Microencapsulation of sacha inchi oil using emulsion-based delivery systems

Accepted Manuscript Microencapsulation of sacha inchi oil using emulsion-based delivery systems

Juarez Vicente, Taylana de Souza Cezarino, Luciano José Barreto Pereira, Elisa Pinto da Rocha, Guilherme Raymundo Sá, Ormindo Domingues Gamallo, Mario Geraldo de Carvalho, Edwin Elard Garcia-Rojas PII: DOI: Reference:

S0963-9969(17)30293-4 doi: 10.1016/j.foodres.2017.06.039 FRIN 6767

To appear in:

Food Research International

Received date: Revised date: Accepted date:

15 March 2017 9 June 2017 17 June 2017

Please cite this article as: Juarez Vicente, Taylana de Souza Cezarino, Luciano José Barreto Pereira, Elisa Pinto da Rocha, Guilherme Raymundo Sá, Ormindo Domingues Gamallo, Mario Geraldo de Carvalho, Edwin Elard Garcia-Rojas , Microencapsulation of sacha inchi oil using emulsion-based delivery systems, Food Research International (2017), doi: 10.1016/j.foodres.2017.06.039

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Microencapsulation of sacha inchi oil using emulsion-based delivery systems Juarez Vicentea; Taylana de Souza Cezarinob; Luciano José Barreto Pereiraa; Elisa Pinto da

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Rochac; Guilherme Raymundo Sád; Ormindo Domingues Gamalloa,d; Mario Geraldo de

Programa de Pós-graduação em Ciência e Tecnologia de Alimentos (PPGCTA), Universidade Federal Rural de

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a

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Carvalhob; Edwin Elard Garcia-Rojasa,e*.

Rio de Janeiro (UFRRJ), Rodovia BR 465, Km 7, Seropédica/RJ, 23890-000, Brasil. Programa de Pós-Graduação em Química (PPGQ), Departamento de Química-ICE, Universidade Federal Rural

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b

do Rio de Janeiro (UFRRJ), Rodovia BR 465, Km 7, Seropédica/RJ, 23890-000, Brasil. Programa de Pós-graduação em Engenharia Metalúrgica, Universidade Federal Fluminense (UFF), Av. dos

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c

Trabalhadores, 420, Volta Redonda/RJ, 27255-125, Brasil. d

Laboratório de Catálise – Departamento de Engenharia Química-IT, Universidade Federal Rural do Rio de

Laboratório de Engenharia e Tecnologia Agroindustrial (LETA), Universidade Federal Fluminense (UFF), Av.

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e

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Janeiro. Rodovia BR 465, Km 7, Seropédica/RJ, 23890-000, Brasil.

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dos Trabalhadores, 420, Volta Redonda/RJ, 27255-125, Brasil.

*Corresponding author. Telephone number: +55 24 2107 3563; Fax number: +55 24 33443019. e-mail address: [email protected] (E.E. Garcia-Rojas);

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ABSTRACT

In this study, sacha inchi oil (SIO) was microencapsulated by emulsion-based systems using ovalbumin (Ova), pectin (Pec), and xanthan gum (XG), followed by freeze-drying. The

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microencapsulation was confirmed using Fourier transform infrared (FTIR) spectroscopy, Xray diffraction (XRD), thermogravimetric analysis (TGA), and scanning electron microscopy

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(SEM). The stability of omega-3 in SIO alone as well as in microencapsulated SIO was

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assessed by nuclear magnetic resonance (1H NMR) spectroscopy after human gastric simulation (HGS). The SEM results revealed distinct structures for the two types of

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microcapsules. The thermograms showed that the thermal resistance was increased in the

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microencapsulated SIO, indicating that the emulsion-based system may be a way to protect the omega-3 in the SIO. In addition, the microencapsulation conferred an increased

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crystallinity degree, indicating a higher structural organization. Moreover, this method did not

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affect the stability of SIO, as confirmed by 1H NMR. The release of omega-3 acyl units from the SIO was correlated with the decrease of the methynic proton (sn, 2 position) of

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triacylglycerol (TAG). In contrast, the increase of 1,3-diglycerides was negatively correlated

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with the decrease of glyceryl groups (sn, 1,3 positions). The HGS conditions did not significantly alter the stability of omega-3 of SIO over 180 min. The SIO-Ova microcapsules had a similar behavior to the SIO, and the presence of Ova was not enough to prevent the decrease of omega-3 content over 180 min. The SIO-Ova-Pec and SIO-Ova-XG microcapsules were shown to protect the omega-3 content effectively. In conclusion, the microcapsules developed in this study can be used to transport nutraceutical compounds because they are resistant to the human gastric conditions tested in vitro.

Keywords: Sacha inchi oil, omega-3 stability, 1H nuclear magnetic resonance, human gastric 2

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simulation, microencapsulation.

1. Introduction

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Microencapsulation is a technique that aims to ensure the quality and physicochemical stability of heat- or photosensitive components; in particular, omega-3

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(Gaonkar, Vasisht, Khare, & Sobel, 2014; McClements, 2004). Polyunsaturated fatty acids,

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such as omega-3, are important to the food and nutraceutical industries because they have been shown to prevent fatal cardiovascular disease (Arab-Tehrany et al., 2012). The source of

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omega-3 used in this study was Sacha inchi oil (SIO) (Plukenetia volubilis L.). It has a high

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concentration of unsaturated fatty acids (approximately 90%), including 48–50% linolenic acid (omega-3), 32–37% linoleic acid (omega-6), and 9–12% oleic acid (omega-9) (Guillén,

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Ruiz, Cabo, Chirinos, & Pascual, 2003; Follegatti-Romero, Piantino, Grimaldi, & Cabral,

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2009; Vicente, Carvalho & Garcia-Rojas, 2015). SIO is a rare vegetable source (along with flaxseed oil) that has an omega-3 concentration greater than or equal to 50% (Maurer, Hatta-

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Sakoda, Pascual-Chagman, & Rodriguez-Saona, 2012; Fanali et al., 2011). Therefore, it is

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known as “oro Inca” or Incan gold (Follegatti-Romero, Piantino, Grimaldi, Cabral, 2009; Guillén, Ruiz, Cabo, Chirinos, & Pascual, 2003). One of the most inexpensive and practical ways to encapsulate substances is by

simple emulsion formation following by freeze-drying. It is a useful tool to encapsulate lipophilic components that naturally have a low water solubility. Thus, this method can be used for food fortification and/or hydrophilic systems with hydrophobic components. In addition, this technique can be used in a variety of food applications that are thermodynamically unfavorable (McClements, 2004). There is a growing interest in the formulation of added-value products containing healthy ingredients (i.e., omega-3) for the 3

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functional food market (Achouri, Zamani & Boye, 2012). Emulsions are used on a large scale in the food industry and have many applications, such as the microencapsulation of lipophilic components, thus adding value and ensuring the quality of sensitive compounds. The use of polysaccharides such as pectin (Pec) and/or xanthan gum (XG) aims to reduce the

