhan, Liu, & Perez, 2000; Grompone et al., 2013; Litwinienko & Kasprzycka- 366 Guttman, 1998). RI PT 361 369 370 3.2.3 Thermogravimetric analysis (TGA) Figure 5 presents the TGA thermogram and its derivative for chia oil and oil-loaded M AN U 368 SC 367 microparticles. (Figure 5) 371 The onset oxidation temperature for the oxidation of the chia oil and the oil-loaded 373 microparticles were 426.15 K and 497.55 K, respectively. Oil oxidation is a complex chain 374 reaction involving a multi-step mechanism (Ostrowska-Ligeza et al., 2010). As pointed out by 375 Qi and coworkers (Qi et al., 2016), oxidation follows a first order reaction when excess of 376 oxygen is used. The first stage of thermal decomposition of vegetable oils is the most 377 important in the thermal stability study because is in this step that the unsaturated fatty acid 378 decomposition begins (Santos et al., 2002). Mass loss was detected in all samples while the 379 temperature in which degradation took place was lower for the oil before encapsulation than 380 for the microparticles, demonstrating the gain in stability due to the encapsulation. It is worth 381 reminding that the application of the isoconversional method is not immediately invalidated 382 due to the occurrence of multi-step processes (Vyazovkin et al., 2011). AC C EP TE D 372 383 384 3.3 Accelerated oxidative stability tests 17 ACCEPTED MANUSCRIPT Samples of oil and microparticles containing chia oil were evaluated in oven 386 accelerated stability tests (Schaal oven test). Results of Multivariate Curve Resolution with 387 Alternating Least Squares (MCR-ALS) are presented in Figures 6 (UV-Vis spectra recovered 388 by the MCR-ALS for the chia oil and for the oil-loaded microparticles are presented in the 389 Supplementary Material, Figure 4S (a) and Figure 4S (b), respectively). RI PT 385 (Figure 6) 391 It is worth noting that the test temperature (353.25 K) was lower than the melting 392 temperatures of the blank and oil-loaded microparticles (361.55 and 357.35 K) meaning that 393 they remained solid during all the duration of the experiments. M AN U SC 390 The oxidation of polyunsaturated fatty acids is followed by an increase in absorption 395 in ultraviolet region (Vieira and Regitano-d’Arce, 1999) with conjugated dienes and trienes 396 presenting intense absorption at the 234 nm and 270 nm, respectively. For chia oil, this 397 absorption profile is consistent with the spectral profile recovered by MCR-ALS. The relative 398 concentration profiles showed that oxidation started at the beginning of the oven test for the 399 oil but started after 54 hours for the encapsulated oil demonstrating the protection against 400 degradation due to the encapsulation. The dashed lines in the spectral profiles may be 401 attributed to tocopherol (Gonçalves et al., 2014) which relative concentration decreased from 402 the beginning of the exposure at 353.15 K for the oil but started after 48 hours of for the 403 encapsulated oil. The encapsulant played in important role protecting the oil from the oxidant 404 atmosphere thus preventing degradative oxidation. Migration of the oil from the interior of the 405 capsules to the surface after long exposure times may be responsible for the degradation at 406 the later stages of the experiment. 