*2.7. Statistical Analysis*

One-way analyses of variance were carried out. The software SPSS version 11.5 (SPSS Inc., Chicago, IL, USA) was used. Differences in pairs of mean values were evaluated by the Tukey b-test at a confidence interval of 95%. Data were represented as the average ± standard deviation.

#### **3. Results and Discussion**

#### *3.1. Evaluation of Polyphenolic Content and Antioxidant Capacity of Açaí Extracts*

Total phenolic content (TPC) and antioxidant capacities of açaí fruit extracts measured through Folin–Ciocalteu, TEAC, DPPH, and FRAP methods are listed in Table 1. The extraction of natural active compounds was highly influenced by the solubility of these compounds in the extractive solvent. In this case, hydroalcoholic extraction achieved the greatest performance by extracting the highest amount of phenolic and antioxidant compounds with different polarities related to both ethanol and water [35]. A better solvation of the compounds, as a result of the hydrogen bond interactions between the polar sites of the antioxidant molecules and both solvents, in comparison to whether each solvent is used separately for extraction, has been demonstrated [36]


**Table 1.** Polyphenolic content and antioxidant capacities results of açaí fruit extracts.

Letters a–c indicate significant differences among the extracts of the same method.

The Folin–Ciocalteu method is broadly used to measure the content of total phenolic compounds in plant products. It is based on the fact that the phenolic compounds react with the Folin–Ciocalteu reagen<sup>t</sup> at basic pH, giving rise to a blue coloration that can be easily spectrophotometrically determined [37]. The resulting TPC values indicated that the type of solvent directly affected the total number polyphenols extracted. Other works have shown lower TPC values for açaí fruit pulp extracts, e.g., 31.7 ± 0.6 and 26.7 ± 0.5 mg GAE g<sup>−</sup><sup>1</sup> açaí when extractions were carried out by using 1% acetic acid aqueous solution and a solvent mixture acetone/water 70:30, respectively [36,38]. Açaí fruit has been revealed to be the fruit with the highest total polyphenolic content, followed by murtilla, calafate, and maqui, whose TPC values were 34.9, 33.9, and 31.2 mg GAE g<sup>−</sup>1, respectively, according to the antioxidant database directed by Speisky et al. [39]. Nevertheless, a reliable comparison of TPC values between studies is very difficult to achieve because the lack of standardization of this assay and the extraction conditions can imply several orders of significant difference in detected phenols [30].

The highest phenolic content and antioxidant capacity of açaí fruit pulp was obtained for the Aç2 extract, except for the TEAC assay, where the hydroalcoholic extract Aç2 and aqueous Aç3 values did not present significant differences. This fact was due to the scavenging capacity of the compounds that were extracted and their affinity with the radical. This assay demonstrated that radical ABTS<sup>+</sup>• presented grea<sup>t</sup> affinity for hydrophilic systems [40], principally aqueous systems, and very poor affinity for ethanolic extraction. The antioxidant capacity of extracts is the expression of the different phenolic components, which behave through different mechanisms of interactions with oxidative species. Therefore, it is necessary to perform more than one antioxidant method to reflect both lipophilic and hydrophilic capacities. TEAC assay is based on the generation of the cationic radical ABTS<sup>+</sup>• with blue–greenish coloration, which is applicable to both hydrophilic antioxidant systems as to the lipophilic ones, while the DPPH assay uses the radical DPPH• dissolved in organic medium such as ethanol and, therefore, is more applicable to hydrophobic systems [41]. Thus, in agreemen<sup>t</sup> with Floegel et al. (2001), which compared both antioxidant methods by measuring antioxidant activities of different groups of fruit, vegetables, and beverages, TEAC results were higher than DPPH values [42]. On the other hand, FRAP assay revealed that Aç2 presented the highest antioxidant activity measured through SET. Schauss et al. (2006) also demonstrated that antioxidant activity of this fruit measured through ORAC method resulted in the highest reported scavenging activity values for a fruit or berry [43].

#### *3.2. Characterization of the Zein Extract Solutions*

Zein concentration of electrospinning solutions was one of the main parameters that determined the morphology of the fibers or capsules because they directly affected the viscosity and the conductivity values. Table 2 shows viscosity and conductivity results of zein solutions. As expected, viscosity significantly increased as the concentration of zein increased because this parameter is closely related to the polymeric chain entanglement and intercalation [44].

**Table 2.** Viscosity and conductivity of polymer solutions with and without açaí extract used during electrospinning process.


Letters a–c indicate significant differences among the different zein concentration samples. Letters x, y indicate significant differences between sample with and without açaí extract at the same zein concentration.

