*3.5. Structure and Morphology of the Powder*

The confocal laser scanning microscopy technique allows the simultaneous identification of several compounds, at the surface of the particles using specific samples that usually discharge light at different wavelengths. This type of analysis also permits the visualization of the internal morphology of any type of particle by different fluorophores labels, hence displaying the compositional evolution of the targeted molecules, which represents the main aim of any study that regards the protection of valuable molecules such as antioxidant compounds, mainly polyphenols, through an encapsulation process. Therefore, it can be applied as a nondestructive visualization technique for microparticles.

By using a confocal laser scanning Carl Zeiss 710 microscope with the ZEN 2012 SP1 software (Black Edition), the images of V1 and V2 powders (Figure 2) were captured, both in the native state without any other additional dye added (Figure 2a,c) and stained with Congo Red (Figure 2b,d, respectively). In the native state, the powders showed an autofluorescence (in the range of 520–580 nm) due to the rich content of polyphenols among which quercetin predominates [39]. The biologically active compounds were captured in the WPI–X matrix (with a displayed autofluorescence showed in blue). Several irregular scaly formations could be observed with larger dimensions for the V2 (199.6–253.3 μm), compared to V1 (94.5–104.8 μm), probably due to the WPI–X conjugates. The microstructure was rather similar to that reported by Horincar et al. [41], who used different polymers as the encapsulating matrices such as chitosan, maltodextrin and pectin.

Through the fluorescent staining with Congo Red, a fluorophore usually used to highlight the fluorescence of proteins, several spherosomes were revealed, highlighting the encapsulated flavonoids in the WPI–X matrix. Nonetheless, the displayed formations were larger (up to 51.5 μm) and less numerous in the V1 sample where a significant amount of nonencapsulated flavonoids was visualized (Figure 2b).

The interaction of the WPI with the small carbohydrates via the Maillard reaction favored a better incorporation of the biologically active compounds from the onion extract into finer spherosomes (approximately 20 μm) or in the form of scales with digitiform extensions. The fluorophore bound to the conjugated proteins and generated a fine, orange wall with a fluorescent emission between 600 and 620 nm around the bioactives.

.**-**2

.2