407 408 409 AC C EP TE D 394 Oil stability was also evaluated by the specific extinction coefficients (K) as showed in Figure 6. (Figure 7) 18 ACCEPTED MANUSCRIPT It is worth noting that monitoring the oil sample after 48 hours was not possible due to 411 intense deterioration of the oil which became insoluble in the cyclohexane. Blank 412 microparticles were also evaluated, presenting an extinction coefficient of 15.5 at 232 nm 413 and 13.2 at 270 nm, which was used in the calculation of the coefficient value of the 414 oil-loaded microparticles. This value was constant over the storage time which may be 415 attributed to the encapsulating material as already demonstrated by Freitas and colaborators 416 (2016) and Villalobos-Hernández and Müller-Goymann (2006). Variations in the extinction 417 coefficients were observed after 36 storage hours for oil while the oil-loaded microparticles 418 started to degrade after 54 hours. 420 M AN U 419 SC RI PT 410 4. Conclusions Microencapsulation of chia oil using carnauba wax as wall material showed to be 422 effective since encapsulation efficiencies around 97% were found for important fatty acids 423 such as omega 3 and omega 6. TE D 421 Encapsulation led to gains in thermal stability which was verified by Differential 425 Scanning Calorimetry, both in isothermal and non-isothermal modes, by the increase in the 426 activation energy of the degradation reaction due to the encapsulation. During the 427 accelerated oven stability test the microencapsulated oil presented greater stability as 428 detected by the extinction coefficient, disfavoring the appearance of degradation products. 429 Multivariate analyses also showed the effectiveness of encapsulation in protecting 430 tocopherols present in the oil. AC C 431 EP 424 The effective protection of the oil by the encapsulation process was demonstrated by 432 thermal, spectroscopic and advanced mathematical methods since all analyses agreed that 433 the encapsulated oil presented improved oxidative stability. 434 435 5. Acknowledgments 19 ACCEPTED MANUSCRIPT 436 Authors thank CNPq, CAPES and Fundação Araucária for the support. 437 439 6. Bibliography Adhvaryu, A., Erhan, S. Z., Liu, Z. S., & Perez, J. M. (2000). Oxidation kinetic studies of oils RI PT 438 derived from unmodified and genetically modified vegetables using pressurized 441 differential scanning calorimetry and nuclear magnetic resonance spectroscopy. 442 Thermochimica Acta, 364(1–2), 87–97. https://doi.org/10.1016/S0040-6031(00)00626-2 443 American Oil Chemists Society. (2009). Official Methods and Recommended Practices of the 445 AOCS (6th ed.). Chicago. Blake, A. I., Co, E. D., & Marangoni, A. G. (2014). Structure and physical properties of plant M AN U 444 SC 440 446 wax crystal networks and their relationship to oil binding capacity. Journal of the 447 American Oil Chemists’ Society, 91(6), 885–903. https://doi.org/10.1007/s11746-014- 448 2435-0 450 Codex Standard for Edible Fats and Oils Not Covered By Individual Standards. (1999). TE D 449 Codex Alimentarius Commission. https://doi.org/10.1017/CBO9781107415324.004 Flynn, J. H., & Wall, L. A. (1966). A quick, direct method for the determination of activation 452 energy from thermogravimetric data. Journal of Polymer Science Part B: Polymer 453 Letters, 4(5), 323–328. https://doi.org/10.1002/pol.1966.110040504 455 456 457 458 Freitas, C. A. S., Vieira, Í. G. P., Sousa, P. H. M., Muniz, C. R., Gonzaga, M. L. D. C., & AC C 454 EP 451 Guedes, M. I. F. (2016). Carnauba wax p-methoxycinnamic diesters: Characterisation, antioxidant activity and simulated gastrointestinal digestion followed by in vitro bioaccessibility. Food Chemistry, 196, 1293–1300. https://doi.org/10.1016/j.foodchem.2015.10.101 459 Gardette, J. L., & Baba, M. (2013). FTIR and DSC studies of the thermal and photochemical 460 stability of Balanites aegyptiaca oil (Toogga oil). Chemistry and Physics of Lipids, 170– 461 171, 1–7. https://doi.org/10.1016/j.chemphyslip.2013.02.008 462 Gonçalves, R. P., Março, P. H., & Valderrama, P. (2014). Thermal edible oil evaluation by 20 ACCEPTED MANUSCRIPT 463 UV-Vis spectroscopy and chemometrics. Food Chemistry, 163, 83–6. 