Fluorescence microscopy test was used to investigate cell membrane damage after microencapsulation procedure or after exposure of samples to GI conditions and heat treatments. Furthermore, the test showed possible structural changes of microcapsules. Cells were dyed by using LIVE/DEAD® BacLight™ Bacterial Viability Kit (Molecular Probes, Eugene, Oregon) according to the procedure previously described [25]. Briefly, a stock solution of the two fluorochromes was prepared with 0.7 µL of SYTO 9 (green dye) and 1 µL of propidium iodide (red dye) in 330 µL of sterile deionized water. Microorganisms cultures and microcapsules exposed to GI conditions or heat treatments were ready for dyeing. Freshly prepared microcapsules, instead, were threefold diluted by adding quarter-strength Ringer. Fluorochromes stock solution (6 µL) was applied to 10 µL of each sample and incubated in the dark for 15 min at room temperature. After staining, samples were observed using a Nikon Eclipse E400 epifluorescence microscope (Nikon, Tokyo, Japan) equipped with an UV lamp and a 40× magnification objective. Images were captured by a Nikon Coolpix 4500 Digital Camera equipped with a microscope adapter.

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