*3.3. β-Carotene Encapsulation*

The encapsulation of the β-carotene supposes a technical challenge due to its high instability to light, its hydrophobicity and its low solubility in common organic solvents [25–27]. The grea<sup>t</sup> interest of the food industry in this compound has motivated researchers to try the encapsulation of β-carotene by several methods. Tan and Nakajima [25] suggested the nanodispersion of β-carotene by the solvent evaporation method. Ribeiro et al. [26] proposed the encapsulation in Polylactide(PLA) and Poly lactic-*co*-glycolic acid (PLGA) by the solvent displacement method and Astete et al. [27] proposed the encapsulation in calcium alginate. However, some of these proposals presented the disadvantage of using organic solvents such as acetone [26], hexane [25] or chloroform [27]. Traces of these solvents would make the encapsulates unsuitable for food applications, and, therefore, it is of grea<sup>t</sup> interest to find a methodology to obtain nanocapsules based on the use of eco-friendly ingredients with a high encapsulation efficiency, which could reach the status of "generally recognized as safe (GRAS)" granted by the FDA.

Our first proposal was to use a solution to encapsulate the β-carotene. Nevertheless, the low solubility of β-carotene in conventional solvents prevented the obtaining of capsules with β-carotene concentrations over 0.1%. The second attempt was to use an emulsion, but for that option, the use of dichloromethane was required. The residual organic solvent concentration in the capsules was evaluated by headspace-solid-phase microextraction–gas chromatography (HS-SPME-GC) according to Camelo-Méndez et al., [28] with some modifications, and no traces of dichloromethane were detected in samples. Despite this good result, the concentration of the β-carotene in the particles was less than 1%. On the other hand, β-carotene encapsulated by the EC method allowed a higher concentration of β-carotene and consequently, the whole study was focused on this option.

#### *3.4. FTIR Analysis of the Encapsulation Structures*

Interactions between β-carotene and HDPAF nanocapsules were evaluated by infrared spectroscopy (FTIR) according to Peinado et al. [29]. The FTIR spectra of β-carotene showed a broad peak at 3411 cm<sup>−</sup><sup>1</sup> that represents the presence of O–H stretching of the hydroxyl group, which is likely due to the interaction of β-carotene with oxygen in the air [30]. The peaks at 2929 cm<sup>−</sup><sup>1</sup> and 2869 cm<sup>−</sup><sup>1</sup> indicate the CH2 asymmetry and symmetry stretching, respectively (Figure 2a). The presence of carbonyl groups and the stretching symmetry of the C–H bond group was evidenced in peaks at 1717 cm<sup>−</sup><sup>1</sup> and 1366 cm<sup>−</sup>1, respectively. The sharp peak at 965 cm<sup>−</sup><sup>1</sup> marks the deformation mode of trans-conjugate alkenes as the specific areas of trans=CH (1 in Figure 2a) used for identification of β-carotene [30,31].

**Figure 2.** FTIR spectra. β-carotene (**a**), HDPAF nanocapsules (**b**) and HDPAF/β-carotene nanocapsules produced by the electrospraying coating (EC) process(**c**).

The FTIR spectra of HDPAF nanocapsules (Figure 2b) showed the most intensive broad band, with the maximum at 1050 cm<sup>−</sup><sup>1</sup> and two shoulders at 940 cm<sup>−</sup><sup>1</sup> and 1130 cm<sup>−</sup>1. The bands in the region 900–1153 cm<sup>−</sup><sup>1</sup> have been assigned to C–O and C–C stretching modes (2 in Figure 2b,c). These bands are characteristic of carbohydrates. Moreover, the two overlapped bands at 2930 cm<sup>−</sup><sup>1</sup> and 2870 cm<sup>−</sup><sup>1</sup> are characteristic of carbohydrates too [32]. The band from 2800 cm<sup>−</sup><sup>1</sup> to 3000 cm<sup>−</sup><sup>1</sup> is similar to the inulin spectra reported by Grube et al. [32] and Apolinário et al. [33]; this band is attributed to C–H stretching. The broad stretching peak around 3492 cm<sup>−</sup><sup>1</sup> indicated the presence of hydroxyl groups (–OH) of carbohydrates [33].