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electrophoretic mobility of the formed emulsion, which prevents coalescence and consequently provides greater stability to the system (Dickinson, 2009). Biopolymers

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(proteins and polysaccharides) are widely used to encapsulate bioactive compounds, such as

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omega-3, carotenoids, vitamins, and others lipophilic compounds with nutraceutical purposes. Previously, Carneiro, Tonon, Grosso, and Hubinger (2013) used gum Arabic, maltodextrin,

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whey protein concentrate, and two types of modified starch; whereas Fioramonti, Martinez,

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Pilosof, Rubiolo, and Santiago (2015) used whey protein isolate and sodium alginate to microencapsulate flaxseed oil, which is rich in omega-3, similar to SIO. The main reasons to

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encapsulate bioactive compounds are as follows: (i) for protection and to avoid exposure to

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factors such as oxygen, light, heat, metals, and water (to ensure the properties of the bioactive compounds); (ii) to mask flavors, colors, or odors; (iii) to facilitate manipulation; (iv) to

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prevent undesirable reactions between the bioactive compound and food ingredients; and (v)

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to ensure that the release of the substance is controlled in the human gastrointestinal tract (Gaonkar, Vasisht, Khare, & Sobel, 2014). Bioactive compounds, such as omega-3, are commonly referred to as nutraceuticals

and have constituents that typically occur in small amounts in food as well as have the ability to modulate one or more metabolic processes (Ajila, Jaganmohan Rao, & Prasada Rao, 2010). Many bioactive compounds are lipophilic, which hinders their addition to aqueous foods. Their low solubility in water also results in less absorption in the gastrointestinal tract, therefore limiting their bioavailability (Donsì, Annunziata, Sessa, & Ferrari, 2011). The fortification of foods with polyunsaturated fatty acids, such as omega-3, is of great interest not 4

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only because of the increasing awareness of nutrition throughout the world, but mainly for the prevention of malnutrition in developing countries. However, the main difficulty for food fortification with functional lipids is to maintain their physicochemical stability due to their high susceptibility to undergo oxidation (McClements, Decker & Weiss, 2007; Hur, Lim,

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Decker, & McClements, 2011). Emulsion-based systems as a way to encapsulate substances can be a strategy to prevent changes in the oil compositions (e.g., omega-3) when submitted

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to human gastric conditions. According to McClements (2015), the most commonly used

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encapsulation technology for this type of lipid is oil-in-water emulsions. In the human gastrointestinal tract, foods are digested by a combination of physical

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and chemical processes. Chemical digestive processes are catalyzed by digestive enzymes that

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are secreted in the stomach, thereby degrading the foods to a molecular scale. Physical processes are mainly induced by peristalsis, which plays an important role by promoting food

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digestion in the human gastrointestinal tract (Kozu et al, 2014). Several research groups have

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reported that the stomach fluid movements promote emulsification of the oil components and

2005).

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the release of these compounds (Schwizer, Steingoetter, & Fox, 2006; Abrahamsson et al.,

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The first aims of this paper were to microencapsulate SIO by a simple emulsionbased method and to characterize SIO by thermogravimetric analysis (TGA), X-ray diffraction (XRD), Fourier transform infrared (FTIR) spectroscopy, nuclear magnetic resonance (1H NMR) spectroscopy, and scanning electronic microscopy (SEM). The crystallinity degree, loaded oil content, encapsulation efficiency, and stability of the SIO alone and SIO microcapsules under human gastric simulation (HGS) conditions was assessed. The use of 1H NMR was used to understand how changes occur in the oil in the free and microencapsulated structure when subjected to gastric conditions as well as to evaluate the chemical stability and effectiveness of omega-3 release in the system. 5

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2. Materials and methods

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2.1 Materials

Ovalbumin (Ova) from chicken egg white, A5253, containing 62–68% of ovalbumin,

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XG, and Pec were purchased from Sigma-Aldrich (St. Louis, MO, USA). SIO (Plukenetia

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volubilis L.) was purchased from a local market in Lima, Peru. Pepsin (1/1000 proteolytic activity) was purchased from Proquimios (Rio de Janeiro, RJ, Brazil). Deuterated chloroform

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(CDCl3, 99%) was used as a solvent for NMR analysis. HCl was purchased from Vetec (Rio

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de Janeiro, RJ, Brazil). Analytical grade reagents and ultrapure water (Gehaka-Master P&D,

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Brazil) with a conductivity of 0.05 μS·cm-1 were used in all experiments.

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2.2 Methods

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2.2.1 Preparation of microencapsulated SIO

First, the emulsions were prepared and divided into four systems (according to the

descriptions detailed in Table 1). The emulsions were prepared with 0.5, 1.0, 1.5, and 2.0% (w/w) Ova; 8% (w/w) SIO (control), and added of either 0.25, 0.5, and 1.0% (w/w) XG or 1.0, 2.0, and 3.0% (w/w) Pec, depending on the system. The pH of the ultrapure water utilized to prepare the emulsions was adjusted to 7.0 in phosphate buffer (10 mM). The emulsions were homogenized using an UltraTurrax-T25 homogenizer (IKALabortechnik, Germany) at 18,000 rpm for 1 min. Subsequently, they were submitted to sonication with an ultrasound probe (Ultrassonic Processor, Hilscher, Germany) for 12 min at 6

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a frequency of 30 kHz (100% amplitude and 0.5 cycles.min-1). Then, the samples were freezedried to form the microencapsulated products. The oil content (expected) values after freezedrying (AISI 304 Enterprise, Terroni, Brazil) are shown in Table 1.

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2.2.2 Microcapsule characterization

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The microencapsulated freeze-dried products were characterized by TGA, NMR,

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SEM, FTIR spectroscopy, and XRD. The thermograms of the microencapsulated products as well as XG, Pec, SIO, and Ova were obtained on a TA Instruments SDT-Q600

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thermogravimetric analyzer (USA). Each sample (10 mg) was heated on a platinum pan (as

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the reference) between 25 °C and 600 °C at a rate of 10 °C·min-1 under a N2 flow of 100 mL·min-1. The FTIR spectra of the microencapsulated samples and all components (XG, Pec,

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SIO, and Ova) used were obtained on a Bruker FTIR spectrometer (Vertex 70, Germany)

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using KBr pellets and a range of 4000 to 500 cm-1. The NMR spectra were acquired on a 400 MHz Bruker Advance II spectrometer, dissolving the sample in CDCl3 in a 5-mm tube. The

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crystallinity assays (or XRD patterns) were recorded over a 2θ range of 1–60° using an X-ray

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diffractometer (Rigaku, MiniFlex II, Japan) operating at 30 kV and 15 A, with a step angle of 0.02°·min-1 and acquisition time of 2 s. The microencapsulated freeze-dried samples were deposited in flesh-colored mica, and they were metalized with gold-palladium using a sputter coater (Emitech, K550X, United Kingdom). The morphology of the samples and all components (XG, Pec, SIO, and Ova) used was observed by SEM (ZEISS, EVO MA 10, Germany) at an acceleration of 5–10 kV.