The incorporation of the açaí extract significantly increased the viscosity owing to the presence of more solutes in the solution, which could increase entanglements of zein protein molecules [45]. When the highest concentration of zein (20% *w*/*v*) was processed, the chains remained entangled enough to resist the electric charges that tend to break the jet during the electrospinning process, resulting in fiber formation. On the other hand, when using 16% (*w*/*v*) zein solution, the electric charge broke the chains, resulting in dispersed droplets whose evaporation originated spherical particles or capsules (see Supplementary Material Figure S1), thereby being the zein concentration selected.

The conductivity values of the solutions slightly decreased with increasing zein concentration and significantly increased when the extract was added. Possibly, this fact could be related to the presence of polyphenols solved in the hydroalcoholic extract that enhanced the conductivity [46]. When conductivity increased, the difference in electric charges between the Taylor cone and the collector plate increased, which promoted capsule development during the formation of droplets [44].

#### *3.3. Morphological Studies of Açaí-Containing Zein Capsules*

Once the zein concentration was fixed, samples S1–S6 were processed through an electrospinning process to obtain particles with grea<sup>t</sup> homogeneity. The size and homogeneity are highly dependent on electrospinning process parameters. The particle size distribution plots and Scanning Electronic Microscopy (SEM) micrographs of the samples at different electrospinning encapsulation conditions are shown in Figure 1. SEM micrographs of the electrosprayed zein–açaí capsules demonstrated particles with cavities and similar shape to those found in previous works [47,48].

Samples S1, S2, S4, and S6 were found to have a smaller average particle size: 882, 924, 899, and 896 nm, respectively, without significant differences between them. According to Duque et al. (2013), when increasing the injection flow, there is less solvent evaporation time, so there can be agglomerations of droplets and an increase in the diameter of particles [44]. This fact occurred with samples S1 and S3, where, by increasing the flow from 0.3 to 0.5 mL <sup>h</sup>−1, the particle average diameter increased from 0.88 to 1.13 μm. On the other hand, the distance between the tip of the capillary and the collector plate also influenced on the homogeneity and morphology of the capsules. The sample with the highest homogeneous particle size, based on the smaller standard deviation, turned out to be sample S2. Hence, the parameters selected were 0.4 mL h−<sup>1</sup> flow rate and 10 cm height.

**Figure 1.** Histograms of electrosprayed zein–açaí structures with size distribution information as average and deviation (μm) and SEM micrographs of samples: S1, S2 and S3) 10 cm height and flow rates 0.3, 0.4, and 0.5 mL <sup>h</sup>−1, respectively; and S4, S5, and S6) 12 cm height and flow rate 0.3, 0.4, and 0.5 mL <sup>h</sup>−1, respectively. Values a, b, and c indicate significant differences between the samples, determined through a one-way analysis of variance (ANOVA) (*p* < 0.05). SD corresponds to the standard deviation.

## *3.4. Structural Characterization*

The infrared spectra of electrosprayed zein particles (ZNe), freeze-dried açaí extract (AÇEXT) and electrosprayed zein-containing açaí (ZN/AÇEXT) are displayed in Figure 2. Zein particles obtained through the electrospinning process presented characteristic peaks of zein protein at 3309 cm<sup>−</sup><sup>1</sup> which represented the N–H stretching of amide A, and peaks at 2966 and 2872 cm<sup>−</sup><sup>1</sup> derived from the stretching of the C–H aliphatic groups. The peak at 1655 cm<sup>−</sup><sup>1</sup> represented the stretching vibration of C=O group from amino acids and the bands at 1543 and 1450 cm<sup>−</sup><sup>1</sup> illustrated the flexion vibration of N–H and vibrational stretching of the C–N peptide bonds, respectively [49,50].

**Figure 2.** FTIR spectra of: electrosprayed zein particles, ZNe (black line); açaí extract, AÇEXT (red line); and electrosprayed zein particles containing açaí, ZN/AÇEXT (green line).

Lyophilized açaí extract spectra exhibited a similar pattern to other berries, such as maqui and murta [51,52]. A broad sign with peak at 3383 cm<sup>−</sup><sup>1</sup> could represent some hydroxyl groups O–H and aliphatic C–H from the polyphenolic compounds. The peak centered at 1615 cm<sup>−</sup><sup>1</sup> was assigned to the C=C vibrations from aromatic systems. The region between 1500 and 1340 cm<sup>−</sup><sup>1</sup> (centered at 1413 cm<sup>−</sup>1) represented the deformation vibrations of phenolic O–H groups. A peak was distinguished in the region of 1150 and 1040 cm<sup>−</sup>1, which was attributed to C–O stretching vibrations [53]. Although ZN/AÇEXT presented similar bands to ZNe spectra, a certain displacement of these peaks was observed. This fact indicated the presence of a certain intermolecular interaction between zein protein and phenolics from açaí fruit extract. Zein protein, which contains mostly non-polar amino acids, favored chemical interactions with phenolic functional groups, increasing the protection of active compounds. Non-covalent hydrophobic interactions and hydrogen bonds were probably the main mechanisms of interaction between zein and polyphenols [54,55].

