*2.1. Materials*

High polymerization degree Agave fructans (HDPAF) were purchased from Campos Azules Co., (Ciudad de Mexico, Mexico). TEGO SML (sorbitan fatty acid ester) was purchased from Evonik Inc., (Essen, Germany). HPLC grade methanol, absolute ethanol and β-carotene were purchased from Sigma-Aldrich (Steinheim, Germany). Deionized water was used throughout the study.

#### *2.2. Preparation of Formulation*

In order to demonstrate the ability of HDPAF to form nanocapsules, different solutions and emulsions were prepared. Solutions contained different concentrations of HDPAF (5%, 10%, 20%, 30%, 40% and 50% *w*/*w*), TEGO SML (1%) as a surfactant and a hydroalcoholic solution (water–ethanol, 9:1) as a solvent. They were prepared under magnetic stirring at 350 RPM for 5 min (Agimatic-S model 7000242). Oil in water emulsions (O/W) were formulated at a ratio of 10:90. The continuous phase consisted of different HDPAF concentrations (4%, 9%, 19%, 29%, 39% and 49%) dissolved in the hydroalcoholic solution (water–ethanol, 9:1). The dispersed phase consisted of extra virgin olive oil and was used without further processing. TEGO SML (5% of total emulsion volume) was used as a surfactant to aid the emulsion stability and decrease surface tension. The two phases were first mixed in a high-shear mixer at 16,800 RPM for 2 min (Ultra Turrax T25, IKA, Staufen, Germany) in order to prepare the pre-emulsion. The emulsion process was carried out with an ultrasonic homogenizer model

Sonopuls 2200 (Bandelin Electronic Gmbh & Co., Berlin, Germany) at 20 kHz for 1 min, according to Paximada et al. [16]. The temperature was maintained at 25 ± 1 ◦C using an ice bath.

The ability to electrospray the solutions and emulsions was evaluated, parameters (flow-rate (30–50 μL/h), voltage (10–20 KV) and the tip-to-collector distance (10–25 cm)) were varied one at a time until the Taylor's cone was visible, and then particle morphology and size were analyzed to select the most adequate solution and emulsion for the photoprotection study.

For solutions containing β-carotene, β-carotene (0.1%) was incorporated in ethanol and then mixed with the solution containing water, TEGO SML and HDPAF. The mixtures were homogenized under continuous stirring at 350 RPM for 30 min. For the emulsions, β-carotene (1%) was previously incorporated in dichloromethane (1 mL) and was gradually added to the olive oil. When the oily phase was saturated with β-carotene, dichloromethane was separated for 24 h by natural evaporation in the extraction chamber, and then the mix was incorporated into the ethanol. The oily phase was added to the solution containing water, TEGO SML and HDPAF, following the same emulsion preparation procedure previously stated.

#### *2.3. Characterization of Different Solutions and Emulsions*

The apparent viscosity (η) was determined using a rotational viscosimeter Visco Basic Plus L from Fungilab S.A. (San Feliu de Llobregat, Spain) with a Low Viscosity Adapter (LCP). The LCP spindle was placed in the runner bar of the viscometer and 50 mL of sample was placed in a Falcon tube and put in contact with the spindle to obtain the η value. The surface tension was measured using the Wilhemy plate method in an EasyDyne K20 tensiometer (Krüss GmbH, Hamburg, Germany). Twenty-five milliliters of sample was placed in a vessel, then the Wilhemy plate is burned and suspended from the pendulum; the vessel is then placed on the platform to be analyzed. The conductivity was measured using a conductivity meter XS Con6 (Labbox, Barcelona, Spain). The probe was submerged in 10 mL of sample in a Falcon tube until the sensors were covered and stabilized. All measurements were made at 25 ◦C in triplicate.

