## 4 Discussion
The calculated concentration of \(\beta\)-LG in ready-to-drink IFs produced from the H1 and H2 hydrolysates was significantly lower compared to cow's milk. The results correspond with previous studies, showing that ready-to-drink partially hydrolyzed IF contained 31,200 \(\mu\)g/L, 48,180 \(\mu\)g/L, and 96,250 \(\mu\)g/L of \(\beta\)-LG, and extensively hydrolyzed IF--below 200 \(\mu\)g/L of \(\beta\)-LG [11; 12]. Conventional IFs, containing nonhydrolyzed bovine milk proteins (both caseins and whey proteins) have shown similar residual \(\beta\)-LG values to ready-to-drink IFs based on the H3 and H4 hydrolysates (Table 4) with \(\beta\)-LG level of 1,320,000 \(\mu\)g/L [12]. A total of 287 IgE-binding allergenic epitopes (T- and/or B-cell responses) have been previously identified on bovine \(\beta\)-LG and \(\alpha\)-LA sequences and included in the Immune Epitope Database [40]. Among these, several epitopes have been reported as major, which were recognized by the majority of IgE antibodies from human sera of patients with IgE-mediated milk allergy: f124-134 [20]; f97-108 [21]; f95-113 [22]; f1-8, f25-40, f41-60, f102-124, f149-162 [23]; f75-86, f127-146 [24]. The majority of IgE-binding epitopes from \(\beta\)-LG and \(\alpha\)-LA range from 10 to 20 amino acids in length [40]. However, in this study, most of the identified epitopes were 5-15 amino acids long (Table 3). The more that peptides are hydrolyzed to smaller than 10-amino-acid-long fragments, the lesser the probability that they will bind with a specific IgE antibody to induce an allergenic reaction. Nevertheless, the results of the present study indicated that the presence of IgE-binding allergenic epitopes was not directly related to the extent of hydrolysis of the WPHs. As described earlier, 31, 45, 27, and 13 peptides from \(\beta\)-LG were found to be associated with 46 IgE-binding allergenic epitopes in the H1, H2, H3, and H4 hydrolysates, respectively (Figures 4 and 5, Table 3). Furthermore, two major epitopes known to induce an IgE reaction, f25-40 (in H3 and H4) and f124-134 (in H1, H2, and H3) [20; 23], were identified. The total intensity of peptides related to the epitopes comprised 17%, 39%, 24%, and 13% of all identified peptides in the H1, H2, H3, and H4 hydrolysates, respectively. Despite the fact that H1 did not contain intact \(\alpha\)-LA and \(\beta\)-LG proteins, and was more hydrolyzed than the other WPHs, it still contained a significant proportion of peptides related to the IgE-binding epitopes. Indeed, in this study, we demonstrated the absence of intact whey proteins in H1 (MW > 13,500 Da, Table 1) along with a higher content of \(\alpha\)-LA- and \(\beta\)-LG-derived small peptides revealed using LC-HRMS (Figures 4 and 5). The complete hydrolysis of intact whey proteins with trypsin was shown by [PERSON] et al. [4]. Also, it was shown that the application of high pressure was efficient to achieve complete hydrolysis of both major whey proteins by pepsin [47]. The inactivity of chymotrypsin against \(\alpha\)-LA was revealed by [PERSON] et al. [4], who showed that the chymotrypsin used to produce WPHs mainly affects \(\beta\)-LG. \(\alpha\)-LA seemed to be resistant to chymotrypsin activity [4], and this could be the case in the production of the H3 and H4 hydrolysates, where few \(\alpha\)-LA related peptides were identified. It is noteworthy that the enzyme specificity is very important in the hydrolysis of epitopes, and can decrease the WPH allergenicity. The cleavage sites with the production of peptides with amino acids Leu (L), Ile (I), and Met (M) at the C-terminal end were shown to be preferred by the enzymes chymotrypsin and elastase in \(\beta\)-LG [17]. These cleavages were found among the most frequent cleavage sites in the H1 and H3 hydrolysates (Figure 4), while trypsin was known to produce peptides with Lys (K) and Arg (R) at the C-terminal end [17]. Corresponding cleavage sites were identified among the abundant ones in the H2 and H4 hydrolysates (Figure 4).