#### *3.5. Loading Capacity (LC) and Encapsulation E*ffi*ciency (EE)*

The encapsulation efficiency (EE) concept has given rise to different definitions in various works relating to compound encapsulation. In this study, EE was defined as the precise amount of active compounds that was actually protected in the capsule. On the other hand, the performance of the encapsulation process was distinguished as loading capacity (LC). Loading capacity (%LC) resulted in (98.6 ± 1.6)% value, since theoretical phenolic content of ZN/AÇEXT capsules was 2104 mg gallic acid g −1, and PT value after dissolution of ZN/AÇEXT was 2075 mg gallic acid g<sup>−</sup><sup>1</sup> ZN/AÇEXT. This value justified the efficiency of this technology to encapsulate bioactive compounds without compromising its activity. Similar loading capacity values between 85% and 95% were observed to encapsulate other natural extracts by using the electrospinning technique, such as green tea extract in zein and carotenoids from tomato peel extract in gelatin [45,48].

Encapsulation efficiency value is essential to study the number of active compounds trapped in the capsule and the ability of the material to retain them [56]. EE value indicated (72.1 ± 1.7)% of phenolic content from açaí fruit extract was efficiently encapsulated. EE depends to a large extent on the affinity between the polymer matrix and active compounds. During the encapsulation process, the açaí fruit extract as core material was mixed with the substance of zein and the generated droplets were solidified by the evaporation of the ethanol and water [57]. Yao et al. (2016) also indicated that the EE would also be influenced by variations in the morphology that arise in the fibers or capsules due to the concentration of the solution and process conditions. In this study, the result indicated that more than 70% of the extract was efficiently trapped and distributed inside the capsule [58]. A similar value was found in the Flores et al. study of physical and storage properties of cranberry pulp encapsulated in whey protein by spray drying [59]. In addition, it is important to consider the methodology used to determine the efficiency, since EE results depend greatly on the methodology and, principally, the solvents used for the extraction of components.

#### *3.6. Thermal Studies of Zein-Containing Açaí Extract Capsules*

#### 3.6.1. Thermal Stability Test

Figure 3 shows the thermogravimetric curves (TGA) (Figure 3A) and their respective derivatives (DTGA) (Figure 3B) of the samples. An initial stage of weight loss between 30 and 100 ◦C was observed for all the compounds, which indicated a loss of water and some volatile compounds. The thermogram of the electrosprayed zein particles presented a second degradation process between 270 and 450 ◦C, with a peak of maximum degradation at 332.8 ◦C. This second stage is attributed to the main degradation of the protein, causing changes in the structure due to the breakdown of low-energy intermolecular bonds that maintain their conformation [26,27,60]. Lyophilized açaí fruit extract presented an early degradation that started at approximately 100 ◦C, showing a maximum degradation at 162.5 ◦C. This extract was mainly composed of anthocyanins, flavonoids highly sensitive to temperature [16]. The anthocyanins of the extract were totally unprotected and exposed to degradation due to the increase in temperature. When the extract was encapsulated, the thermogram presented a similar degradation profile to zein. The incorporation of açaí fruit extract did not affect the protein stability. The degradation of açaí fruit was not exhibited because zein effectively protected the extract, delaying its degradation.

**Figure 3.** (**A**) Weight loss; and (**B**) derivative of the weight loss of electrosprayed zein (Zne), dried açaí fruit (AÇ), lyophilized açaí fruit extract (AÇEXT), electrosprayed zein capsules containing açaí (ZN/AÇEXT).

The main degradation of dehydrated fruit AÇ occurred at higher temperatures, approximately at 180–190 ◦C, and displayed two peaks of maximum degradation at 320.5 and 406 ◦C. This fact can be possibly explained because the dehydrated fruit contained a food matrix based on husk and pulp that could exert some protection to the active compounds, while the extract is a concentrated sample of antioxidants totally exposed to heating [12,61].

#### 3.6.2. Thermal Protection of Açaí Phenolic Compounds Encapsulated in Zein

The thermal stability of the encapsulated extract was also evaluated by determining the loss of polyphenols when exposed to two thermal treatments of high-temperature processing: sterilization and baking. In addition, the stability of the phenolic content of dehydrated açaí fruit (AÇ) and lyophilized açaí extract (AÇEXT) was also analyzed. Figure 4 shows the loss of the phenolic content after each heat treatment.

