*3.3. Determination of Mealworm Allergenic Protein Precursors from Hydrolysates*

Table 1 presents the determination of mealworm allergenic protein precursors from peptides generated after 120 min of Alcalase® hydrolysis or 240 min of pepsin hydrolysis in vitro digestion. The total spectrum count (TSC) value, defined as the total number of spectra identified for a protein, is of particular interest, since this parameter is a semi-quantitative measure for a given protein abundance in proteomic studies [31]. Indeed, previous studies have demonstrated that the spectral counts of proteins correlate linearly with protein abundances in complex samples [32–34]. High throughput mass spectrometry identified a total of 110 proteins and close to 2800 spectrum counts. From these proteins, 19 allergenic proteins in mealworm meal were identified from the 161 unique peptides that were generated after Alcalase® or pepsin in vitro digestion of the mealworm proteins. The coverage and TSC ranged from 3 to 70% and 0 to 106, whatever the conditions (control, pretreatment, and simultaneous) and enzyme (Alcalase® and pepsin). The 19 allergenic proteins were identified according to the publication of Barre et al. in 2018 [35]. The majority of protein MWs ranged from 11 to 51 kDa (~74%), but five of the identified proteins ranged from 84 to 123 kDa. Most of the identified proteins had good peptide coverage, with ~31% of proteins having >10% of the sequence coverage and ~31% of proteins having >20% sequence coverage.




\* The probability of protein identification was over 95%. \*\* The peptide identification probability was ranging from 96 to 100%. 1 Control, 2 Pretreated, and 3 Simultaneous conditions.

Globally, the in vitro digestion of mealworm allergenic proteins by pepsin was more efficient than Alcalase®, since higher TSC values were obtained for all 19 allergenic proteins (Table 1). Regardless of the condition tested (control, pretreated, and simultaneous), allergenic proteins #5, 8, 14, 15, 16, and 17 were more susceptible to in vitro digestion by Alcalase®, as illustrated by high TSC values (TSC>10). Specifically, allergenic proteins #1, 2, and 6 were more resistant to hydrolysis by Alcalase® (TSC<10), whereas proteins #3, 4, 7, 9, 10, 11, 12, 18, and 19 were not digested by this enzyme (TSC values closed to 0). However, some differences were observed in TSC values as a function of the hydrolysis conditions. Indeed, while proteins #1, 2, 6, 8, 14, 18, and 19 were similarly digested by Alcalase®, as shown by the TSC detected for all conditions, the in vitro digestion of proteins #5, 13, 15, 16, and 17 was more efficient for the simultaneous treatment than for control and pretreated conditions. Only proteins #4, 9, 10 and 18 were resistant to pepsin hydrolysis, with TSC values < 10, regardless of the hydrolysis conditions (control and pressurization treatments). Protein #1 was not hydrolyzed by any treatment, since similar TSC values were obtained for all conditions. When compared to conventional hydrolysis at atmospheric pressure (0.1 MPa), the application of pressurization treatments (380 MPa for 1 min) before or simultaneous to hydrolysis improved the in vitro digestion of proteins #6, 11, 12, 13, 14, 15, 16, 17, and 19. The hydrolysis of proteins #2, 5, 8, 15, and 19 was enhanced by pretreatment, as compared to control and simultaneous conditions, but the hydrolysis of protein #3 was unaffected.