2.2.3 Loaded oil content and microencapsulation efficiency

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According to Karaca, Nickerson, and Low (2013), approximately 20–30 mg of microcapsules were weighed in a Falcon tube (15 mL) to which 5 mL of isopropanol, 2 mL of ultrapure water, and 2 mL of hexane were added. The components were homogenized on a Vortex (Phoenix, AP 56, Brazil) and centrifuged (Orto Alresa, Digicen 21 R, Spain) at 4000

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rpm for 30 min. Then, the supernatant phase was removed (washed twice with hexane), the oil

𝑤𝑐

. 100

(Eq. 1)

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𝑤𝑜𝑖𝑙

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OC (%) =

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mass was measured, and the oil content (OC) was given by Eq. 1.

where 𝑤𝑜𝑖𝑙 is the oil mass after centrifugation and 𝑤𝑐 is the capsule mass utilized in this

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analysis. The microencapsulation efficiency (ME) is the percentage of loaded oil content

% 𝐿𝑜𝑎𝑑𝑒𝑑 𝑜𝑖𝑙 𝑐𝑜𝑛𝑡𝑒𝑛𝑡

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% 𝑜𝑖𝑙 𝑐𝑜𝑛𝑡𝑒𝑛𝑡 (𝑡ℎ𝑒𝑜𝑟𝑖𝑐𝑎𝑙)

. 100

(Eq. 2)

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ME (%) =

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divided by the percentage of oil content (theoretical), given by Eq. 2.

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2.2.4 Omega-3 stability from SIO

After the microcapsule formation, the omega-3 stability was evaluated in the

microcapsule control (SIO-Ova) as well as in microcapsules containing XG (SIO-Ova-XG) and Pec (SIO-Ova-Pec). Approximately 100 mg of each sample was measured and solubilized in CDCl3 to separate the oil phase. The oil phase was analyzed by 1H NMR to quantify the omega-3 content, as described previously by Vicente, Carvalho, and Garcia-Rojas (2015).

2.2.5 Preparation of gastric juice 8

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The gastric juice was prepared according to the method of Kozu et al. (2014), in which NaCl (8.775 g·L-1) and pepsin (1.0 g·L-1) were dissolved in 0.5 M HCl, and the pH was kept at 2.0.

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2.2.6 Human digestion simulation (HGS)

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The HGS was performed in a distillation flask with four rings (Fig. 1), with a

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volumetric capacity of 500 mL. To the distillation flask on a Hotplate Stirrer (AccuPlate, Labnet International Inc, USA), 200 mL of gastric juice (corresponding to the stomach gastric

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juice volume) was added, and the mixture was stirred at 400 rpm. The temperature was

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controlled at 36.0–37.0 °C by an alcohol thermometer (-10 °C to 110 °C), and the pH (Analyser® pHmeter 300M, Brazil) ranged from 1 to 3, as described previously by Kozu et al.

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(2014).

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Approximately 1000 mg of SIO (~500 mg of omega-3) and equivalent amounts of microcapsules (oil mass fraction) were inserted into the HGS. Aliquots (10 mL) of gastric

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juice were taken at 10, 20, 30, 40, 50, 60, 90, 120, and 180 min. After withdrawal of each

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aliquot, 10 mL of gastric juice was added to continue the digestion process. The oil phase was extracted with hexane (Augustin et al., 2014) and dried under N2. The omega-3 content as well as the compounds formed after digestion (and their possible correlation) were evaluated by 1H NMR for each sample (SIO, SIO-Ova, SIO-Ova-Pec, and SIO-Ova-XG), separately.

2.2.7 Triacylglycerol (TAG) analysis by 1H NMR

The NMR spectra were recorded on a Bruker Advance II spectrometer (Billerica, MA, USA) operating at 400 MHz for 1H. The acquisition parameters for the one-dimensional 9

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NMR experiments were as follows: time domain data = 65536, acquisition time = 3.98 s, number of scans = 16, spectral width = 8224 Hz, relaxation delay between successive scans/transients = 1.0 s, and exponential line broadening prior to Fourier transformation = 0.3 Hz.

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Aliquots of SIO, SIO-Ova, SIO-Ova-Pec, and SIO-Ova-XG samples were collected for each time point (from 10 to 180 min) under HGS conditions and solubilized in CDCl3 with

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tetramethylsilane as the reference. The mixture was added to a 5-mm-diameter tube, and the

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experiments were conducted at 25 °C. The spectra were processed using the ACD/NMR Processor Academic Edition Program (Version 12.0). The omega-3 content after HGS was

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2.2.8 Statistical analysis

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assessed by 1H NMR according to the method of Vicente, Carvalho, and Garcia-Rojas (2015).

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All analyses were performed in triplicate, and the results were expressed as the average ± confidence interval (CI) (Eq. 3). Correlation coefficients were calculated from the

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different signals obtained by 1H NMR by the Pearson correlation coefficient equation (Eq. 4)

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(Moore, 2007).

𝐶𝐼 =

𝜎

√𝑛

.𝑡

(Eq. 3)

where CI is the confidence interval, σ is the standard deviation, n is the number of repetitions, and t is the Student’s t-test inverse function (significance level of 5%).

𝑟=

1 𝑛−1

𝑥𝑖 −𝑋̅

∑(

𝑠𝑥

𝑦𝑖 −𝑌̅

)(

𝑠𝑦

)

(Eq. 4)

10

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where r is the Pearson correlation coefficient, n is the number of repetitions, sx,y is the standard deviation of variables x, y; xi is the observed value I of the variable x; yi is the observed value I of the variable y; 𝑋̅ is the average value of the variable x, and 𝑌̅ is the

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average value of the variable y.

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3. Results and discussion

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3.1 Microencapsulation characterization

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For structural characterizations, the samples with the highest efficiency of encapsulation for each composition were chosen (Table 1). Therefore, the following samples

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were chosen: S.3-control (SIO-Ova); S.3.3, SIO-Ova-1% XG; and S.3.4, SIO-Ova-1% Pec.

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The efficiency (%) to this samples were 94.2±0.4, 92.3±1.2 and 91.7±0.2, respectively (All values are shown in Table 1). The oil content (theoretical value; *) after freeze-drying, the

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loaded oil content (%, experimental value; **), and the efficiency (the percentage of oil

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loaded divided by oil content; ***) are shown in Table 1.

3.1.1 TGA

The thermograms of the microcapsules as well as XG, Pec, SIO, and Ova are shown in Fig. 2a. In addition, the first derivative of the TGA curve is shown in Fig. 2b. The first step of weight loss from 40 °C to 120 °C refers to the loss of moisture (adsorbed and bound water). The second step from 200 °C to 340 °C (to Ova, Pec, and XG, lines 2, 3, and 4, respectively, in Fig. 2a) is attributed to dehydration of the saccharide rings, depolymerization, 11

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and decomposition of the acetylated and deacetylated units of the polymers. For SIO, SIOOva, SIO-Ova-Pec, and SIO-Ova-XG, this second step occurs at a temperature of 350–480 °C (Hosseini, Zandi, Rezaei, & Farahmandghavi, 2013; Kakkar, Verna, Manjubala, & Madhan, 2014).