464 https://doi.org/10.1016/j.foodchem.2014.04.109 González, A., Martínez, M. L., Paredes, A. J., León, A. E., & Ribotta, P. D. (2016). Study of 466 the preparation process and variation of wall components in chia (Salvia hispanica L.) 467 oil microencapsulation. Powder Technology, 301, 868–875. 468 https://doi.org/10.1016/j.powtec.2016.07.026 469 RI PT 465 Grompone, M. A., Irigaray, Bruno. Rodríguez, D., & Sammán, N. (2013). Assessing the Oxidative Stability of Commercial Chia Oil. Journal of Food Science and Engineering, 3, 471 349–356. SC 470 Hayati, I. N., Aminah, A., & Mamot, S. (2000). Melting Characteristic and Solid Fat Content of 473 Milk and Palm Stearinm Blends before and after Enzymatic Interesterification. Journal of 474 Food Lipids, 7, 175–193. 476 477 Instituto Adolfo Lutz. (2008). Métodos Físico-Químicos para Análise de Alimentos. Métodos Físico-Químicos para Análise de Alimentos. Ixtaina, V. Y., Julio, L. M., Wagner, J. R., Nolasco, S. M., & Tomás, M. C. (2015). TE D 475 M AN U 472 Physicochemical characterization and stability of chia oil microencapsulated with 479 sodium caseinate and lactose by spray-drying. Powder Technology, 271, 26–34. 480 https://doi.org/10.1016/j.powtec.2014.11.006 EP 478 Ixtaina, V. Y., Martínez, M. L., Spotorno, V., Mateo, C. M., Maestri, D. M., Diehl, B. W. K., … 482 Tomás, M. C. (2011). Characterization of chia seed oils obtained by pressing and 483 484 485 AC C 481 solvent extraction. Journal of Food Composition and Analysis, 24(2), 166–174. https://doi.org/10.1016/j.jfca.2010.08.006 Ixtaina, V. Y., Nolasco, S. M., & Tomás, M. C. (2012). Oxidative Stability of Chia (Salvia 486 hispanica L.) Seed Oil: Effect of Antioxidants and Storage Conditions. Journal of the 487 American Oil Chemists’ Society, 89(6), 1077–1090. https://doi.org/10.1007/s11746-011- 488 1990-x 489 Joseph, J. D., & Ackman, R. G. (1992). Capillary column gas chromatography method for 490 analysis of encapsulated fish oil and fish oil ethyl esters: collaborative study. Journal 21 ACCEPTED MANUSCRIPT 491 Association of Official Analytical Chemists, 75(3), 488–506. Julio, L. M., Ixtaina, V. Y., Fernández, M. A., Sánchez, R. M. T., Wagner, J. R., Nolasco, S. 493 M., & Tomás, M. C. (2015). Chia seed oil-in-water emulsions as potential delivery 494 systems of ω-3 fatty acids. Journal of Food Engineering, 162, 48–55. 495 https://doi.org/10.1016/j.jfoodeng.2015.04.005 496 RI PT 492 Litwinienko, G., & Kasprzycka-Guttman, T. (1998). A DSC study on thermoxidation kinetics of mustard oil. Thermochimica Acta, 319(1–2), 185–191. https://doi.org/10.1016/S0040- 498 6031(98)00410-9 499 SC 497 Mahboubifar, M., Hemmateenejad, B., Javidnia, K., & Yousefinejad, S. (2017). Evaluation of long-heating kinetic of edible oils using ATR-FTIR and chemometrics tools. Journal of 501 Food Science and Technology, 54(3), 659–668. 502 M AN U 500 Micić, D. M., Ostojić, S. B., Simonović, M. B., Krstić, G., Pezo, L. L., & Simonović, B. R. 503 (2015). Kinetics of blackberry and raspberry seed oils oxidation by DSC. Thermochimica 504 Acta, 601, 39–44. https://doi.org/10.1016/j.tca.2014.12.018 Milanovic, J., Levic, S., Manojlovic, V., Nedovic, V., & Bugarski, B. (2011). Carnauba wax 506 microparticles produced by melt dispersion technique. Chemical Papers, 65(2), 213– 507 220. https://doi.org/10.2478/s11696-011-0001-x Milinsk, M. C., Matsushita, M., Visentainer, J. V, Oliveira, C. C., & Souza, N. E. de. (2008). EP 508 TE D 505 Comparative analysis of eight esterification methods in the quantitative determination of 510 vegetable oil fatty acid methyl esters (FAME). Journal of the Brazilian Chemical Society, 511 512 513 AC C 509 19(8), 1475–1483. Moros, J., Roth, M., Garrigues, S., & Guardia, M. de la. (2009). Preliminary studies about thermal degradation of edible oils through attenuated total reflectance mid-infrared 514 spectrometry. Food Chemistry, 114(4), 1529–1536. 