In the case of the commercial dehydrated açaí fruit, a phenolic content reduction greater than 40% was displayed after both thermal processes without significant differences between both treatments. In the case of the encapsulated extract, phenolic content loss values were 5% and 20% approx. after sterilization and baking, respectively. Encapsulated açaí phenolic compounds presented a greater stability against both treatments compared to two other samples of açaí fruit, principally during a sterilization simulation process. Otherwise, during the baking process, the amount of phenolic loss correlated with the amount of phenols on the surface (shown in Section 3.5) which were the phenolic content available to degradation. This fact confirmed the protective effect of the encapsulation in zein by the electrospinning technique, which was clearly demonstrated when comparing with AÇEXT phenolic loss. The sterilization process (121 ◦C) was a lesser influence than the baked (180 ◦C) over lyophilized açaí extract, exhibiting phenolic content reductions of 10 and 55%, respectively. Although sterilization did not cause a large degradation rate, in the case of baking, it turned out to be the sample that suffered the highest phenolic decrease, possibly due to higher temperature and longer exposure time. This fact is in agreemen<sup>t</sup> with AÇEXT TGA thermogram (Figure 3B) that indicates AÇEXT present an early degradation that starts at 100 ◦C and the maximum degradation temperature occurs at approximately 160 ◦C. Thus, the sterilization process clearly affects the phenolic content to a lesser extent than baking (180 ◦C), which occurs at a higher temperature than the maximum degradation. Other works have already shown that temperature is a determining factor in the degradation of polyphenols. Pacheco et al. studied the phytochemical composition and thermal stability of two commercial açaí species and concluded that the changes in antioxidant capacity during warming were highly related to the loss of anthocyanins because their polyphenols, such as phenolic acids and flavone glycosides, were not significantly altered [62]. The thermal degradation of anthocyanins can lead to a variety of species depending on the severity and nature of the heating. High temperature causes losses in the glycosidant sugar of the molecules and the opening of the ring, producing the so-called "colorless chalcones".

**Figure 4.** Loss of polyphenolic content (%) of the samples when subjected to thermal treatments. Values a, b, and c indicate significant differences between samples for the same thermal treatment. On the other hand, x and y indicate significant differences between thermal treatments for the same sample, determined through a one-way analysis of variance (ANOVA) (*p* < 0.05).

#### *3.7. In Vitro Bioaccesibility Study*

Phenolic content (PC) of AÇ, AÇEXT, and ZN/AÇEXT were determined after each stage of in vitro digestion (gastric and intestinal stages) to evaluate phenolic content release within gastrointestinal tract (GI) and to assess bioaccessibility at the end of digestion process. Bioaccessibility can be defined as the fraction of a compound that is soluble in the gastrointestinal (GI) tract that is available for absorption, or the fraction of a compound released from its matrix in the GI tract [63]. Therefore, in this study, bioaccessibility was defined as phenolic content recovered from the intestinal phase after in vitro digestion. Table 3 shows phenolic content of gastric and intestinal stages per gram of each sample. Thus, significant differences to these values were because of their intrinsic phenolic content between samples. In general, polyphenols from açaí samples without encapsulation showed a moderate stability under gastric conditions since the phenolic content decreased between 20% and 35% with respect to intrinsic content (Table 3).


**Table 3.** Phenolic content as (mg gallic acid per gram) of açaí samples after in vitro digestive processes.

Letters a, b indicate significant differences among the different digestive stages of a sample. Letters x, y indicate significant differences between samples during the same digestive process.

As with during thermal analyses (Section 3.6.1), pulp matrix from açaí fruit generated some protection to açaí polyphenols during the gastric digestion phase. Other works have also shown flavonoid oligomers were degraded to smaller units at low pH values [64]. These results agreed with Gullón et al. (2015) who observed that the total phenolic recovery from pomegranate peel flour (35.8%) decreased after gastric digestion. Results after intestinal stage demonstrated phenolic content depended on açaí sample. Although AÇ polyphenols degraded to a lesser extent during the gastric phase, the dried fruit pulp showed the greatest PC decrease after intestinal digestion phase, remaining approx. 20% from initial phenolic content. On the other hand, although AÇEXT suffered a higher reduction after gastric phase, the total PC loss was shorter than AÇ, with a PC reduction close to 60% with respect to non-digested samples. In general, these results suggested that several changes in phenolic compounds as a chemical structure modification, reduction of their solubility due to pH, and/or interaction with other compounds might have occurred during the duodenal stage [65,66]. On the other hand, encapsulated zein-containing açaí presented an interesting behavior because phenolic content values increased after both in vitro digestion processes. During the gastric phase, the phenolic components released from the zein capsules was approx. 60% with respect their intrinsic content, and this value increased after the intestinal stage. This fact is probably due to the breakage of the zein structures, thanks to the digestion of protein matrix, allowing the release of polyphenols. Gómez-Mascaraque et al. (2019) also revealed an increase to antioxidant capacity derived from catechin from zein and gelatin electrosprayed systems.