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The maximum temperatures of degradation (Td) for each component alone as well as for the microcapsules are clearly shown in the DTGs depicted in Fig. 2b. In this analysis, it

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was observed that SIO was incorporated and interacted harmoniously (by hydrophobic

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interaction) with the biopolymer to form the microcapsules. The formation of microcapsules led to an increase in the Td (from 95 °C to 175 °C, approximately) when compared to the Td of

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the biopolymers alone, nearing the Td of SIO (411.56 °C), however when occurs the

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encapsulation process the Td (of encapsulated) tend to decrease due the interaction with the Ova. The Td values of the control microcapsules (SIO-Ova; 401.43 °C), those with Pec (SIO-

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Ova-Pec; 397.17 °C), and those with XG (SIO-Ova-XG; 407.29 °C) did not differ much from

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each other; however, they did differ from those of the initial biopolymers, Ova (302.86 °C), Pec (232.53 °C), and XG (276.76 °C). The fact that the Td values of the microcapsules are

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similar to the Td of SIO can be explained by two different reasons. First, there is an interaction

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between SIO and Ova (in the formation of the microcapsule by an emulsion-based method); second, the high concentration of oil (about 73–80%) causes the characteristics of the SIO to prevail (McClements, 2004; Dickinson, 2009).

3.1.2 FTIR spectra

The FTIR spectra of the encapsulated SIO as well as all components (XG, Pec, SIO, and Ova) are shown in Fig. 3a–b. The principal observed signals (max (cm-1)) of SIO were as follows: 3010 (w, =CH), 2962–2853 and 1463 (C-H of CH2 and CH3), 1746 (C=O of ester), 12

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and 1648 (w, C=C). The additional signals in the spectra corresponded to the strong absorptions of the biopolymers (Ova, Pec, and XG) used to form the encapsulated SIO, as shown by Hosseini, Zandi, Rezaei, and Farahmandghavi (2013) and Rosa et al., (2013). The letter w represents a weak signal. This analysis showed that there was a formation of the

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capsule and the presence of the SIO was confirmed mainly by the band in 1746 cm -1 (relative to the ester) in all systems (SIO-Ova, SIO-Ova-1% Pec and SIO-Ova-1% XG ) shown in Fig.

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3a. An indication of the formation of the capsule and therefore improvement in the structural

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organization is the reduction of the band between 3500 and 3000, referring to OH stretching. When this decrease occurs, it indicates that the interaction between the components is

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stronger. This fact can be observed when Pec and XG are added compared with control SIO-

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Ova.

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3.1.3 XRD

The XRD patterns are shown in Fig. 3c. The structures of SIO, Ova, Pec, and XG

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were evaluated separately. All three polymers showed similar values to each other at their

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characteristic 2θ of 18.9° (XG), 19.6° (Ova), and 21.2° (Pec), indicating a low crystallinity degree. All of the utilized components (XG, Pec, SIO, and Ova) in this study were observed to have an amorphous structure. However, the microcapsules formed among SIO-Ova-Pec, SIO-Ova-XG, as well as the microcapsule-control (SIO-Ova) showed a higher degree of crystallinity; consequently, they showed a more defined structure than the compounds alone. The signals were observed between 5° and 14°. Thus, the formation of the microcapsule can provide a more organized structure of the biopolymers, compared to the initial structure. The amorphous characteristics are linked directly to the hygroscopicity and hydrophilicity of the biopolymers. It is known, however, that the peak width is related to the crystallinity degree of 13

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the components (Pickup et al, 2014; Souza & Garcia-Rojas, 2015). As shown in Fig. 3c, the counts per sec (cps) value corresponds to the peak intensity, and theta (θ) is the inclination degree. It is important to point out that the improved structural organization that was

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observed with the increase in the degree of crystallinity may possibly be related to the low OH stretching frequency (around 3470 cm-1) shown in the FTIR spectra (Fig. 3a–b). In the

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SIO and the encapsulated SIO, the frequency was lower than that seen in Ova, Pec, and XG.

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These results suggest that when the frequency is smaller, the hydrogen bonding between the components is larger, which indicates greater interaction between the hydrogen bonding and

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consequently, confirming a greater degree of structural organization in this case.

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3.1.4 SEM

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The SEM images of XG, Pec, and Ova as well as SIO-Ova, SIO-Ova-1% Pec, and SIO-Ova-1% XG are shown in Fig. 4. The images revealed that SIO-Ova-1% Pec and SIO-

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Ova-1% XG had distinct and characteristic structures, compared to the control microcapsules

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containing only SIO and Ova (SIO-Ova-Control). It is possible perceive clearly the polyssacaride presence in capsule formation and the distindible between their, shown in Fig 4e (with Pec) e 4f (with XG).

3.1.5. 1H NMR spectra

The 1H NMR spectra (Fig. 3d) were used to evaluate the oxidative stability of encapsulated SIO in the freeze drying process. The signals (θ) with a range of chemical shifts at δH 0.80–0.97 correspond to CH3, and γ (δH 1.23–1.37) refers to the (CH2)n.

Other 14

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assignments were as follows: δH 1.49–1.68, CH2-CH2-CO; δH 2.25–2.32, CH2-C=O; δ (δH 1.96–2.09), allylic protons; ε (δH 5.27–5.40), HC=CH; ζ (δH 2.73–2.83), doubly allylic protons (=C-CH2-C=) of omega-3 (δH 2.78 ppm); δH 2.74 ppm, the omega-6 fatty acids; δH 4.10–4.29 and 5.22–5.26, the methylene and methynic protons of the glycerol unit,

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respectively. There were no additional signals (Fig. 5b–d) suggesting oxidation of the encapsulated oil, such as the formation of epoxides or peroxides (Fang, Goh, Tay, Lau, & Li,

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2013; Barison et al., 2010). Thus, encapsulation of the emulsion formation followed by

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3.1.6. Loaded oil content and ME

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freeze-drying is an effective way to keep the omega-3 contained in the SIO oxidatively stable.

The ME of SIO ranged from 86.6 ± 0.7% to 96.6 ± 0.2%, which is in the range for

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Ova (0.5–2.0%) studied, indicating the high capacity of the biopolymers used to form

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encapsulated products (Table 1). However, in comparison to the influence of Pec and XG at the same Ova concentration, it was observed that the encapsulated SIO containing Pec had a

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greater efficiency to hold the SIO than those using XG, although the range of XG

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concentrations utilized was smaller. It can happen due to the presence of methoxyl in the pectin structure, causing greater interaction with the oil phase. The presence of Pec or XG caused a lower ME, compared to the encapsulated-control (SIO-Ova) with the same Ova concentration.

