**1. Introduction**

In the last decade, the market for nutraceuticals has grown enormously due to accentuated interest by consumers in their therapeutic e ffects for health disorders, including neurodegenerative and cardiovascular diseases [1,2]. A broad variety of active compounds from natural sources has been researched. The main active compounds from plants are polyphenols, secondary metabolites that make up a large family of substances, from simple molecules to complex structures [3]. Numerous studies have shown that certain fruit contains high levels of antioxidant active compounds. Specifically, one fruit with grea<sup>t</sup> antioxidant capacity is açaí (*Euterpe oleracea* Mart.), which has recently emerged as a promising source of energy, and nutritional and antimicrobial properties [4,5]. Preventing oxidative stress in human endothelial cells and the therapeutic e ffect on neurodegenerative diseases have emerged as bioactivities related to this fruit [6–9]. Açaí is a palm native to South America that grows mostly in the Amazon estuary, in the north of Brazil. Some studies have also revealed this fruit significantly reduces

the risk of atherosclerosis through antioxidant and anti-inflammatory activities [10,11]. The principal flavonoid responsible for this anti-inflammatory activity is the flavone velutin [12]. Açaí fruit is also composed by high content of polyphenolic compounds, especially anthocyanins, as major components cyanidin-3-glucoside and cyanidin-3-rutinoside, and phenolic acids [13–15]. Açaí is commonly sold as dehydrated powder to be added to food, in dietary supplements or beverages. However, its current applications are limited to certain foods that do not include such high thermal processes as baking and cooking due to the low stability of the polyphenols, principally the anthocyanins. These molecules can be degraded at increased temperatures, leading to the loss of their functional properties [16]. Recently, a kinetic study of anthocyanin's açaí thermal degradation by Costa et col. (2018) revealed their degradations fitted a kinetic model of the first order [17]. On the other hand, it is also well known that for natural bioactive compounds to present real benefits, they must be available for absorption after the process of gastrointestinal digestion [18,19]. Thus, encapsulation of natural bioactive compounds is an interesting alternative for performing a double purpose of extending possibilities of incorporation into broader food matrices and enhancing bioavailability [20].

Although several procedures have been used to encapsulate active compounds, such as spray drying, lyophilization, and emulsification, these techniques present disadvantages, such as the complexity of the equipment, use of high temperatures, non-uniform conditions in the drying chamber and lack of particle size control [21]. In recent years, a technology that has received special attention is electrospinning and/or electrospraying [22]. Research studies have clearly shown electrospraying and electrospinning are techniques with functional advantages such as sustained release property, high encapsulation e fficiency, and enhanced stability of encapsulated food bioactive compounds [22]. This technique consists of spinning polymeric solutions through high electric fields that exceed the forces of surface tension in the solution of charged polymers. At a certain voltage, fine jets of solution are expelled from the capillary to the collector plate. The solvent evaporates and the segments of fibers or particles are deposited randomly on a substrate. Depending on the specific conditions of polymer solution and the equipment, the process can result in a stretched jet or dispersion of droplets [23]. Several bioactive substances have been successfully encapsulated into electrosprayed particles by using a wide variety of natural polymers as encapsulating materials, depending on the compound to be encapsulated [24]. Among the edible materials, carbohydrates, lipids, and protein have gained the most interest. The latter have numerous advantages, such as increasing the bioavailability of the encapsulated compounds and high binding capacity with active compounds [25]. Corn zein protein has been shown to be a protein resistant to temperatures above 200 ◦C and has been used as an encapsulation material for some compounds, such as curcumin, improving stability against di fferent values of pH and ultraviolet (UV) radiation [26,27]. Thus, this work presents the selection of a powerful açaí fruit extract, based on its highest phenolic content and antioxidant activities, to be further encapsulated into electrosprayed zein capsules. Although the limited use of açaí fruit in food formulations is evident due to its thermal instability, few works have developed alternatives to protect its active compounds. These encapsulated structures were morphological and structurally characterized and considered a suitable shell to impart thermal protection and enhance the bioavailability of phenolic compounds.

#### **2. Materials and Methods**

#### *2.1. Test Materials and Reagents*

Freeze-dried and milled organic açaí fruit was obtained from "Healthy Foods". Zein (Z 3625), 2,2-diphenyl-1-picrylhydrazyl (DPPH), 2,2--azinobis(3-ethylbenzothiazoline-6-sulphonate) (ABTS), Folin–Ciocalteu phenol reagent, anhydrous sodium carbonate, gallic acid (GA), ferric 2,4,6-tripyridyls-triazine (TPTZ) and 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox) were obtained from Sigma–Aldrich (Santiago, Chile). Lipase (L3126) and pancreatin (P1750) from porcine pancreas, pepsin (P6887) from porcine gastric mucosa, and porcine bile extract (B8631) were purchased from Sigma–Aldrich (Sigma–Aldrich S.A., USA). NaOH, HCl and di fferent salts to prepare simulated

digestion fluids (KCl, KH2PO4, NaHCO3, NaCl, MgCl2.(H2O)6, (NH4)2CO3, and CaCl2.(H2O)2) were purchased from Merck (Merck KGaA S.A., Darmstadt, Germany).

#### *2.2. Selection of Açaí Fruit Extract*

#### 2.2.1. Preparation of Açaí Fruit Extracts

Active compounds from açaí fruit were extracted using absolute ethanol, ethanol 50%, and distilled water in a 1:300 (solid (g):solvent (mL)) ratio to study the e ffect of the solvent polarity on the extraction capacity of the most relevant antioxidant compounds. The extractions were carried out at 40 ◦C for 3 h with an agitation of 150 rpm. Samples were centrifuged, filtered, and used for the antioxidant assays and the analysis of polyphenolic content (PC). Extracts obtained were named "Aç1, Aç2, Aç3" for extracts under ethanol, ethanol 50% and water, respectively.