515 https://doi.org/10.1016/j.foodchem.2008.11.040 516 Morselli, M. D. M., Barrera, D., & Ferreira, C. R. (2012). The effect of adding oleic acid in the 517 production of stearic acid lipid microparticles with a hydrophilic core by a spray-cooling 518 process, 47, 38–44. https://doi.org/10.1016/j.foodres.2012.01.007 22 ACCEPTED MANUSCRIPT 519 Müller, R. H., Radtke, M., & Wissing, S. A. (2002). Nanostructured lipid matrices for improved 520 microencapsulation of drugs. International Journal of Pharmaceutics, 242, 121–128. 521 Musialik, M., & Litwinienko, G. (2007). DSC study of linolenic acid autoxidation inhibited by BHT, dehydrozingerone and olivetol. Journal of Thermal Analysis and Calorimetry, 523 88(3), 781–785. https://doi.org/10.1007/s10973-006-8507-0 524 RI PT 522 Noello, C., Carvalho, A. G. S., Silva, V. M., & Hubinger, M. D. (2016). Spray dried microparticles of chia oil using emulsion stabilized by whey protein concentrate and 526 pectin by electrostatic deposition. Food Research International2, 89, 549–557. 527 Ostrowska-Ligeza, E., Bekas, W., Kowalska, D., Lobacz, M., Wroniak, M., & Kowalski, B. 528 (2010). Kinetics of commercial olive oil oxidation: Dynamic differential scanning 529 calorimetry and Rancimat studies. European Journal of Lipid Science and Technology, 530 112(2), 268–274. https://doi.org/10.1002/ejlt.200900064 533 M AN U 532 Ozawa, T. (1970). Kinetic analysis of derivative curves in thermal analysis. Journal of Thermal Analysis, 2(3), 301–324. https://doi.org/10.1007/BF01911411 Pardauil, J. J. R., Souza, L. K. C., Molfetta, F. a, Zamian, J. R., Rocha Filho, G. N., & da TE D 531 SC 525 Costa, C. E. F. (2011). Determination of the oxidative stability by DSC of vegetable oils 535 from the Amazonian area. Bioresource Technology, 102(10), 5873–7. 536 https://doi.org/10.1016/j.biortech.2011.02.022 538 539 540 541 Poyato, C., Ansorena, D., Navarro-Blasco, I., & Astiasarán, I. (2014). A novel approach to monitor the oxidation process of different types of heated oils by using chemometric AC C 537 EP 534 tools. Food Research International, 57, 152–161. https://doi.org/10.1016/j.foodres.2014.01.033 Poyato, C., Navarro-Blasco, I., Calvo, M. I., Cavero, R. Y., Astiasar??n, I., & Ansorena, D. 542 (2013). Oxidative stability of O/W and W/O/W emulsions: Effect of lipid composition and 543 antioxidant polarity. Food Research International, 51(1), 132–140. 544 https://doi.org/10.1016/j.foodres.2012.11.032 545 546 Qi, B., Zhang, Q., Sui, X., Wang, Z., Li, Y., & Jiang, L. (2016). Differential scanning calorimetry study—Assessing the influence of composition of vegetable oils on 23 ACCEPTED MANUSCRIPT 547 548 oxidation. Food Chemistry, 194, 601–607. Révérend, B. J. D., Fryer, P. J., Coles, S., & Bakalis, S. (2010). A method to qualify and 549 quantify the crystalline state of cooa butter in industrial chocolate. Journal of the 550 American Oil Chemists Society, 87, 239–246. Sandoval-Oliveros, M. R., & Paredes-López, O. (2013). Isolation and characterization of 552 proteins from chia seeds (Salvia hispanica L.). Journal of Agricultural and Food 553 Chemistry, 61(1), 193–201. https://doi.org/10.1021/jf3034978 RI PT 551 Santos, J. C. O., Santos, I. M. G., Souza, A. G., Prasad, S., & Santos, A. V. (2002). Thermal 555 Stability and Kinetic Study on Thermal Decomposition of Commercial Edible Oils by 556 Thermogravimetry. Journal of Food Science, 67(4), 1393–1398. M AN U 557 SC 554 Segura-Campos, M. R., Ciau-Solís, N., Rosado-Rubio, G., Chel-Guerrero, L., & Betancur- 558 Ancona, D. (2014). Physicochemical characterization of chia (Salvia hispanica) seed oil 559 from Yucatán, México. Agricultural Sciences, 5(3), 220–226. 