3.2 HGS

3.2.1. Omega-3 content of SIO and microencapsulated SIO

15

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As shown in Fig. 5g, the HGS procedure did not significantly alter the omega-3 intensity (acyl units) and did not cause epoxide or hydroperoxide formation in SIO over 180 min. The omega-3 acyl unit content decreased by 2% (from 50.7% to 48.7%); this result shows the stability of the SIO under the HGS conditions. A very similar behavior was

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observed with SIO-Ova, in which the omega-3 acyl unit content ranged from 50.3% to 48.6%. In this case, the presence of Ova did not prevent the release of omega-3 acyl units. Also

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shown in Fig. 5g, in the microcapsules formed with SIO-Ova-Pec and SIO-Ova-XG, the

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release of omega-3 acyl units did not occur instantaneously as occurred with the microcapsules without polysaccharide (SIO-Ova). The omega-3 acyl units of the SIO-Ova-

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Pec and SIO-Ova-XG microcapsules showed a similar release profile. In both cases, there was

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a slow and growing trend, which can be estimated by Eq. 5 (SIO-Ova-Pec) and 6 (SIO-Ova-

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XG) with R2 = 0.992 and 0.948, respectively.

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SIO-Ova-Pec (10-180min): 𝑦 = 0.0338 (±0.0011)𝑥 + 8.99 (±0.09)

(Eq. 6)

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SIO-Ova-XG (10-180min): 𝑦 = 0.046 (±0.004)𝑥 + 10.9 (±0.3)

(Eq. 5)

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The omega-3 acyl unit release rates are shown in Fig. 5g for a given time (tx) in relation to its previous time. The change in this rate was measured from 10 to 180 min, with the time 0 min indicating the time that the samples were subjected to the HGS conditions. For SIO-Ova-Pec and SIO-Ova-XG, there were greater variations of these release rates; but after 120 min of HGS, they showed an increasing trend. However, for SIO and SIO-Ova, the rate showed a decrease in omega-3 acyl units over time. The global rate of decrease of omega-3 acyl units after contact with HGS conditions was -0.20 ± 0.01%·min-1 for SIO and -2.64 ± 0.16%·min-1 for SIO-Ova. The negative sign indicates a decrease in the omega-3 acyl group content. The global release rates of omega-3 from 10 to 180 min were 1.19 ± 0.08%·min-1 for 16

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SIO-Ova-Pec and 1.91 ± 0.29%·min-1 for SIO-Ova-XG. Although the omega-3 release rates were similar, the microcapsule formed with SIO-Ova-Pec retained almost 4% more omega-3 acyl unit content than that formed with SIO-Ova-XG at the end of HGS. Fig. 5g shows the

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release rate of omega-3 acyl units during the HGS.

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3.2.2 1H NMR spectra of SIO and microencapsulated SIO

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Based on the omega-3 structure (α linolenic acid) (Fig. 5e), the signals present in Fig. 5a–d were identified. The signals (θ) with shifts of δH 0.80–0.90 ppm and δH 0.92–0.98 ppm

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refer to terminal methyl groups and aliphatic CH3 and CH3 bonded to allylic CH2,

related to the CH2 intermediate.

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respectively. The signals at δH 1.15–1.35 ppm represent methylene protons (γ); this signal is

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In the β position, protons appeared with chemical shifts between δH 1.50 and 1.70

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ppm. As shown in Fig. 5a–b, the anisotropy effect occurred for SIO and SIO-Ova with an increasing contact time under HGS conditions. However, this effect was not visible for SIO-

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Ova-Pec (Fig. 5c) or SIO-Ova-XG (Fig. 5d) because the microcapsules better protect the

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omega-3 structure compared to SIO alone and SIO-Ova. As the process of hydrolysis led to the generation of “free” acid and not the ester (such as TAG), a lower scattering of this signal was observed in the β-position (Karupaiah & Sundram, 2007). The δ indicates the signal of allyl protons (neighboring double bonds) and is

represented by the δH 1.95–2.10 ppm signals. The proton signals located at the α carbonyl position were observed with chemical shifts of δH 2.25–2.35 ppm. The ζ signals of doubly allylic protons (double double bonds, ddb) refer to omega-6 (2.68–2.75 ppm) and omega-3 (2.77–2.83 ppm) acyl units. Meanwhile, the signals at 4.10–4.29 ppm refer to methylenic protons bonded at the 1 and 3 positions of the glyceryl group, and the signal at δH 5.20–5.25 17

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ppm refers to the methynic proton (sn, 2 position) of TAG. Based on these data, the signal at δH 4.00–4.07 ppm (sn-1,3-diglycerides) increases over time, while the glyceryl protons (δH 4.10–4.29 ppm) decrease proportionally in the same period. The ε signal at δH 5.30–5.40 ppm represents the protons of the double bonds (unsaturated), while the protons at δH 4.10–4.29

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3.2.3. Analysis of the Pearson correlation coefficient

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ppm (shown in Fig. 4) refer to the glyceryl groups at the 1 and 3 positions attached to TAG.

The Pearson correlation coefficient (r) was determined by applying Eq. (4) for the

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signals shown in Fig. 5a–d for SIO, SIO-Ova, SIO-Ova-Pec, and SIO-Ova-XG, respectively.

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Although the HGS did not promote the formation of oxidized compounds (epoxides and hydroperoxides) in the SIO and the SIO-Ova, the slight decrease in the omega-3 acyl unit

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content observed in Fig. 5g was evaluated by determining the Pearson correlation coefficient

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(r), compared to the increase of the other signals. As shown in Fig. 5a, the intensity of the 1,3-diglyceride signals (δH 4.00–4.07 ppm)

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of SIO increased over time and was directly correlated (r = -1.000) to the reduction of the

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protons attached to the glyceryl group at the 1 and 3 positions (δH 4.10–4.29 ppm). The same result occurred, albeit at a lower intensity (r = -0.635), with the signals of omega-3 acyl units (δH 2.77 to 2.83 ppm) and 1,3-diglyceride. These findings, however, cannot be related directly because the omega-3 signals of TAG as well as “free” acids have the same chemical shift (δH 2.77 to 2.83 ppm). Thus, only omega-3 release can be assigned for the sn-1,3 positions of TAG. The methynic protons (sn, 2-positions) of TAG (δH 5.20–5.25 ppm) decrease, while the omega-3 acyl units of doubly allylic protons at 2.77–2.83 ppm decrease (r = 0.741). This phenomenon can be attributed to the steric hindrance of the sn-2 position compared with the 18

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other positions (sn-1 and sn-3), which are more easily hydrolyzed (Karupaiah & Sundram, 2007). Thus, it is easier for the protons at the sn-2 position to remain attached to the omega-3 acyl units. In other words, as soon as a signal decreases, the other also decreases. Although there was an anisotropic effect on the acyl groups (δH 1.51–1.65 ppm), they were not

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correlated with the omega-3 acyl units (r = -0.030) (Fig. 5a). For SIO-Ova, the intensity of omega-3 acyl units (δH 2.77–2.83 ppm) was negatively

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correlated with the 1,3-diglyceride signals (r = -0.873) (Fig. 5b). For the same reason that

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occurred with SIO, the correlation cannot be assigned in this case, since both the “free” acid and the TAG form have the same chemical shift, thus blocking this interpretation. The

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anisotropic effect (on acyl groups at δH 1.51–1.65 ppm) was lower than that observed in SIO

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due to the presence of Ova. Another signal that was strongly positively correlated with the omega-3 acyl groups was the methynic protons (sn, 2-positions) of TAG (r = 0.915), meaning that the decrease in omega-3 acyl units was almost proportional to the decrease in the signal

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intensity at δH 5.20–5.25 ppm, thus indicating that omega-3 is in the TAG form (attached at sn, 2-position).

