Glycerol

The protective agents (Table 1) were purchased from Sigma-Aldrich except d-mannose, d-xylose, d-fructose, d-galactose (Duchefa, Haarlem, The Netherlands), and glycerol, magnesium sulfate heptahydrate (Duksan, Ansan, Korea), and l-fucose (Carbosynth, Newbury, UK), and peptone, skim milk, yeast extract (Difco, Detroit, MI, USA), and taurine (Daejung, Siheung, Korea), and isomaltooligosaccharide, l-rhamnose (WAKO, Osaka, Japan), and d-maltose (YAKURI, Osaka, Japan).
To prepare the cells, S. cerevisiae 88-4 was cultured in YPD broth (1% inoculum) at 30 °C for 18 h, corresponding to approximately 10⁷ CFU/mL. Cells were centrifuged at 3000× g at 4 °C for 15 min and washed with 0.85% NaCl solution. Prior to the experiments, all protective agents were sterilized under UV for 18 h. The concentrations of each protectant were described in Table 1. Cells were resuspended in 1 mL protective agent solution as the freeze-dried sample, or 1 mL distilled water as a blank. Suspensions were frozen in a deep freezer at −80 °C for 3 h. Subsequently, freeze-drying was conducted under vacuum at 5 Pa for 24 h with a condenser temperature of −51 °C. Freeze-dried cells were immediately used to determine the survival rate. All protective agents were used directly with the yeast pellet.

L5 larvae were challenged with nematode (80 IJs/larva) for 8 h. Midgut tissues of the challenged larvae were dissected carefully in TC-100 insect medium and incubated in a wet chamber for 10 min. After removing TC-100, it was then incubated with 3 µL Fura-8AM (1 mM) in RT for another 10 min. Then midgut was washed three times with PBS and midgut cells were permeabilized with 0.2% Triton X-100 in PBS for 20 min at RT. Following washing three times with PBS, the midgut was incubated for 5 min with DAPI (1 μg/mL) in PBS for staining the nucleus. Finally, the midgut was washed three times with PBS and fixed with 50% glycerol (Duksan, Seoul, Korea). Fura signals in the midgut were observed under a fluorescence microscope (DM500, Leica, Wetzlar, Germany) at 200× magnification. Fluorescence changes were analyzed by using ImageJ software (https://imagej.nih.gov/ij). Each treatment was replicated three times with independent sample preparations.

Chitosan (low molecular weight), EDTA (99%), (3-aminopropyl)triethoxysilane (APTES, 98%), isopropanol (C₃H₈O, 99.7%), acetic acid (C₂H₄O₂, 99.7%), 1-ethyl-3-(3-(dimethylamino)propyl) carbodiimide (EDC), N-hydroxysuccinimide (NHS, 98%), and copper (II) nitrate trihydrate (Cu(NO₃)₂.3H₂O, 99%) were purchased from Sigma-Aldrich Co. (St Louis, MO, USA). Deionized water was produced by Biosesang Co. (Seongnam-Si, Korea). Glycerol (C₃H₈O₃, 99%) was purchased from Duksan Pure Chemicals Co. (Ansan, Korea), and polydimethylsiloxane (PDMS) was purchased from Dow Corning Co. (Hemlock, ML, USA).