*3.2. Correlation between Browning Intensity, Grafting Degree and Microencapsulation Efficiency*

An effective method to improve the functional properties of proteins, including the ability to include and protect low molecular bioactives, is based on the interaction with polysaccharides and smaller carbohydrates, via Maillard conjugation [29]. The Maillard reaction is a complex reaction occurring between amines and carbonyls [30], which involves, first, to consumption of the free amino group by the carbonylation reaction, mainly coming from the free amino group on the side chain such as lysine and arginine, or the free amino group on the N-terminus of the peptide chain of the protein molecule. Therefore, the carbonyl condensation between the reducing sugar and nucleophilic amino group could be analyzed by the loss of amino acids in the reaction [31]. Ideally, as reported by Jiménez-Castanõ et al. [32], to produce a glycol conjugate destined for incorporation into food, to avoid the formation of the highly colored, insoluble, nitrogen-containing polymeric compounds, referred to as melanoidins, the Maillard reaction needs to be performed under carefully controlled conditions to prevent the later stage changes. As reported by Jia et al. [13], protein glycation by the Maillard reaction might be favored in alkali conditions, while glycation is inhibited by the partial denaturation of the protein in acid conditions. These authors suggested a higher grafting degree and lower browning intensity at pH 9.0 after heating for 3 h. Therefore, these parameters were selected in our study to promote Maillard-based conjugates between WPI and X.

The brown-colored pigment formation in foods is caused by the Maillard reactions or caramelization [16]; therefore, the browning index is generally accepted as an indicator of the Maillard reaction. The brown pigment formation in the microencapsulated powders was evaluated by absorbance measurements at 420 nm and 600 nm, respectively. As expected, the browning intensity was higher (0.12 ± 0.01) for the heat-treated variant (Variant 2) than for the untreated variant (0.09 ± 0.01) (Variant 1). A proportional increase was observed between the browning intensity and antioxidant activity of the powders, indicating the strong antioxidant potency of the glycated variant due to the heating process. The powders showed significant differences in antioxidant activity (*p* < 0.05), with values of 179.7 ± 4.5 mMol TE/g DW for V1 and 184.4 ± 0.7 mMol TE/g DW for V2. Suminar et al. [33] explained that this was probably caused by reducing sugar reacting more easily with amino acids in heating conditions and producing antioxidant activity. The formation of the conjugate was also confirmed by grafting degree, which is able to reflect the level of glycation [13]. In the present study, a correlation between the grafting degree and encapsulation efficiency can be observed in both variants. Thereby, a grafting degree of 22.6 ± 2.5% and an encapsulation efficiency of 86.7 ± 1.4% were observed for V1. A significantly higher (*p* < 0.05) values were estimated for Variant 2, with a grafting degree of 30.4 ± 1.6% and an encapsulation efficiency of 90.5 ± 0.3%. Therefore, it can be appreciated that the Maillard-based conjugates showed a higher ability to entrap the flavonoids from yellow onion skin extract. These results indicate that the glycated form of the powder has a positive effect on the encapsulation efficiency.

In the conditions applied in our study, the Maillard-based conjugates between WPI and X caused structural changes in proteins that improved the ability to entrap flavonoids, in good agreement with reports of Liu et al. [34], Xu et al. [35] and Liu et al. [36]. The glycation degree can be correlated with the decrease in available -NH2 groups. For example, Shang et al. [37] suggested a dramatic loss in Lys and Arg, whereas a significant decrease in Tyr and Cys was also found, due to the formation of the dehydroalpropyl side chain. These authors also reported a transition toward a higher molecular weight distribution of WPI heated in the presence of X, at 90 ◦C and 95 ◦C and pH 9.0, whereas the contents of protein polymers larger than 40 kDa increased with the reaction time, thus indicating a protein crosslinking phenomenon. The heat-induced glycation reaction between WPI and X molecules might induce the formation of hydrogen bonds, thus weakening the interaction between molecules, and result in a reduction in the β-sheet and β-turns but an increase in the random coil [37].

In another study, lycopene was encapsulated in whey protein isolate and xylooligosaccharides conjugates and presented values ranging from 10.0 ± 0.4% to 27.0 ± 0.5%, depending on the other parameters of the reaction (temperature, time, pH). Muhoza et al. [38] evaluated the possibility of glycating the casein by the Maillard reaction with dextran for delivering coenzyme Q10. These authors reported that when the reaction time was less than 8 h, the grafting degree of the mixture about 20%. Ghatak and Iyyaswami [39] encapsulated quercetin from dry onion peels and obtained an encapsulation yield between 40.4% and 96.4%. They found that the highest encapsulation yield was achieved under the following process conditions: casein concentration of pH 7.09 for the crude extract containing the quercetin concentration of 16.27 M. Akdeniz et al. [40] reported the encapsulation efficiency values of phenolic from onion skins as being between 55.6 and 89.2% for different coating material combinations, with a maximum value found for maltodextrin:casein in a ratio of 6:4.
