*2.3. High Hydrostatic Pressure-Assisted Enzymatic Hydrolysis of Mealworm Proteins*

Enzymatic hydrolysis of mealworm proteins was performed prior to (pretreated) or during (simultaneous) pressurization while using a laboratory scale HHP system (Mini FoodLab FPG5620, Stansted Fluid Power LTD, Essex, UK) with a rate of pressurization of 50 MPa/min. The pressure system has a maximum capacity of 900 MPa and a glycol/water solution (30:70) was the pressure transfer fluid. Alcalase® (Subtilisin from *Bacillus licheniformis*, Sigma Aldrich, St. Louis, MO, USA) and pepsin (BD Difco, Franklin Lakes, NJ, USA) were the enzymes used for hydrolysis. The pressure and duration parameters applied during enzymatic hydrolysis for both pretreated and simultaneous

conditions were 380 MPa for 1 min. The specific value of 380 MPa was chosen because Zhang et al. [24] demonstrated a significant decrease of Alcalase® activity when the pressure reached 400 MPa, which indicated that this level of pressurization induced partial denaturation of the enzyme. Similarly, Curl and Jansen [25] observed that the activity of pepsin that was retained when diluted in a buffer solution at pH 1.9 was close to 100% at 400 MPa, but decreased drastically at higher pressures.

For pretreatment experiments, the mealworm meal suspension was first pressure-treated at 380 MPa for 1 min and then hydrolyzed at atmospheric pressure (0.1 MPa) while using Alcalase® or pepsin. The choice of these enzymes was based to their ability to hydrolyse the chitin-protein complex from arthropods and, consequently, to improve the recovery of separate protein and chitin separately, as described by Le Roux et al. [26] and De Holanda and Netto [27], respectively. Moreover, Alcalase® and pepsin were already used to decrease protein allergenicity in edible insect matrices [5,19]. The hydrolysis parameters for Alcalase® were an enzyme/substrate (E/S) ratio of 0.03% w/w at pH 8.5 and 60 ◦C for 120 min. The pepsin hydrolysis conditions were an E/S ratio of 0.25% w/w at pH 2.0 and 40 ◦C for 240 min. Hydrolysis durations of 120 and 240 min were determined as optimal for inducing the separation of the chitin-protein complexes in initial edible insect meals (results not shown) and consequently improved the recovery of protein/peptide fraction in the hydrolysates. For the simultaneous treatment condition, Alcalase® or pepsin enzyme, at the same E/S ratio, pH, and temperature as the pretreated condition, were added to the insect meal suspensions (5% w/V) before pressurization. The mixture of mealworm suspension and enzyme was also pressure-treated at 380 MPa for 1 min. An external thermoregulation system was used to maintain the enzyme's optimal temperature (60 and 40 ◦C for Alcalase® and pepsin, respectively) during pressurization. At the end of pressure treatment, decompression was instantaneous and mealworm samples were recovered. The remaining duration of hydrolysis (total hydrolysis of 120 and 240 min for Alcalase® and pepsin, respectively) was carried out at 0.1 MPa (atmospheric pressure) with constant pH and temperature control. The pH was controlled during control and pretreatment conditions, but not during simultaneous treatment (1 min), since it was not possible to open the reactor pressure vessel. However, it was controlled after pressure treatment and until the end of the hydrolysis. The control condition consisted of mealworm suspension (5% w/V) that was digested at atmospheric pressure (0.1 MPa) for 120 and 240 min with Alcalase® and pepsin, respectively. During the hydrolysis step, and for all conditions (control, pretreated, and simultaneous digestions), a sample of mealworm hydrolysate was collected every 2 min for the first 10 min of hydrolysis and then every 30 min until the end of digestion. Samples that were collected during and at the end of hydrolysis were immediately immersed in a 90 ◦C water bath for 5 min to inactivate the enzyme and then centrifuged at 9000× *g* for 10 min. Supernatants, corresponding to soluble peptide fractions and potential non hydrolyzed soluble proteins, were recovered and stored at 5 ◦C until analysis.
