*2.6. In Silico Investigations*

In agreement with the experimental approach, the molecular modeling tools were used to simulate X binding by the two major whey proteins. The three-dimensional molecular models of the α-lactalbumin (α-LA, PDB ID: 1F6S) [17] and β-lactoglobulin monomers (β-LG, PDB ID: 4DQ3) [18] from the RCSB Protein Data Bank were optimized and relaxed at 25 ◦C using GROMACS 4.6 software [19], in agreement with the protocol previously described by Aprodu et al. [20]. The equilibrated protein models were used as receptors for the X binding. The PatchDock algorithm [21], which is very efficient for performing protein–small ligand docking, was used to identify the most probable binding site of X molecules to the WP. Matching the receptor and ligand molecules was carried out through rigid body docking, which is based on the shape complementarity principles. The algorithm employed involves the following major stages: the surface of the receptor was first segmented to identify the so-called hot spot residues on the concave, convex or flat geometric patches, which were selected for a further surface patch matching step with the ligand. The resulting complexes were filtered to disqualify the solutions, involving steric clashes, and finally ranked based on the geometric shape complementarity score. The best three WPI–X fits were selected based on the binding energy values among the potential docking models generated by the PatchDock algorithm [21]. An in-depth analysis of the binding pockets was carried out using the PDBePISA [22] tools and DoGSiteScorer web server [23] to identify the extent to which protein glycation affects flavonoids binding.

#### *2.7. Confocal Laser Microscope Spectroscopy*

A confocal laser scanning microscopy analysis of the samples was employed to assess the structural appearance of the microencapsulated powders. The CLSM images were captured with a Zeiss confocal laser scanning system (LSM710) equipped with several types of lasers such as a diode laser (405 nm), Ar laser (458, 488, 514 nm), DPSS laser (diode pumped solid state—561 nm) and HeNe laser (633 nm). The powders were observed with a 20× apochromatic objective, at zoom values of 1 and 0.6, respectively. The obtained 3D images were rendered and processed by ZEN 2012 SP1 software (Black Edition).