#### 2.2.2. Determination of Total Phenolic Content and Antioxidant Activity Studies

Total phenolic content (TPC) of the extracts was determined following the Folin–Ciocalteu method [28]. 100 μL of each extract was mixed with 3100 μL of distilled water and 200 μL of Folin–Ciocalteu reagent. The samples were taken to darkness for 5 min and 600 μL of anhydrous sodium carbonate at 20% (w/v) was added [29]. The samples were shaken and brought back into darkness for 2 h. The absorbance readings were performed at 765 nm. Results were expressed as mg of gallic acid equivalent (mg GAE) g<sup>−</sup><sup>1</sup> of dried açaí.

Antioxidant evaluation of extracts was carried out through three antioxidant assays: Trolox Equivalent Antioxidant Capacity (TEAC), 2,2-diphenyl-1-picrylhydrazil (DPPH), and Ferric Reducing Antioxidant Power (FRAP). All antioxidant results were expressed as mg Trolox g<sup>−</sup><sup>1</sup> dried açaí. Both TEAC and DPPH methods measure the antioxidant power of extracts by the percentage inhibition of ABTS<sup>+</sup>• and DPPH• radicals, respectively, via both single-electron transference (SET) and hydrogen-atom transference (HAT) mechanisms [30]. The cationic radical ABTS<sup>+</sup>• was generated from an oxidation reaction of the ABTS reagen<sup>t</sup> with potassium persulfate incubated in the dark at room temperature for 16 h. ABTS<sup>+</sup>• working solution was obtained by dilution of the concentrated solution until an absorbance value of 1 at 734 nm. 3 mL of working ABTS<sup>+</sup>• radical solution was mixed with 300 μL of each extract and three controls were prepared with the addition of 300 μL of water. DPPH radical-scavenging activity of açaí extracts was evaluated according to the method described by Okada and Okada with some modifications [31,32]. 5 mL of extracts were incubated with 0.5 mL of 6.4 × 10−<sup>4</sup> DPPH solution for 30 min in the dark at room temperature, and absorbance was determined at 517 nm. FRAP assay measures the antioxidant activity through reduction of ferric 2,4,6-tripyridyl-s-triazine (TPTZ) to a colored product via SET mechanism. FRAP reagen<sup>t</sup> was prepared by mixing 25 mL of 0.3 M acetate bu ffer (pH 3.6) with 2.5 mL of 10 mM TPTZ (2,4,6-tripyridyl-s-triazine) and 2.5 mL of 20 mM FeCl3. 2850 μL of FRAP reagen<sup>t</sup> was mixed with 150 μL of each extract and the absorbance was measured at 593 nm after 30 min of reaction at room temperature. The assays were performed in triplicate and results were expressed as mg Trolox/g fruit.

#### *2.3. Encapsulation of Açaí Extract with Highest Phenolic Content*

#### 2.3.1. Determination of Zein–Açaí Extract Solution Properties

The açaí extract to be encapsulated (Aç2) was selected according to the highest concentration of active compounds by means of highest polyphenolic content and antioxidant capacities. Aç2 was obtained following the same procedure as described in Section 2.2.1 and subsequently, this extract was concentrated to a final concentration of 0.4 g dried açaí mL−<sup>1</sup> using a rotary evaporator. This concentrated extract was named AÇCC. Electrospinning solutions were prepared with 2 mL of AÇCC, 8 mL of ethanol and zein was added at di fferent concentrations (16, 18 and 20% *w v*<sup>−</sup>1). The mixtures were gently stirred at room temperature for 1 h until homogeneous solutions were obtained

(ZN16-AÇCC, ZN18-AÇCC, and ZN20-AÇCC, respectively). Additionally, three control solutions of zein using 80% ethanolic solution were prepared at the same concentrations without the extract to study the e ffect of the incorporation of açaí extract on the properties of the polymer solutions (ZN16, ZN18, and ZN20, respectively).

The zein–açaí extract and control zein solutions were characterized by determination of viscosity and conductivity. Viscosity was evaluated using the SC4-18 spindle at a deformation rate of 79.2 s<sup>−</sup>1. In addition, the conductivity was measured using a conductivity meter from 0.01 to 1000 mS cm<sup>−</sup>1. Both studies were performed in triplicate at room temperature.

#### 2.3.2. Electrospinning Process of the Zein–Açaí Extract Solutions

The encapsulation was carried out using electrospinning equipment (Spraybase ®power SupplyUnit, Maynooth, Ireland) with a vertical standard configuration equipped with a capillary connected to a high-voltage source. The technique was carried out at room temperature and 40% relative humidity. Initially, the purpose was to reveal the optimal concentration of zein to obtain electrosprayed capsules. In this process, the capillary was located 10 cm from the collector plate using a voltage of 13 kV. Each zein–açaí extract solution was introduced in a 5 mL syringe, which was expelled by the capillary during the process with an injection flow of 0.15 mL h−1. The first samples were collected on a slide for easy observation by optical microscopy, and to obtain an initial and fast appreciation of the morphology of the electrospun structures. Once the zein concentration was fixed, the electrospinning parameters were studied to be able to fix the best conditions to obtain electrosprayed particles through a homogeneous and stable process. Flow rate and distance between capillary and collector were studied as follows: Samples S1, S2, and S3 with 10 cm distance and flow rates 0.3, 0.4, and 0.5 mL <sup>h</sup>−1, respectively; and samples S4, S5, and S6 with 12 cm as distance and 0.3, 0.4, and 0.5 mL <sup>h</sup>−1, respectively.

#### *2.4. Characterization of Electrosprayed Açaí-Containing Capsules*
