*2.1. Insect Samples*

LM, provided by Protifarm (Ermelo, The Netherlands), were reared as described in our previous work [14]. Larvae were killed by liquid nitrogen, packed under vacuum sealed and frozen at −20 ◦C. After one week, samples were freeze-dried for 36 h (Christ, gamma 1−16 LSC, Osterode am Harz, Germany) and stored at −20 ◦C for the future analysis. Samples were grinded for 2 min with a laboratory grinder (Microtron MB 550, Kinematica, Luzern, Switzerland) at maximum speed before each analysis.

#### *2.2. Enzymatic Hydrolysis for Protein Extraction*

#### 2.2.1. Preliminary Enzymatic Assisted Extraction

The enzymatic assisted extraction of proteins was carried out by commercial protease from *Bacillus licheniformis* (≥2.4 U/g; EC Number 3.4.21.62) at optimal conditions for hydrolysis as suggested by the supplier (Sigma-Aldrich, St. Louis, MO, USA). More specifically, 200 g of dried ground larvae were mixed with 1 L of a buffer solution (Na2HPO4 10 mM) and 0.25% of enzyme ina2Lreactor, combined with the pH-STAT system (Metrohm, Varese, Italy) to control the pH during the reactions by the addition of NaOH 1 M. By knowing the amount of NaOH added, it was possible to back-calculate the degree of hydrolysis as described by Butrè et al. [19]. The hydrolysis reaction was performed for

five hours and after this time the solution was heated at 90 ◦C for 5 min for enzyme inactivation. The hydrolysates were then centrifuged (Eppendorf, 5810/5810 R, Milano, Italy) at 2683 g at 4 ◦C for 30 min. The supernatant was separated from the pellet and lyophilized with LIO-5PDGT freeze-dryer (5pascal, Milano, Italy). The freeze-dried protein hydrolysate was then defatted with diethyl ether, followed by quantification for protein content with Kjeldhal analysis, as described by Leni et al. [14].

### 2.2.2. Set of Protein Hydrolysates Collected at Different Time-Points

The enzymatic hydrolysis as previously described, was performed again in order to obtain a set of hydrolysates collected at different time-points. More specifically, 1.5 kg of dried ground larvae were mixed with 7.5 L of a buffer solution (Na2HPO4 10 mM) in a 10 L flask at pH 7.5 and at 60 ◦C. Before addition of enzyme, the mixture was homogenized in an incubator (New Brunswick, Innova 42 shaker, Eppendorf, Milano, Italy) at 130 rpm for 30 min, while checking the pH (ProfiLine pH 3310, WTW, Xylem Analytics LLC, Weilheim, Germany). The pH fluctuations were adjusted by adding NaOH 50%. After this period, 1.5 L of solution was subsampled and collected as control (time 0). Next, 0.25% of enzyme was added and the hydrolysis was performed for 180 min, with sub-sampling of 1.5 L aliquots of hydrolysate at different time points, being after 30 min, 60 min, 120 min, and 180 min. Each sub-sample, control included, was heated at 90 ◦C for 5 min to inactive the endogenous and exogenous enzymes and subsequently centrifuged at 4 ◦C for 30 min at 3220 g. The supernatant was separated from the pellet and the lipid upper layer using a 500 μm sieve and were freeze-dried to generate hydrolysates for the further analysis. Each hydrolysis reaction was performed in duplicate.