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For SIO-Ova-Pec, there was a strong positive correlation between the decrease in the

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omega-3 acyl units and increasing signals of the methynic protons (sn, 2-positions) (r = 0.799) as well as another positive correlation (r = 0.526), although weak, with an increase of methylenic protons (1,3-diglycerides). In SIO-Ova-XG, the correlation was almost zero (r = 0.041) between the formation of methylenic protons and the decrease of omega-3 acyl units. However, there was a weak positive correlation (r = 0.554) between the decrease in omega-3 acyl units and the increase of the methynic protons (sn, 2-positions) (Guillén & Uriarte, 2012). The positive correlation between the decrease of omega-3 acyl units and the increase in signal related to the methynic protons (sn, 2-positions) showed a downward trend in 19

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microcapsules as follows: SIO-Ova > SIO-Ova-Pec > SIO-Ova-XG, wherein r = 0.915, 0.799, and 0.554, respectively. In this case, the presence of Ova, Ova with Pec, and Ova with XG proportionally decreased the signals involving the methynic groups (sn, 2-position); thus, it can be inferred that the release of omega-3 acyl groups at the 2-position is affected by the

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presence of biopolymers (Hur, Lim, Decker & McClements, 2011).

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3.2.4. Analysis of TAG positions

Lipid digestion occurs primarily in the small intestine (as with most components)

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because of the presence of pancreatic lipase. Although the gastric conditions are appropriate for protein digestion, McClements (2015) reports that the absorption of bioactive components

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can occur in smaller proportions at any part of the gastrointestinal tract, i.e., mouth, esophagus, or stomach, before reaching the small intestine.

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The analysis of 1H NMR spectra for different reaction times indicates that there was

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no significant consumption of triglyceride. However, the appearance of signals at δH 4.00–

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4.07 ppm (CH-OH) can be attributed to diglyceride (sn-1,3) formation, which is justified by the conversion (hydrolysis) of triglycerides (sn-1,2,3) to (sn-1.3). Thus, the hydrolysis

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occurred (preferably) at the sn-2 position of the ester derived from glycerol (Fig. 5f). The spatial orientation of the methynic carbon, shown in Fig. 5f, demonstrates the hydrolysis in sn-2. Another relevant consideration is the absence of signals that justify oxidation product formation, revealing the continuing source of omega-3 for future metabolism. Note that the red signal in Fig. 5f indicates the proton at the sn-2 position. Therefore, it can be seen that most esters were hydrolyzed at the other positions (sn-1 and 3), shown in blue. This interpretation is in accordance with the report by Zeeb, Weiss & McClements (2015), who observed that the rate is slower. In the emulsions formed with biopolymers, the conversion of 20

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triglycerides to diglycerides occurs, maybe even reaching monoglycerides. The coating biopolymers, however, improve the lipid stability because there is a decrease in the surface area of the lipid droplets exposed to gastric conditions, thus reducing hydrolysis and consequently the release of fatty acids.

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Zhang, Zhang, Zhang, Decker, and McClements (2015) observed (as expected) that the digestion of polyunsaturated fatty acids containing TAG, such as SIO, is slower than that

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of monounsaturated fatty acids and that the rate as well as extent of lipid digestion were also

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lower for the emulsions (containing oil) than for TAG oils. This phenomenon can be explained by the fact that the structure of the colloidal particles formed in the emulsion may

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have been resistant to digestion (Verrijssen, Verkempinck, Christiaens, Loey, & Hendrickx,

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2015; Zhang, Zhang, Zhang, Decker, and McClements, 2015). The hydrolysis of TAG sequentially generates diacylglycerol and monoacylglycerol (Guillén & Uriarte, 2012) before

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being absorbed (McClements, 2015). In oil-in-water emulsions of olive oil enriched with β-

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carotene and containing L-α-phosphatidylcholine (as an emulsifier), it was observed by Verrijssen, Verkempinck, Christiaens, Loey, and Hendrickx (2015) that the lipid digestion is

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not dependent on the presence of Pec.

4. Conclusions

Encapsulation of SIO by an emulsion-based method was successfully carried out and

confirmed by FTIR, TGA, and XRD. This method provides an increased thermal resistance of the biopolymers used for the microcapsule formation, thus demonstrating that this may be a way to protect the omega-3 contained in the SIO. The encapsulation also conferred an increased crystallinity degree, which indicates a higher degree of organization of their structures. The emulsion-based system and freeze-drying as the encapsulation method did not 21

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affect the oxidative state of SIO, as confirmed by 1H NMR. The HGS conditions did not significantly alter the omega-3 acyl unit content of the SIO over 180 min. Moreover, they did not promote the development of oxidized compound. The SIO-Ova-Pec and SIO-Ova-XG microcapsules were shown to protect omega-3 effectively (assured during the HGS).

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Furthermore, the microcapsules showed a very slow omega-3 release profile, which is good because it prevents omega-3 loss before reaching the intestine. In conclusion, the

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microcapsules developed in this study can be used to transport nutraceutical compounds due

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to their resistance to the human gastric conditions tested in vitro, and finally, the digestion

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priority of triglycerides in sn-2 position.

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Conflict of interest

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The authors declare no conflict of interest.

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Acknowledgment

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The authors thank to CNPq and FAPERJ for the financial support.

5. References

Abrahamsson, B., Pal, A., Sjoberg, M., Carlsson, M., Laurell, E., & Brasseur, J.G. (2005). A novel in vitro and numerical analysis of shear-induced drug release from extendedrelease tablets in the fed stomach. Pharmaceutical Research, 22, 1215-1226. Achouri, A., Zamani, Y., & Boye, J. I. (2012). Stability and Physical Properties of Emulsions Prepared with and without Soy Proteins. Journal of Food Research, 1, 254-267. 22

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Ajila, C. M., Jaganmohan Rao, L., & Prasada Rao, U. J. S. (2010) Characterization of bioactive compounds from raw and ripe Mangifera indica L. peel extracts. Food and Chemical Toxicology, 48(12), 3406-3411. Arab-Tehrany, E., Jacquot, M., Gaiani, C., Imran, M., Desobry, S., & Linder, M. (2012).

PT

Beneficial effects and oxidative stability of omega-3 long-chain polyunsaturated fatty acids. Trends in Food Science & Technology, 25, 24-33.