560 https://doi.org/10.4236/as.2014.53025 562 563 Simopoulos, A. P. (2002). Omega-3 fatty acids in inflammation and autoimmune diseases. TE D 561 Journal of the American College of Nutrition, 21(6), 495–505. Simopoulos, A. P. (2008). The omega-6/omega-3 fatty acid ratio, genetic variation, and cardiovascular disease. Asia Pacific Journal of Clinical Nutrition, 17(SUPPL. 1), 131– 565 134. 567 568 569 570 Tamjidi, F., Shahedi, M., Varshosaz, J., & Nasirpour, A. (2013). Nanostructured lipid carriers AC C 566 EP 564 (NLC): A potential delivery system for bioactive food molecules. Innovative Food Science & Emerging Technologies, 19, 29–43. https://doi.org/10.1016/j.ifset.2013.03.002 Tan, C. P., Man, Y. B. C., Selamat, J., & Yusoff, M. S. A. (2002). Comparative studies of 571 oxidative stability of edible oils by differential scanning calorimetry and oxidative stability 572 index methods, 76, 385–389. 573 574 Timilsena, Y. P., Adhikari, R., Barrow, C. J., & Adhikari, B. (2016). Microencapsulation of chia seed oil using chia seed protein isolatechia seed gum complex coacervates. 24 ACCEPTED MANUSCRIPT 575 International Journal of Biological Macromolecules, 91, 347–357. 576 https://doi.org/10.1016/j.ijbiomac.2016.05.058 577 Valderrama, P., Março, P. H., Locquet, N., Ammari, F., & Rutledge, D. N. (2011). A procedure to facilitate the choice of the number of factors in multi-way data analysis 579 applied to the natural samples: Application to monitoring the thermal degradation of oils 580 using front-face fluorescence spectroscopy. Chemometrics and Intelligent Laboratory 581 Systems, 106(2), 166–172. https://doi.org/10.1016/j.chemolab.2010.05.011 582 Vecchio Ciprioti, S., Paciulli, M., & Chiavaro, E. (2017). Application of different thermal SC RI PT 578 analysis techniques to characterize oxidized olive oils. European Journal of Lipid 584 Science and Technology, 119(1), 1–16. https://doi.org/10.1002/ejlt.201600074 585 M AN U 583 Vieira, T. M. F. S., & Regitano-d’Arce, M. A. B. (1999). Ultraviolet Spectrophotometric 586 Evaluation of Corn Oil Oxidative Stability during Microwave Heating and Oven Test. 587 Journal of Agricultural and Food Chemistry, 47(6), 2203–2206. 588 https://doi.org/10.1021/jf981033p Villalobos-Hernández, J. R., & Müller-Goymann, C. C. (2006). Sun protection enhancement TE D 589 of titanium dioxide crystals by the use of carnauba wax nanoparticles: The synergistic 591 interaction between organic and inorganic sunscreens at nanoscale. International 592 Journal of Pharmaceutics, 322(1–2), 161–170. 593 https://doi.org/10.1016/j.ijpharm.2006.05.037 595 596 597 Vyazovkin, S., Burnham, A. K., Criado, J. M., Pérez-Maqueda, L. A., Popescu, C., & AC C 594 EP 590 Sbirrazzuoli, N. (2011). ICTAC Kinetics Committee recommendations for performing kinetic computations on thermal analysis data. Thermochimica Acta, 520(1–2), 1–19. https://doi.org/10.1016/j.tca.2011.03.034 598 Wood, J. ., Richardson, R. ., Nute, G. ., Fisher, a. ., Campo, M. ., Kasapidou, E., … Enser, 599 M. (2004). Effects of fatty acids on meat quality: a review. Meat Science, 66(1), 21–32. 600 https://doi.org/10.1016/S0309-1740(03)00022-6 601 Zhang, Q., Liu, C., Sun, Z., Hu, X., Shen, Q., & Wu, J. (2012). Authentication of edible 602 vegetable oils adulterated with used frying oil by Fourier Transform Infrared 25 ACCEPTED MANUSCRIPT 603 Spectroscopy. Food Chemistry, 132(3), 1607–1613. 604 https://doi.org/10.1016/j.foodchem.2011.11.129 605 Zhang, W., Li, N., Feng, Y., Su, S., Li, T., & Liang, B. (2015). A unique quantitative method of acid value of edible oils and studying the impact of heating on edible oils by UVâ€“Vis 607 spectrometry. FOOD CHEMISTRY, 185, 326–332. 