Cryodos lyophilizer

After the synthesis of the nanovesicles, maltodextrin at a concentration of 20% (v/v) was added while applying a gentle stirring. The suspensions were then stored at −80 °C for 24 h. Then, the lyophilization process was performed under vacuum conditions (0.1 mbar) using a Telstar Cryodos Lyophilizer (Terrassa, Spain). The obtained powders were afterward resuspended in their corresponding aqueous phases, and their morphology and EE were assessed.

The bacteria that yielded better results as potential BCAs against toxigenic fungi were cultured in flasks with 15 mL of PDB or TSB, depending on whether they were actinobacteria or bacteria, and incubated for 48 h at 25 °C. Subsequently, 5 mL of these cultures were concentrated by centrifugation of the sample and following removal of the supernatant, carrying out 2 biological replicates of each microorganism. This biomass was mixed with 125 μL of sterile 20% Sveltesse^® skim milk (Nestlé, Barcelona, Spain) and 125 μL of PDB or TSB in lyophilization tubes. After homogenization, the lyophilization tubes were frozen for 2 h at –20 °C and then at –80 °C for a minimum of 4 h. Finally, these tubes were lyophilized in a Cryodos lyophilizer (Telstar, Barcelona, Spain) for 24 h. The lyophiles were stored at 4 °C until needed. The biomass of the third Eppendorf was resuspended in 1 mL of saline to be used as a preliminary control.
To check the loss of viability from freeze-drying, viable counts (CFU/mL) were carried out before and after. For this purpose, a bank of decimal dilutions in saline solution and subsequent colony count in PDA and TSA plates, as appropriate, was performed, making three replicates for each experimental condition and replicate. The plates were incubated at 25 °C for 72 h. Loss of viability was obtained from the difference in CFU/mL pre-lyophilization.

The synthesis was carried out through a simple batch method, which does not require hazardous reagents. It consists on mixing 100 mL of an aqueous solution (A) containing 0.2 M Ca(NO₃)₂, 0.2 M Na₃Cit and 8 g of urea with a solution (B) of an equal volume containing 0.12 M K₂HPO₄ and 0.1 M Na₂CO₃. The mixture was then kept at 37 °C for 5 min. After that, the precipitates were repeatedly washed with ultrapure water by centrifugation (5000 rpm for 15 min), and then freeze-dried (Cryodos lyophilizer, Telstar) overnight under vacuum.