Cryodos 80

Gelzan CM (Sigma, St. Louis, MO, USA) was dissolved in distilled water at 90 °C for 30 min. with stirring. Lactoferrin (Lf, Bovine origin, New Zealand) was dissolved in distilled water. Subsequently, to the solution of Gellan Gum (GG), Lactoferrin (Lf), and Hydroxyapatite (HAp, Plasma Biotal, Buxton, UK) were added at 50 °C, as described in Table 1. After complete homogenization, the crosslinking solution CaCl₂ (Sigma, USA) was added and it was allowed to stabilize at room temperature for 30 min. Posteriorly, the hydrogels were cut in discs of 5 mm of diameter and 2 mm of thickness and replenished with phosphate-buffered saline solution (PBS, Sigma, USA) for 30 h to enable complete crosslinking, frozen at −80 °C for 18–20 h, and then freeze-dried (CryoDos -80, Telstar, Terrassa, Barcelona, Spain) for at least three days. Materials were sterilized by ethylene oxide.

The UAE was carried out using an ultrasonic device (Sonic Vibracell, model VC 750, Newtown, CT, USA), comprising a 13 mm diameter tip with amplitude, temperature and time controller. The amplitude employed was 50%. The powdered samples (5 g) were extracted with 100 mL of water into the ultrasonic device at different times and temperatures, as defined by the RSM design. After ultrasonic extraction, the extracts were filtered through Whatman n° 1 paper, centrifuged (Sigma 3-30KS, Sigma, Osterode am Harz, Germany) at 16,000× g for 10 min and frozen at –80 °C for subsequent lyophilization (Telstar, model Cryodos –80, Barcelona, Spain). Samples were stored at 4 °C until analysis. For the subsequent analyses, the final residue was dissolved in water.

The UAE was carried out in an ultrasonic probe processor (Sonic Vibracell, model VCX50, Newtown, CT, USA) associated with a probe tip No. 630-0219 with 13 mm of diameter. Water was used as an extractor solvent and the experiments were carried out according to the RSM design (Section 2.4). After ultrasonic extraction, the extracts were filtered through Whatman n° 1 paper and frozen at −80 °C for subsequent lyophilization (Telstar, model Cryodos–80, Barcelona, Spain). Then, samples were stored at room temperature until further analysis.

The petals of one hundred twenty-five fresh flowers from 52 different families and 102 species were collected from a botanical garden (Real Jardín Botánico de Córdoba, Córdoba, Spain) and local greenhouses in Madrid and Seville (Spain). These places ensure the traceability in the growth of the floral species by providing identification. After measuring the color of the petals, the samples were freeze-dried (Cryodos-80, Telstar, Terrasa, Spain) and the humidity was calculated.

The preparation of the multicomponent bionanocomposites is shown in Figure 1. Two sets of aqueous mixtures of chitosan (CHI) and different proportions (Table 2) of SEP/HNTs/GNPs/MWCNTs were prepared at overall concentrations of 0.2% w/v and 8% w/v, respectively. First, the appropriate amounts of both nanoclays and GNPs/MWCNTs were dispersed in bi-distilled water and exposed to pulsed ultrasonic waves (VC750 Sonics Vibra-Cell, operating at 20 kHz) using a 13 mm standard probe. Separately, chitosan was slowly dissolved in an aqueous solution of 1% v/v acetic acid at 70 °C and added to the SEP/HNTs/GNPs/MWCNTs dispersion under magnetic stirring.
The bionanocomposite films were processed by solvent-casting from the 0.2% w/v dispersion on polyester Petri dishes and dried at 30 °C and 60% relative humidity (RH) in a CLIMACELL EVO Stability Chamber (Incubator model 111L).
The bionanocomposite foams were prepared by freeze-drying (Cryodos-80, Telstar) of the 8% w/v dispersion, which was cast in cylindrical plastic containers and plunged in liquid nitrogen.

The extraction of S. ramosissima was conducted in a home-made subcritical batch-type extractor of 1.7 L, according to Švarc-Gajić et al. [20 (link)]. In order to determine the most efficient temperature to extract antioxidants from S. ramosissima, different temperatures (ranging from 110 °C to 180 °C) were tested, during a 30 min period, applying a pressure of 20 bars and maintaining a sample-to-solvent ratio of 1:20. The movements of the vibrational platform (3 Hz) were supported by housing the extraction vessel during the extraction process. Afterwards, a flow-through water bath (20 ± 2 °C) was used for cooling the extraction vessel, followed by depressurization through the valve opening. Afterwards, the extracts were filtered through Whatman n° 1 paper and frozen at −80 °C. Subsequently, the extracts were subjected to lyophilization (Telstar, model Cryodos −80, Barcelona, Spain) and stored at 4 °C. For the further experiments, the final residue was dissolved in distilled water. The extraction yield corresponding to the recovery of antioxidants was determined for each extract, resulting as the ratio between the total weight of the lyophilized extract and the total weight of the extract obtained after the SWE.