608 https://doi.org/10.1016/j.foodchem.2015.04.005 609 AC C EP TE D M AN U SC 610 RI PT 606 26 ACCEPTED MANUSCRIPT FIGURE CAPTIONS 611 612 Figure 1. Chromatograms (a) of the chia oil and (b) oil-loaded microparticles. RI PT 613 614 Figure 2 – (a) Scanning Electron Microscopy of the oil-loaded microparticles, (b) Infrared 616 spectra (FTIR-ATR) of the (red line) oil-loaded microparticles, (black line) blank 617 microparticles and in natura chia oil (before encapsulation, blue line) and (c) X-Ray 618 diffractograms of the (black line) oil-loaded microparticles and (red line) blank microparticles. M AN U SC 615 619 620 Figure 3 – Non-isothermal DSC for the (a) chia oil and (b) oil-loaded microparticles (onset 621 oxidation temperatures are indicated). TE D 622 Figure 4 – Isothermal DSC for the (a) chia oil and (b) oil-loaded microparticles (oxidation 624 induction times are indicated). EP 623 625 Figure 5 - Thermogravimetric analyses (TGA) and its derivative for the chia oil and oil-loaded 627 microparticles. 628 AC C 626 629 Figure 6. Relative concentration of (- - -) tocopherol and (—) degradation products for (a) 630 chia oil and (b) oil-loaded microparticles. 631 27 ACCEPTED MANUSCRIPT Figure 7. Specific extinction coefficients at 232 nm (K232, ●: chia oil; ▲: oil-loaded 633 microparticles) and 270 nm (K270, ○: chia oil; ∆: oil-loaded microparticles) obtained during the 634 accelerated stability test AC C EP TE D M AN U SC RI PT 632 ACCEPTED MANUSCRIPT Table 1. Physicochemical parameters of chia oil. Maximum values Parameter Result (Codex alimentarius 0.050 ± 0.002 Peroxide index (meq.kgoil-1) 0.784± 0.195 Humidity (%) 0.381± 0.125 0.4 15 * SC Acidity (goleic acid.100goil-1) RI PT comission, 1999) AC C EP TE D M AN U *Value not established. ACCEPTED MANUSCRIPT Table 2. Quantification of major fatty acids present in the chia oil and in the oil-loaded microparticles. Concentration (mgFA.goil-1) Fatty acid Oil-loaded microparticles C16:0 72.8 ± 1.6 72.5 ± 2.1 C18:0 30.1 ± 0.5 C18:1 n-9 59.1 ± 1.3 C18:2 n-6c 200.8 ± 5.4 C18:3 n-3 643.6 ± 18.9 Total* 1006.7 ± 27.6 RI PT Chia oil in natura 33.9 ± 0.6 57.1 ± 2.2 179.0 ± 6.6 SC M AN U SFA (%) MUFA (%) PUFA (%) PUFA: SFA 955.1 ± 33.6 11.8 18.2 5.1 6.0 83.1 75.8 7.1 4.1 0.3 0.3 TE D n6:n3 545.3 ± 20.6 *Sum of all fatty acids identified in the samples (some not shown in the table). AC C EP SFA: Saturated Fatty Acid; MUFA: Monounsaturated Fatty Acid; PUFA: Poliunsaturated Fatty Acid. ACCEPTED MANUSCRIPT Table 3. Activation energy and adjusted coefficient of determination of the adjusted curves for the non-isothermal and isothermal Differential Scanning Calorimetry experiments. Non-isothermal mode Oil-loaded microparticles Ea (kJ.mol-1) 87.4 ± 2.1 97.3 ± 2.9 R2 0.993 Chia oil 73.5 ± 1.5 R2 0.991 Oil-loaded microparticles AC C EP TE D M AN U Ea (kJ.mol-1) 0.991 SC Isothermal mode RI PT Chia oil 91.8 ± 1.6 0.999 AC C EP TE D M AN U SC RI PT ACCEPTED MANUSCRIPT AC C EP TE D M AN U SC RI PT ACCEPTED MANUSCRIPT AC C EP TE D M AN U SC RI PT ACCEPTED MANUSCRIPT AC C EP TE D M AN U SC RI PT ACCEPTED MANUSCRIPT AC C EP TE D M AN U SC RI PT ACCEPTED MANUSCRIPT AC C EP TE D M AN U SC RI PT ACCEPTED MANUSCRIPT AC C EP TE D M AN U SC RI PT ACCEPTED MANUSCRIPT AC C EP TE D M AN U SC RI PT ACCEPTED MANUSCRIPT AC C EP TE D M AN U SC RI PT ACCEPTED MANUSCRIPT AC C EP TE D M AN U SC RI PT ACCEPTED MANUSCRIPT AC C EP TE D M AN U SC RI PT ACCEPTED MANUSCRIPT AC C EP TE D M AN U SC RI PT ACCEPTED MANUSCRIPT ACCEPTED MANUSCRIPT Oxidative stability gains from microencapsulation of edible oils must be correctly assessed. Chia oil encapsulated in carnauba wax showed improved oxidative stability. RI PT Accelerated stability tests corroborated the improved stability verified in the thermal tests. AC C EP TE D M AN U SC MCR-ALS showed the effectiveness of encapsulation in protecting tocopherols present in the oil.