2.4.2. Structural Analysis

Functional chemical group analysis of the samples was performed through spectrometer equipment Bruker Alpha (Ettlingen, Karlsruhe, Germany) with transmission spectra accessory mode. Concentrated açaí extract was previously lyophilized for further analysis (AÇEXT). Electrosprayed zein particles without açaí(ZNe) were also analyzed to study possible chemical interactions between both components. Pellets with samples and potassium bromide were prepared by pressure and the spectra were obtained in a range from 4000 to 400 cm<sup>−</sup><sup>1</sup> with a resolution of 2 cm<sup>−</sup><sup>1</sup> and 64 scans.

#### 2.4.3. Phenolic Loading Capacity (LC) and Encapsulation E fficiency (EE)

Loading capacity (%LC) was measured as the mass ratio between the total polyphenols content (PT) determined in the ZN/AÇEXT capsules and the theoretical phenolic content incorporated during the preparation of the capsules. 0.015 g of ZN/AÇEXT was dissolved with 1.2 mL of 80% ethanol for PT measurement. Samples were filtered through a 0.22 μm filter and the supernatant was analyzed following the Folin–Ciocalteu method described previously. It is worth mentioning that zein was also analyzed and did not present phenolic content interference. The encapsulation e fficiency (%EE) was determined following the procedure of Idham, Muhamad & Sarmidi (2012) with some modifications [33]. This test consisted of the determination of total polyphenols (PT) and surface polyphenols (PS) of the encapsulated extract. 0.015 g of ZN/AÇEXT was weighed and mixed with 1.2 mL of distilled water in an Eppendorf tube to measure PS. On the other hand, 0.015 g of ZN/AÇEXT was mixed with 1.2 mL of 80% ethanol for PT measurement. In both cases, the samples were vortexed for 30 s. Finally, each sample was filtered through a 0.22 μm filter and the supernatants were analyzed following the Folin–Ciocalteu method described previously. %EE was calculated following Equation (1):

$$\% \text{EE} = (\text{PT} - \text{PS}) \cdot \text{PT}^{-1} \cdot 100 \tag{1}$$

#### *2.5. Thermal Properties of Zein–Açaí Capsules*

#### 2.5.1. Thermal Stability Test

Thermogravimetric analyses (TGA) of açaí fruit (AÇ), lyophilized hydroalcoholic açaí extract (AÇEXT), and encapsulated (ZN/AÇEXT) were carried out using a Mettler Toledo Gas Controller GC20 Stare System (Schwerzenbach, Switzerland) TGA/DSC. Samples were heated from 30 to 600 ◦C at 10 ◦C/min under nitrogen atmosphere.

#### 2.5.2. Stability of Total Phenolic Content of AÇ by Encapsulation

Phenolic content of AÇ, AÇEXT and ZN/AÇEXT samples were determined before (at room temperature) and subsequently exposed to high-temperature conditions. These parameters were selected in order to simulate common heat-treatment processes that these active capsules could suffer when being used as nutraceuticals incorporated into food: (A) sterilization process: autoclaving at 121 ◦C and 15 psi for 15 min; and (B) baked process: 180–185 ◦C for 25 min.

#### *2.6. In Vitro Digestion*

Dispersions of açaí (AÇ), lyophilized hydroalcoholic açaí extract (AÇEXT), and encapsulated (ZN/AÇEXT) were subjected to in vitro digestibility assays to evaluate their bioaccesibilities through the analysis of phenolic content after a gastric and intestinal phase. In vitro gastric and intestinal phases were prepared based on the consensus of the protocol for simulating static digestion method described by the COST Action InfoGest [34]. Simulated gastric fluid (SGF) consisted of a stock gastric solution (6.9 mM KCl, 0.9 mM KH2PO4, 25 nm NaHCO3, 47.2 mM NaCl, 0.12 mM MgCl2\*6H2O, 0.5 mM 2NH4CO3), water, 0.3 M CaCl2, 1.0 M HCl, lecithin, and pepsin. At this stage, the different dispersions were mixture with SGF in a 1:1 proportion and incubated at 37 ◦C for 90 min with continuous agitation at 200 rpm, where pH of the mixture was monitored and controlled to a value of 2 through the addition of 0.5 M HCl, and by using an automatic titrator (902 Titrando, Metrohm, USA). Simulated intestinal fluid (SIF) consisted of a stock gastric solution (6.8 mM KCl, 0.8 mM KH2PO4, 85 nM NaHCO3, 38.4 mM NaCl, 0.33 mM MgCl2.6H2O), water, 0.3 M CaCl2, 1.0 M NaOH, bile, pancreatin, and lipase. At this stage, the mixtures obtained from the gastric phase were mixed with SIF in a 1:1 proportion and incubated at 37 ◦C for 120 min with continuous agitation at 200 rpm. During this second stage, pH was monitored and maintained at pH 7 by adding 0.5 M NaOH. The resulting aliquots after each stage of in vitro digestion were collected and frozen at −18 ◦C, and phenolic content were evaluated following the Folin–Ciocalteu method. All experiments were carried out in duplicate.