#### *2.4. Preparation of Capsules by Electrospraying*

The electrospraying apparatus, equipped with a variable high-voltage 0–30 kV power supply, was a Fluidnatek® LE-10 from BioInicia S.L. (Valencia, Spain). Solutions and emulsions with and without β-carotene were introduced in a 12 mL plastic syringe and were electrospun under a steady flow rate using a stainless-steel needle of 700 μm diameter. The needle was connected through a Polytetrafluoroethylene (PTFE) tube to the syringe. The syringe was lying on a digitally controlled syringe pump while the needle was horizontal towards the collector. The electrospraying conditions of the solutions and emulsions for obtaining the capsules were optimized and fixed at 0.1 mL/h of flow-rate, 17 kV of voltage and a tip-to-collector distance of 22 cm. The samples were stored in darkness until analysis.

Additionally, a different encapsulation strategy, named electrospraying coating (EC), patented by Lagaron et al. [17] and reported by Librán et al. [18], was used. The coating was a three-step process carried out at room temperature. In the first step, an initial layer of fructans were electrosprayed over the collector. Secondly, 2% of β-carotene with respect to the solution electrosprayed was spread out over the initial electrosprayed material layer. Finally, a top coating layer of fructans was electrosprayed directly over the material to achieve full encapsulation. The capsules were then collected and mechanically mixed and homogenized. The basic setup of a Fluidnatek ™ LE10 (Bioinicia S.L., Valencia, Spain) was used to conduct the electrospraying process. The collected nanocapsules were stored in a desiccator at 0% relative humidity (RH) and protected from light for subsequent analysis.

#### *2.5. Scanning Electron Microscopy (SEM)*

The morphology and size of the encapsulation structures were examined using SEM on a Hitachi microscope (Hitachi S-4100, Tokyo, Japan) after having been sputtered with a gold–palladium mixture under vacuum for 3 min (SC7640, Polaron, Kent, UK). All SEM experiments were carried out with 1–2 mg of sample at 10 kV, obtaining three micrographs per sample. Capsule diameters were measured by means of the Adobe Photoshop CS3 software from the SEM micrographs in their original magnification.

#### *2.6. Fourier Transform Infrared Spectroscopy*

Attenuated Total Reflectance Fourier Transform Infrared spectroscopy (ATR-FTIR) (Thermo Scientific Nicolet, iS5 iD5, Waltham, USA) was used to evaluate β-carotene, empty HDPAF nanocapsules and nanocapsuled β-carotene. The samples were placed onto the ATR crystal and all the spectra were recorded from 600 cm<sup>−</sup><sup>1</sup> to 4000 cm<sup>−</sup><sup>1</sup> with a resolution of 8 cm<sup>−</sup>1.

#### *2.7. Thermogravimetric Analysis (TGA)*

Thermogravimetric analyses of free β-carotene and HDPAF nanocapsules without and with β-carotene were done in triplicate using TGA 550 equipment (TA Instruments, New Castle, USA) and TRIOS 4.3.0.38388 was the analysis software used. The analyses were conducted under the following conditions: 3–6 mg of sample, heating from 25 ◦C to 500 ◦C, at a heating rate of 5 ◦C/min under nitrogen flow.

#### *2.8. Ultraviolet (UV) Photostability*

With the aim of accelerating the oxidation of β-carotene and simulating the radiation of natural sunlight, an Osram Ultra-Vitalux (300 W) lamp (OSRAM, Múnich, Germany) was used. This blend of radiation is generated by a quartz discharge tube and a tungsten filament [19]. Nanocapsules with β-carotene and free β-carotene were exposed to the UV radiation (13.6 W) at 37 ◦C. After irradiation at different times (0 h, 6 h, 12 h, 24 h and 48 h), extraction of β-carotene from 2.5 mg of nanocapsules was carried out. The polymeric capsule wall was opened with water (1 mL) under magnetic stirring (200 RPM, 1 min). β-carotene was extracted from the mixture by adding 0.75 mL of chloroform and separated by centrifugation (10,000 RPM, 1 min). An aliquot of the organic phase was taken and the absorbance was measured at 466 nm in a spectrophotometer (Spectrophotometer UV/VIS4000, DINKO instruments, Barcelona, Spain). Chloroform was used as a blank. Oxidation was reported as a function of the relative β-carotene content (% absorbance). Analyses were made in triplicate.

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