*4.1. E*ff*ects of High Hydrostatic Pressure-Assisted Enzymatic Hydrolysis on Protein Degradation and Degree of Hydrolysis*

Our results showed that Alcalase® hydrolysis of mealworm proteins under HHP (simultaneous condition-380 MPa for 1 min) improved the degree of hydrolysis by 24 and 22% after 60 min of hydrolysis when compared to the control and pretreated conditions, respectively (Figure 2), despite no major changes being observed in terms of protein degradation during hydrolysis (Figure 1). This non-correlation between SDS-PAGE profiles and degree of hydrolysis results could be explained by the generation of very low molecular weight peptides (largely < to 10 kDa), which could not be detected due to their migration outside the gel. Consequently, and specifically regarding the degree of hydrolysis, the result indicates that HHP treatment might have facilitated the mealworm protein conformational changes that are needed to increase the effectiveness of enzymatic digestion by providing Alcalase® access to the buried cleavage site at the very beginning of hydrolysis (until 10 min). Previous studies using various enzymes demonstrated that HHP-assisted enzymatic hydrolysis induced the exposure of new cleavage sites through protein unfolding, which enhanced enzyme activity, reduced hydrolysis time [36], and improved the degree of hydrolysis and concentration of peptides generated in protein hydrolysates [37–39]. In addition, the DH of mealworm proteins obtained was significantly increased (*p* < 0.05) during HHP-assisted enzymatic hydrolysis by Alcalase®. This increase was drastically improved as compared to the control (0.1 MPa) (Figure 2A), but was lower than similar studies [38,40,41], where DH increases ranged from 17% to 58% for pressure-treated protein hydrolysates as compared to the control (0.1 MPa). The effectiveness of HHP treatment on the enzymatic hydrolysis depends on specific parameters, such as substrate/enzyme ratio, pressure level, and treatment duration. Consequently, different hypotheses could explain the lack of influence of HHP on the in vitro digestion of mealworm protein. First, the duration of HHP treatment (1 min) was too short to induce optimal unfolding

of mealworm proteins to improve Alcalase® access to buried sites. Second, irreversible protein aggregation occurring during the commercial-scale production of mealworm meals could decrease the efficiency of HHP and enzymatic hydrolysis. Indeed, the mealworm powders that were used for this study were roasted at approximately 107 ◦C and ground from fresh larvae [42]. When using the same mealworm powder from Entomo Farm, Stone et al. (2019) observed that the heating step of the insects during processing could result in protein denaturation, exposing hydrophobic groups and leading to protein aggregation [42]. Womeni et al. [43] demonstrated that roasting and grinding enhanced the aggregation of proteins, which makes the insect products unsuitable for food formulations due to their low solubility. A recent study published by Kröncke et al. [44] confirmed that oven drying of *T. molitor* larvae decreased the quality of proteins and reduced their solubility by 74% [44]. Consequently, the large amount of protein aggregates generated during production of mealworm meal rendered HHP ineffective for unfolding the proteins, leaving certain bonds in *T. molitor* proteins buried and inaccessible to Alcalase®, which decreased the proteolysis efficiency [45,46]. This second hypothesis is especially appealing given the large MW protein aggregates that were observed in the sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) wells for control, pretreated, and simultaneous Alcalase® experimental conditions, and could explain why no significant DH was observed between the control and pretreatment conditions. Contrary to Alcalase® hydrolysis, coupling HHP and pepsin had no impact on the DH of mealworm proteins, despite the differences of protein degradation profiles observed in Figure 1. As mentioned for Alcalase®, it is difficult to correlate the results obtained in Figure 1; Figure 2, since very low molecular weight peptides could not be not detected in electrophoresis gels. The short duration of HHP treatment and the presence of protein aggregates in mealworm meal could explain the inability of HHP to improve protein digestion in vitro, as hypothesized for Alcalase®. Cleavage specificities are also important, since Alcalase® has broad specificity, hydrolyzing most peptide bonds. It preferentially hydrolyzes those containing aromatic amino acid residues whereas pepsin is more specific and cleaves peptide bonds following Phe or Tyr residues, as well as other hydrophobic amino acids. The recent study of Dai et al. [47] confirmed the importance of enzyme specificity during hydrolysis of *T. molitor* larva protein, since Alcalase® was the most efficient enzyme in terms of degree of hydrolysis compared to various other commercial enzymes (trypsin, Neutrase, papain, and pepsin) [47].