The comparison of nanocapsules of HDPAF and HDPAF/β-carotene obtained by the EC process (Figure 2b,c) proved HDPAF as the dominating component. The main differences in the nanocapsules of HDPAF and HDPAF/β-carotene spectra appeared in the 1700–1800 cm<sup>−</sup><sup>1</sup> region, which indicates the C=O interaction of fructose molecules with β-carotene, presenting as a stretching of the peak (3 in Figure 2c). The low intensity of β-carotene suggests that only a slight amount is located on the surface of the HDPAF nanocapsules [29]. Nanocapsules with a low surface intensity observed by FTIR (Figure 2c) sugges<sup>t</sup> a centripetal distribution of β-carotene, where the highest concentration is in the core of the nanocapsule. Such confinement, likely due to the hydrophobicity of β-carotene, is desired, as it would create a barrier against oxygen and protection against thermal decomposition processes [29].

#### *3.5. Thermal Stability of β-Carotene and HDPAF Nanocapsules*

The purpose of the thermogravimetric analysis was to evaluate the thermal resistance to degradation of β-carotene encapsulated in HDPAF nanocapsules. Thermograms shown, a termal decomposition of pure β-carotene between 150.58 ◦C and 354.16 ◦C (Figure 3a), similar decomposition temperature range (150–450 ◦C) was reported by Busolo and Lagaron (2015) [34]. HDPAF nanocapsules degraded between 205.48 ◦C and 257.70 ◦C (Figure 3b). These differences in stability can be associated to the structure of the molecules, since the HDPAF is a complex mixture of fructooligosaccharides [1,2] and may have functional properties linked to the degree of polymerization.

**Figure 3.** Thermogravimetric profile. β-carotene (**a**), HDPAF nanocapsules (**b**) and HDPAF/β-carotene nanocapsules obtained by EC (**c**). Curve t represents the thermogram and d the thermogram derivate.

β-carotene encapsulated in HDPAF nanocapsules was decomposed between 208.60 ◦C and 255.43 ◦C (Figure 3c). This result supports the thermal protective effect of the HDPAF nanocapsule on β-carotene, similar to that reported by Peinado et al. [29] for the encapsulation of β-carotene in electrospun nanofibers of poly(ethylene oxide). The thermal stability of antioxidants as β-carotene

depends on whether the molecules are totally encapsulated in the nanocapsules or on the surface [34]. Thermal stability of HDPAF/nanocapsules with and without β-carotene did not show a difference (Figure 3). Therefore, HDPAF exerts a protective role against the thermal degradation of β-carotene.

#### *3.6. Ultraviolet (UV) Photostability of Encapsulated β-Carotene*

β-carotene is highly susceptible to photooxidation (oxidation or isomerization) due to the presence of conjugated double bonds in the molecule [19]. The exposure of β-carotene to UV light led to damage in the molecule, producing a decrease in the absorbance (measured at 466 nm) (Figure 4). Degradation of unprotected β-carotene has been also reported by Fernandez et al. [19] and de Freitas Zômpero et al. [21]. However, the β-carotene encapsulated in HDPAF showed a higher stability to UV light even after 48 h of exposure (Figure 4), attributed to the structure of the fructooligosaccharide mixtures.

**Figure 4.** Relative decay in absorbance percentage (% Abs), as a function of exposure time to UV (a) β-carotene and (b) Nanoapsules with HDPAF and β-carotene by EC.

Photoisomerization under UV light exposure is thought to be able to take place in free bioactive compounds, but not very readily in dried particles [19]. <sup>L</sup>ópez-Rubio and Lagaron [35] produced hydrocolloid films (whey protein concentrate, zein, soy protein and gelatin) containing β-carotene which were able to maintain the β-carotene stability even after 50 h of UV light exposure. De Freitas Zômpero et al. [21] reported that a double encapsulation (nanoliposome + polymeric fiber) by electrospinning was useful to guarantee the β-carotene stability during 6 h of UV light exposure. Therefore, the utilization of the HDPAF as an encapsulating material could be a novel option to be used in nanocapsule manufacture to protect active compounds. In this case, the β-carotene loaded in HDPAF presented with similar behaviors when compared with other polymers/hydrocolloids used before [21,35]. However, this behavior was obtained with a low HDPAF concentration in the particle.
