Freezone freeze dryer

First, 160 mg of BSA were dissolved in 40 mL of 0.1 M PBS buffer (pH 8). Afterward, the solutions of FITC in ethanol (5 mg/mL) or TRITC in DMSO (5 mg/mL) were prepared. Then, 40 mL of BSA solution was added to 5 mL of FITC/TRITC solution under gentle stirring and further the mixture was stirred under 4 °C in the dark for 12 h. Finally, freshly prepared FITC-conjugated (or TRITC-conjugated) BSA was dialyzed for 3 days in deionized water then freeze-dried 24 h after the cooling period using FreeZone Freeze Dryers Labconco (Kansas City, MO, USA).

The effect of oral mastication on the microstructure of the black beans was visualized under light microscope as described previously by Gwala et al. [21 (link)]. Briefly, boluses were lyophilised (Labconco FreeZone freeze dryer, Kansas City, MO, USA) and milled to pass through a 425 µm mesh screen. A small amount of the sample powder was dispersed in distilled water on a microscope slide, covered, and then observed under a light microscope (Ceti, Auckland, New Zealand). Micrographs were viewed at 10× magnification and captured using a camera (Medline Scientific, Oxfordshire, UK) attached to the microscope controlled by a camera control software (ToupTek ToupView, Hangzhou, Zhejiang, China).

WJ-MSCs from three donors at passage 3 were seeded separately into Nunc™ culture flasks (Nunc, Roskilde, Denmark) at a density 3 × 10³/cm² and were cultured till 90% confluence. One day before CM harvesting, the medium was completely changed, and only one half of the original medium volume was added to concentrate the growth factors and other analytes in the CM. In the following 24 h, the secretome was collected, centrifuged at 1500 rpm for 10 min and filtered through a 0.22 µm filter (TPP, Trasadingen, Switzerland). Concurrently, WJ-MSCs were harvested to assess the number of cells producing CM.
The CM from each sample was divided into 9 groups (100 µl CM per sample). One CM sample was fast frozen and stored at − 80 °C (control group). The remaining 8 samples were intended for lyophilization. One half of these CM samples (4 samples) was lyophilized without additional manipulations, whereas the other half (4 samples) was supplemented with trehalose prior to lyophilization. For this, 400 mM trehalose solution was added (1:10) to obtain a final trehalose concentration of 40 mM. The CM samples for lyophilization were fast frozen at − 80 °C and were freeze-dried using a FreeZone freeze dryer (Labconco, Kansas City, MO, USA). The freeze-dried samples were stored at − 80 °C, − 20 °C, + 4 °C and at room temperature (RT) for 3 and 30 months. All samples were prepared in duplicates.

Whole celeries (individually wrapped and labeled) from Australia were purchased from local supermarkets in Singapore, and the stems and leaves were washed with deionized water before freeze-drying in a Labconco FreeZone freeze-dryer (Kansas City, MO, USA). The freeze-dried samples were ground and stored in light-protected centrifuge tubes at −80 °C before extraction. For the preparation of celery stem (CSE) and leaf (CLE) extracts, stem (0.200 g)/leaf (0.100 g) powder were added to 2 mL of 80% ethanol (v/v) adjusted to pH 1, followed by sonication at 80 °C for 2 h. Subsequently, the extract was cooled in water under ambient conditions for 5 min, centrifuged (15,400 rpm) for 10 min and filtered using a 0.2 µm PTFE filter. The supernatant was dried under vacuum, stored at −80 °C and reconstituted with DMEM before cell treatment.

Cod gelatin (G) was extracted from the skin of Atlantic cod following the standard procedure. The cod skin was defrosted, cut into square pieces of 5 × 5 mm and then defatted by washing with ethanol.
The cut cod skin was mixed with distilled water at a ratio of 1:3 (w/w) and stirred for 10 min. Gelatin extraction was carried out at different pH values of the aqueous phase (pH 3.0, 4.0, 5.0, 8.0 and 9.0) for 3 h at a temperature of 50 ± 1 °C with constant stirring at a speed of 80–100 rpm. The samples of gelatin (G3, G4, G5, G8, and G9, respectively) were obtained. Glacial CH₃COOH and 4 M NaOH were used to adjust the pH of the aqueous phase.
After extraction, the reaction mixture was neutralized to pH 5.5–6.0 and then filtered.
The method of vacuum filtration at a temperature of 30 °C was used, and a paper filter (Ekros, St. Petersburg, Russia) with a pore diameter of 12 μm (Akros, Russia) was used. The filtrate (gelatin solution) was dried in a FreeZone freeze dryer (Labconco, Kansas City, USA) at a temperature of −50 °C and a residual pressure of 3.0 Pa. The gelatin obtained was stored at 5 °C until further use.
The product yield (B, %) was calculated using the following formula:
where m is the mass (g) of dried gelatin, and M is the mass (g) of dried raw material with a moisture content of up to 10%.

The mesquite gum purification methodology described by Moreno-Trejo et al. [24 (link)] was employed to obtain purified mesquite gum to synthetize AuNPs. First, the mesquite exudate pearls were carefully chosen and cleaned using established methods; basically, the lighter pearls were selected, while the darker pearls, rich in tannins, were discarded from the further process, and then any little sticks from the mesquite tree and any present dirt were mechanically removed from the lighter pearls [29 (link),30 ]. Subsequently, the cleaned pearls were pulverized using a mortar. The resulting powder was then dissolved in distilled water at room temperature and left to hydrate for 24 h. After the hydration period, the liquid was filtered using a Whatman no. 2 filter paper to remove impurities. The filtered solution was then frozen for 15 h and subsequently lyophilized using a FreeZone freeze dryer (Labconco, Kansas City, MO, USA) for 26 h [28 (link)].