## 4 Discussion  
In this study, we provided a sequence-based characterization of commercial WPH peptide profiles, which enables the detection of specific peptides related to known IgE-binding allergenic epitopes listed in the Immune Epitope Database [40]. Overall, the results obtained from LC-HRMS (Figures 2-5) were consistent with the findings from SE-UPLC, which showed a higher amount of low MW protein fractions (MW range of 500-3500 Da) in H1, followed by the H2, H3, and H4 hydrolyses (Table 1). This fraction was represented by the peptides identified using LC-HRMS, which also revealed a higher total intensity of peptides in H1 and H2, compared to H3 and H4. Furthermore, the results obtained in this study correspond with the information provided in the specification of the WPHs, where the degree of hydrolysis of the H1, H2, H3, and H4 hydrolyses was reported as 25-30%, 10-16%, 9-15%, and 6.5%, respectively. Several studies have shown a significant decrease in allergenicity in extensively hydrolyzed proteins, based on the binding capacity to IgE of sera from patients with CMA [27; 47]. Nevertheless, the reduced allergenicity of hydrolyses does not depend only on the reduction in the size of peptides, but also on the content of the residual proteins [48]. The analyses showed that both the H3 and H4 hydrolyses contain a large amount of intact whey proteins (the fraction with MW > 13,500 Da) (Figure 1, Table 1). At the same time, the H3 and H4 hydrolyses contain a lower amount of \(\beta\)-LG-derived peptides, and few peptides from \(\alpha\)-LA, compared to H1 and H2, indicating a lower degree of protein hydrolysis. The abovementioned intact protein fraction contains the major cow's milk allergens, such as \(\beta\)-LG (MW 18,300 Da), \(\alpha\)-LA (MW 14,200 Da), BSA (MW 66,400 Da), lactotransferin (MW 76,200 Da), and Ig (MW > 150,000 Da), including a significant amount of IgE-binding epitopes, which can cause an allergenic reaction in infants with CMA [2]. The levels of residual \(\beta\)-LG in the WPHs measured using the ELISA test were in accordance with the MW distribution of protein fractions determined using SE-UPLC, and inversely proportional to the amount of peptides identified using LC-HRMS (Table 4). As expected, high levels of residual \(\beta\)-LG were measured in the H3 and H4 hydrolyses, with a lower extent of protein hydrolysis, and a lower amount of identified peptides. To compare the amounts of \(\beta\)-LG in the WPHs measured using ELISA with cow's milk and ready-for-consumption hypoallergenic IFs, the obtained values were recalculated to the amount of \(\beta\)-LG in ready-to-drink IF (Table 4). The estimated concentration of \(\beta\)-LG in ready-to-drink IFs, if prepared from the less proteolyzed H3 and H4 hydrolyses, was only 2-2.5 times lower than in mature cow's milk, where \(\beta\)-LG concentration was \begin{table}
\begin{tabular}{c c c c} \hline \hline
**WPH** & **\(\mu\)g/g (Mean \(\pm\) SD)** & **\(\mu\)g/g IF Powder** & **\(\mu\)g/L in Ready-to-Drink IF** \\ \hline H1 & 0.13 \(\pm\) 0.07 & 0.02 & 2.45 \\ H2 & 237 \(\pm\) 118 & 39 & 5049 \\ H3 & 86,922 \(\pm\) 43,461 & 13,038 & 1,786,252 \\ H4 & 68,786 \(\pm\) 34,392 & 9380 & 1,285,042 \\ \hline \hline \end{tabular} \({}^{\dagger}\) Calculated concentration of \(\beta\)-LG in IF powder and ready-to-drink IFs based on the H1, H2, H3, and H4 hydrolyses, taking into account that for its preparation, 137 g/L of powder is needed according to the manufacturer’s instructions; the average protein content in powder IF is 12 g/100 g, according to the Commission Directive 2006/141/EC on IF and follow-on formulas; the average protein content in the WPHs is 87% (H1), 77% (H2), 80% (H3), and 88% (H4), according to the specification.  
\end{table} 
Table 4: \(\beta\)-LG content in WPHs, IF powder, and ready-to-drink IFs based on WPHs \({}^{\dagger}\). approximately 4 \(\times\) 10\({}^{6}\)\(\mu\)g/L [11].