RI

Augustin, M. A., Sanguansri, L.., Rusli, J. K., Shen, Z., Cheng, L. J., Keogh, J. & Clifton, P.

SC

(2014) Digestion of microencapsulated oil powders: in vitro lipolysis and in vivo absorption from a food matrix. Food & Function, 5, 2905-2912.

NU

Barison, A., Silva, C. W. P., Campos, F. R., Simonelli, F., Lenz, C. A., & Ferreira, A. G.

MA

(2010). A simple methodology for the determination of fatty acid composition in edible oils through 1H NMR spectroscopy. Magnetic Resonance in Chemistry, 48,

D

642-650.

TE

Carneiro, H. C. F., Tonon, R. V., Grosso, C. R. F., & Hubinger, M. D. (2013). Encapsulation efficiency and oxidative stability of flaxseed oil microencapsulated by spray drying

AC C

443–451.

EP

using different combinations of wall materials. Journal of Food Engineering 115,

Dickinson, E. (2009). Hydrocolloids as emulsifiers and emulsion stabilizers. Food Hydrocolloids, 23 (6), 1473-1482. Donsì, F., Annunziata, M., Sessa, M., & Ferrari, G. (2011) Nanoencapsulation of essential oils to enhance their antimicrobial activity in foods. LWT - Food Science and Technology, 44(9), 1908-1914. Fanali, C., Dugo, L., Cacciola, F., Beccaria, M., Grasso, S., Dachà, M., Dugo, P., & Mondello, L. (2011). Chemical Characterization of Sacha Inchi (Plukenetia volubilis L.) Oil. Journal of Agricultural of Food Chemistry, 59(4), 13043–13049. 23

ACCEPTED MANUSCRIPT

Fang, G., Goh, J. Y., Tay, M., Lau, H. F., & Li, S. F. Y. (2013). Characterization of oils and fats by 1H NMR and GC/MS fingerprinting: Classification, prediction and detection of adulteration. Food Chemistry, 138, 1461–1469. Fioramonti, S. A., Martinez, M. J., Pilosof, A. M. R., Rubiolo, A. C., & Santiago, L. G.

PT

(2015). Multilayer emulsions as a strategy for linseed oil microencapsulation: Effect of pH and alginate concentration. Food Hydrocolloids, 43, 8-17.

RI

Follegatti-Romero, L. A., Piantino, C. R., Grimaldi, R., & Cabral, F. A. (2009). Supercritical

SC

CO2 extraction of omega-3 rich oil from Sacha inchi (Plukenetia volubilis L.) seeds. Journal of Supercritical Fluids, 49(3), 323–329.

NU

Gaonkar, A., Vasisht, N., Khare, A. & Sobel, R. (2014). Microencapsulation in the Food

MA

Industry: A Practical Implementation Guide. San Diego: Academic Press. Guillén, M. D., Ruiz, A., Cabo, N., Chirinos, R., & Pascual, G. (2003). Characterization of

D

Sacha Inchi (Plukenetia volubilis L.) oil by FTIR spectroscopy and 1H NMR.

TE

Comparison with linseed oil. Journal of the American Oil Chemists Society, 80(8), 755–762.

EP

Guillén, M. D. & Uriarte, P. S. (2012) Study by 1H NMR spectroscopy of the evolution of

AC C

extra virgin olive oil composition submitted to frying temperature in an industrial fryer for a prolonged period of time. Food Chemistry, 134, 162–172. Hosseini, S. F., Zandi, M., Rezaei, M., & Farahmandghavi, F. (2013). Two-step method for encapsulation of oregano essential oil in chitosan nanoparticle: Preparation, characterization and in vitro release study. Carbohydrate Polymers, 95, 50-56. Hur, S. J., Lim, B. O.; Decker, E. A., & McClements, D. J. (2011) In vitro human digestion models for food applications. Food Chemistry, 125(1), 1-12. Kakkar, P., Verna, S., Manjubala, I., & Madhan, B. (2014). Development of keratin-chitosangelatin composite scaffold for soft tissue engineering. Materials Science and 24

ACCEPTED MANUSCRIPT

Engineering C, 45, 343-347. Karaca, A. C., Nickerson, M. & Low, N. H. (2013). Microcapsule production employing chickpea or lentil protein isolates and maltodextrin: Physicochemical properties and oxidative protection of encapsulated flaxseed oil. Food Chemistry, 139, 448–457.

PT

Karupaiah, T. & Sundram, K. (2007) Effects of stereospecific positioning of fatty acids in triacylglycerol structures in native and randomized fats: a review of their nutritional

RI

implications, Nutrition & Metabolism, 4 (16), 1-17.

SC

Kozu, H.; Uemura, K.; Nakata, Y.; Sato, S.; Nakajima, M.; Kobayashi, I.; Neves, M. A. & Ichikawa, S. (2014) Development of a Human Gastric Digestion Simulator Equipped

NU

with Peristalsis Function for the Direct Observation and Analysis of the Food

MA

Digestion Process. Food Science and Technology Research, 20 (2), 225-233. Maurer, N. E., Hatta-Sakoda, B., Pascual-Chagman, G., & Rodriguez-Saona, L. E. (2012).

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Characterization and authentication of a novel vegetable source of omega-3 fatty

TE

acids, sacha inchi (Plukenetia volubilis L.) oil. Food Chemistry, 134(2), 1173-1180. McClements, D. J. (2004). Food emulsions: principles, practices and techniques. (2nd ed.).

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Boca Raton: CRC press.

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McClements, D. J., & Decker, E. A., & Weiss, J. (2007) Emulsion-based delivery systems for lipophilic bioactive components. Journal of Food Science, 72 (8), 109–124. McClements, D.J. & Li, Y. (2010) Review of in vitro digestion models for rapid screening of emulsion-based systems. Food & Function, 1, 32-59. McClements, D. J. (2015) Nanoparticle- and Microparticle-Based Delivery Systems. (1st ed.). Boca Raton: CRC Press. Moore, D. S. (2007) The Basic Practice of Statistics. (4th ed.). New York: W. H. Freeman and Company, 2007. Pickup, D. M., Newport, R. J., Barney, E. R., Kim, J.-Y., Valappil, S. P., & Knowles, J. C. 25

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(2014). Characterization of phosphate coacervates for potential biomedical applications. Journal of Biomaterials Applications, 28(8), 1226-1234. Rosa, C. G., Borges, C. D., Zambiazi, R. C., Nunes, M. R., Benvenutti, E. V., Luz, S. R., D’avila, R. F & Rutz, J. K. (2013). Microencapsulation of gallic acid in chitosan, β-

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cyclodextrin and xanthan. Industrial Crops and Products, 46, 138-146. Schwizer, W., Steingoetter, A., & Fox, M., (2006) Magnetic resonance imaging for the

RI

assessment of gastrointestinal function. Scandinavian Journal of Gastroenterology,

SC

41, 1245-1260.