#### 2.2.3. Bulk Composition

The hydrolysates and the intact larvae were characterized in terms of humidity, lipids, total N and ash. The dry matter content was determined after drying of the samples in oven at 105 ◦C for 24 h. Total ash was determined after mineralization at 550 ◦C for two times 5 h. For crude lipid quantification an automatized Soxhlet extractor (SER 148/3 VELP SCIENTIFICA, Monza e Brianza, Italy) was used with diethyl ether. The total N content was measured by Vario EL Cube (Elementar, Langenselbold, Germany) instrument by the supplier. Briefly, the sample was burned in an oxygen rich environment at 1150 ◦C in the combustion tube. All the burning gasses flowed through the reduction tube (helium as support gas) and were reduced to N2, CO2, H2O, and SO2. These different components were adsorbed at Selective Trap Columns and separated liberated (purge and trap technique). The detection of the components was performed with a thermo conductivity detector (TCD) cell. The proteinaceous *N* contribution was separated from the chitinous one, assuming that 87% of total *N* in LM was from protein origin, and then multiplied for the *N* to protein conversion factor 5.67, as we described in our previous work [14]. For each sub-sample, the protein content (g/100 g DM) was calculated, as well as the protein concentration as described in Equation (1):

$$\text{Protein concentration} \left(\frac{\text{g}}{\text{L}}\right) = \frac{\text{g of protein in the sub -- sample after freeze dry expressed on DM}}{\text{L of sub -- sample collected}} \quad (1)$$

#### 2.2.4. Degree of Hydrolysis

The DH, defined as the percentage of cleaved peptide bonds in a protein hydrolysate, was calculated using o-phtaldialdehyde (OPA) as described by Leni et al. [14] with some modification. In particular, the supernatants were diluted in a 2% (*w*/*v*) sodium dodecyl sulphate, stirred for 20 min and stored at 4 ◦C overnight before the assay. The OPA/NAC (N-acetyl-cysteine) reagent (100 mL) was prepared by combining 10 mL of 50 mM OPA (in methanol) and 10 mL of NAC 50 mM, 5 mL of 20% (*w*/*v*) SDS, and 75 mL of borate buffer (0.1 M, pH 9.5). The reagent was covered with aluminum foil to protect it from light and allowed to stir for at least 1 h before use. The OPA assay was carried out (in triplicates) by the addition of 5 μL of sample (or standard) to 215 μL of OPA/NAC reagent in microplates. The absorbance of this solution was measured, after 10 min of shacking, at 340 nm

with Tecan Infinite® 200 PRO spectrophotometer (Tecan, Männedorf, Switzerland) against a control cell containing the reagent and 5 μL of the buffer used for the sample. The intrinsic absorbance of the samples was measured before OPA addition and subtracted. The standard curve was prepared using L-isoleucine (0−2 mg/mL). The DH was calculated as the ratio between the free nitrogen groups after hydrolysis and the total nitrogen groups: DH% = (N free/N total) × 100. The first value was calculated by the OPA reactivity. The total moles of nitrogen atoms involved in peptide bonds before hydrolysis were calculated by considering that the 87% of total N [14] in insects is from protein origin and is involved in peptide bonds.

#### *2.3. Techno Functional Properties*

#### 2.3.1. Solubility

The protein solubility was determined at pH 3, pH 5, and pH 7. The hydrolysates collected at different time points, were solubilized in demineralized water till a final concentration of 1% (*w*/*w*). The pH was adjusted with NaOH and HCl and the solution was mixed with an overhead shaker (Trayster digital, IKA, Königswinter, Germany) for 30 min. The pH was checked and adjusted if needed and the samples were placed on an overhead shaker for another 30 min. After that, the tubes were weighted and centrifuged at 5910 g for 20 min at 4 ◦C. The supernatant was separated and collected for the analysis in order to quantify the soluble N and the ionic strength. The total *N* was determined by a chemiluminescence detector (Multi N/C 3100 Analytik Jena, Jena, Germany). Briefly, the sample was oxidized by catalytic combustion in an oxygen atmosphere at 800 ◦C, to N oxides. The formed measuring gas was dried and entered in the reaction chamber of the chemiluminescence detector. There, the N monoxide present in the measuring gas, was oxidized with ozone into activated N dioxide. By emitting light photons (luminescence) the molecules of the N dioxide returned to their original state. The luminescence, proportional with the N monoxide concentration, was detected using a photomultiplier. The ionic strength was measured with a conductivity meter (ProfiLine Cond 3310, WTW, Weilheim, Germany).

