*3.3. Ratio of* α*-Lac to* β*-Lg*

In this study, the retention of α-Lac and β-Lg after HTST and HPP was measured by ELISA and compared thereafter (Figure 6). Before treatment, the concentration of α-Lac and β-Lg in reconstituted IMF was 1.04 and 6.2 mg/mL, respectively. Conventional HTST (72 ◦C for 15 and 30 s) retained 78% and 70% of α-Lac and β-Lg respectively, which corroborates the results reported previously [27,41]. However, in contrast, the degree of denaturation of β-Lg was more pronounced than α-Lac, as observed with HPP combinations of increased pressure, temperature, and time (Figure 6). This trend is in agreement with those obtained from HPP in skim milk [31,32] and in a protein solution [42].

**Figure 6.** Retention of α-Lac and β-Lg (%) after high-temperate short-time (HTST) pasteurization at 72 ◦C for 15 and 30 s, and HPP applied at ~20 ◦C (**A**), ~30 ◦C (**B**), and ~40 ◦C(**C**). Error bars represent the standard deviations of duplicates.

Figure 7 represents the relative proportions (%) of α-Lac and β-Lg derived from Figure 6. The highest ratio of α-Lac to β-Lg (77:23) was achieved from the HPP treatment of 600 MPa at 51.7 ◦C for 5 min, whereas it was only 24:76 and 23:77 in HTST for 15 and 30 s, respectively (Figure 7). From the results obtained in this work, it is evident that the synergistic effect of HPP at elevated temperature induces a higher ratio of α-Lac to β-Lg, compared to the untreated (22:78) and HTST-treated α-Lac-added reconstituted IMF. The higher baroresistance of α-Lac, compared with β-Lg, is consistent with previous observations in milk [31,32,43]. This difference is considered to be due to the higher number of intramolecular disulfide bonds (4 in α-Lac and 2 in β-Lg) and to the presence of a free sulphydryl group in β-Lg [32,40]. Upon unfolding of β-Lg due to HPP, this free sulphydryl group interacts with proteins containing disulphide bonds (e.g., αs2-casein, k-casein, α-Lac, and β-Lg) through sulphydryl–disulfide interchange reactions resulting in aggregation. Moreover, unfolded

α-Lac and β-Lg, which did not interact with other proteins, refold to their native forms on the release of pressure [31]. Therefore, the mechanistic approach of using HPP followed in this work explains the mechanism to achieve a final product with a massive reduction in the β-Lg portion, which subsequently would result in lowering the allergenicity and protein content.

**Figure 7.** Relative proportions of α-Lac and β-Lg (%) after HTST at 72 ◦C for 15 and 30 s, and HPP applied at ~20 ◦C (**A**), ~30 ◦C (**B**), and ~40 ◦C (**C**). Total refers to the sum of α-Lac and β-Lg content.

The results found in this work show the potential route to develop an HPP-treated pasteurized RTF hypoallergenic formula because of having a higher ratio of α-Lac to β-Lg. In addition to this, such a formula would ensure the required amino acid balance in the treated product due to the α-Lac supplementation, which may also compensate the lower contribution of heavily denatured β-Lg in the amino acid profile. Thus, this work streamlines the possibility of manufacturing a hypoallergenic and

low-protein pasteurized RTF formula. However, further investigation in post-treatment analysis (e.g., bioavailability, digestibility, amino acid profile, etc.) of HPP-treated formula is highly recommended to commercialize this research. Besides, the shorter shelf life at refrigerated conditions of this pasteurized product than that of the sterilized RTF formula would result in slower progress in gaining market. Moreover, HPP is still limited by its batch operation although the recent patent-pending concept of Hiperbaric, the HPP equipment manufacturer, to process liquid foods up to 10,000 L/h before bottling (aseptic packaging) is being considered as a promising innovation to address HPP's batch operation [44].