Souza, C. J. F., & Garcia-Rojas, E. E. (2015). Effect of salt and protein concentration on the

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association and dissociation of ovalbumin-pectin complexes. Food Hydrocolloids, 47,

MA

124-129.

Verrijssen, T. A. J.; Verkempinck, S. H. E.; Christiaens, S.; Loey, A. M. V. & Hendrickx, M.

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E. (2015) The effect of pectin on in vitro β-carotene bioaccessibility and lipid

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digestion in low fat emulsions. Food Hydrocolloids, 49, 73-81. Vicente, J., Carvalho, M. G. D., & Garcia-Rojas, E. E. (2015) Fatty acids profile of Sacha

EP

Inchi oil and blends by 1H NMR and GC–FID. Food Chemistry, 181, 215-221.

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Zeeb, B.; Weiss, J. & McClements, D. J. (2015) Electrostatic modulation and enzymatic cross-linking of interfacial layers impacts gastrointestinal fate of multilayer emulsions. Food Chemistry 180, 257–264. Zhang, R.; Zhang, Z.; Zhang, H.; Decker, E. A. & McClements, D. J. (2015) Influence of lipid type on gastrointestinal fate of oil-in-water emulsions: In vitro digestion study. Food Research International, 75, 71–78.

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Figures

Fig. 1. Schematic presentation of human gastric digestion (HGS) used to experiments.

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Fig. 2. Thermograms of (a) TGA and (b) DTG (derivate of TGA curve) of SIO, biopolymers

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and encapsulated formed by SIO and biopolymers.

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Fig. 3. FTIR spectra of (a) encapsulated formed by SIO and biopolymers and (b) of SIO, Ova, Pec and XG used to produce of encapsulated, (c) XDR patterns of each component (SIO, Ova,

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Pec and XG) and microcapsules formed (SIO-Ova-Pec and SIO-Ova-XG) and (d) 1H NMR

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spectra of encapsulated formed by SIO and biopolymers.

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Fig. 4. SEM images of (a) XG, (b) Pec, (c) Ova, (d) SIO-Ova, (e) SIO-Ova-1% Pec and (f)

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SIO-Ova-1% XG.

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Fig. 5. 1H NMR spectra of (a) SIO, (b) S-O, (c) S-O-Pec, (d) S-O-XG over 180 min (e)

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linolenic acid (omega-3) structure and (f) spatial positions of the protons in the triglyceride structure.

27

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Fig. 1.

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Fig. 2.

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29

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Fig. 3.

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Fig. 4.

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31

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32

SIO

% Omega-3

S-O-XG

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30

S-O-Pec

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50 40

S-O-Cont

g

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60

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20 10 0

0

20

40

60

80 100 Time (min)

120

140

160

180

Fig. 5.

33

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Table

Table 1. Composition, oil content (%), loaded oil content (equation 1) and efficiency of

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microencapsulated (equation 2) studied.

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SIO (%)

Ova (%)

XG (%)

Pec (%)

Oil content (%)*

Control

8.0

0.5

0.0

0.0

94.1

89.8

±

0.5

95.4

±

0.5

S.1.1

8.0

0.5

0.25

0.0

91.4

84.5

±

0.9

92.4

±

0.9

S1.2

8.0

0.5

0.5

0.0

88.9

79.0

±

0.4

88.9

±

0.5

S1.3

8.0

0.5

1.0

0.0

84.2

75.0

±

0.3

89.1

±

0.3

S1.4

8.0

0.5

0.0

1.0

84.2

78.9

±

0.3

93.7

±

0.3

S1.5

8.0

0.5

0.0

2.0

76.2

69.3

±

0.2

90.9

±

0.2

S1.6

8.0

0.5

0.0

3.0

69.6

62.1

±

0.4

89.2

±

0.5

8.0

1.0

0.0

0.0

88.9

85.8

±

0.2

96.6

±

0.2

S2.1

8.0

1.0

0.25

0.0

86.5

78.9

±

S2.2

8.0

1.0

0.5

0.0

84.2

S2.3

8.0

1.0

1.0

0.0

80.0

S2.4

8.0

1.0

0.0

1.0

80.0

S2.5

8.0

1.0

0.0

2.0

S2.6

8.0

1.0

0.0

3.0

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91.2

±

0.5

±

0.3

88.2

±

0.3

69.4

±

0.5

86.8

±

0.7

74.8

±

0.3

93.5

±

0.3

72.7

65.7

±

0.3

90.4

±

0.4

66.7

59.2

±

0.5

88.8

±

0.7

84.2

±

0.3

94.2

±

0.4

82.1

73.9

±

0.6

90.1

±

0.7

80.0

75.0

±

0.6

93.7

±

0.7

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0.5

74.2

0.0

76.2

70.3

±

0.9

92.3

±

1.2

1.0

76.2

69.8

±

0.2

91.7

±

0.2

2.0

69.6

61.6

±

0.5

88.6

±

0.7

3.0

64.0

56.9

±

0.3

89.0

±

0.5

0.0

80.0

76.4

±

0.3

95.5

±

0.4

0.25

0.0

78.0

72.1

±

0.2

92.3

±

0.3

2.0

0.5

0.0

76.2

68.9

±

0.5

90.4

±

0.6

2.0

1.0

0.0

72.7

64.0

±

0.4

88.0

±

0.5

2.0

0.0

1.0

72.7

63.8

±

0.4

87.8

±

0.6

2.0

0.0

2.0

66.7

61.8

±

0.5

92.7

±

0.8

2.0

0.0

3.0

61.5

57.0

±

0.6

92.6

±

0.9

0.0

0.25

0.0

S3.2

8.0

1.5

0.5

0.0

S3.3

8.0

1.5

1.0

S3.4

8.0

1.5

0.0

S3.5

8.0

1.5

0.0

S3.6

8.0

1.5

0.0

8.0

2.0

0.0

S4.1

8.0

2.0

S4.2

8.0

S4.3

8.0

S4.4

8.0

S4.5

8.0

S4.6

8.0

AC C

MA

0.0

1.5

TE

1.5

8.0

System 4 S4-control

ME (%)

79.4

8.0

S3.1

System 3 S3-control

OC (%)

D

System 2 S2-control

EP

System 1

Code

RI

ACCEPTED MANUSCRIPT

* Oil content (is the theorical value) after freeze-drying; OC: Loaded oil content (%) is the oil content (equation 1; ME: Efficiency is the percentage of oil loaded by oil content (equation 2).

Table 1.

35

SC

RI

PT

ACCEPTED MANUSCRIPT

AC C

EP

TE

D

MA

NU

Graphical abstract

36

ACCEPTED MANUSCRIPT

Highlights

Evaluation of linolenic acid content after in vitro human digestion simulation.

PT

Microencapsulation conferred an increased structural organization.

RI

Encapsulation of SIO was assured during the HGS

SC

Sacha Inchi oil encapsulated useful to transport nutraceutical components.

AC C

EP

TE

D

MA

NU

Digestion priority of triglycerides in sn-2 position.

37