The protein solubility was calculated as described in Equation (2):

$$\text{Protein solubility } (\%) = \frac{\text{g } N \text{ in the supernovaant}}{\text{g protein across } N \text{ in the sample}} \times 100\tag{2}$$

The N-content in the supernatant measured by chemiluminescence analysis was assumed to be only from protein origin, whereas the amount of proteinaceous *N* in the sample was determined as described in 2.2.3. The analysis was performed in triplicate. In order to compare the results obtained with food standard proteins, the procedure was repeated with egg white (Sigma-Aldrich, St. Louis, MO, USA).

#### 2.3.2. Emulsification Properties

The emulsification property was determined following the method proposed by Purschke et al. [17] with some modification. Briefly, the hydrolysates were diluted with demineralized water at a concentration of 0.1% (*w*/*w*) and mixed with an overhead shaker for 30 min. The ionic strength was measured with a conductivity meter (ProfiLine Cond 3310, WTW, Weilheim, Germany). The solution was centrifuged at 3220 g for 15 min and the supernatant mixed with commercial corn oil (Vandemoortele, Ghent, Belgium) (1:1 *v*/*v*) and emulsified at 11.000 rpm for 30 s using a homogenizer (ULTRA-TURRAX® T18, IKA, Königswinter, Germany). An aliquot of the emulsion was immediately transferred into scaled tubes and centrifuged at 3220 g for 15 min at 20 ◦C (fixed angle centrifuge). The height of the resulting emulsified layer (Hel), even if not homogeneous in the scaled tube, and the

total height of solution (Hs) were used to calculate the emulsification ability as described below (3). The analyses were performed in triplicate.

$$\text{Eunksifying activity (\%)} = \frac{\text{Hel}}{\text{Hs}} \times 100\tag{3}$$

Casein from bovine milk and egg white (Sigma-Aldrich, St. Louis, MO, USA) were subjected to the same procedure in order to evaluate and compare their emulsification properties with insect protein hydrolysates.

#### 2.3.3. Oil Holding Capacity

For the oil holding capacity (OHC), 1 g of hydrolysate was transferred to a falcon tube and 10 g of commercial corn oil (Vandemoortele, Ghent, Belgium) were added. The solution was mixed with on overhead shaker for 5 min at 55 rpm. After 30 min, the tube was centrifuged at 3000 g for 30 min at 20 ◦C. The sample was re-weighed after 10 min of decantation upside-down (45◦ angle) and the holding capacity calculated as described in the equation below (4):

$$\text{OHC}\_{\text{(g oil/g sample)}} = \frac{\text{W2} - \text{W1}}{\text{W0}} \tag{4}$$

where W0 was the weight of the sample, W1 was the weight of the tube and the sample, W2 was the weight of the tube after decantation. The analyses were performed in triplicate. In order to compare insect protein hydrolysate with standard food proteins, egg white, and casein from bovine milk (Sigma-Aldrich, St. Louis, MO, USA) were subjected to the same procedure and their OHC determined.

#### 2.3.4. Foaming Capacity

The foaming capacity were measured with a homemade apparatus, composed by a graduated glass cylinder, in which was placed the solution, and a pump which fluxed air inside the mixture. The hydrolysate was suspended in demineralized water at a final concentration of 1% and mixed on an overhead shaker for 30 min. The ionic strength was measured with a conductivity meter (ProfiLine Cond 3310, WTW, Weilheim, Germany) and then the solution was transferred in the foam tube and the starting volume (in cm) was noted. The air flow was bubbled through the sample at a flow rate of 2 L/h for 1 min and the final volume reached by the foam was red after pump stopping and reported. Casein from bovine milk and egg white (Sigma-Aldrich, St. Louis, MO, USA) were subjected to the same procedure for evaluating and comparing their foaming capacity with insect protein hydrolysates. The analysis was performed in triplicate. The foam capacity was measured with the following Equation (5):

$$\text{Foam capacity} \left( \% \right) = \frac{\text{Volume foam}}{\text{Volume start}} \times 100 \tag{5}$$
