*3.3. Solubility of Insect Meals and Hydrolysates*

Table 3 shows the protein solubility of cricket and mealworm meals and their hydrolysates (control and generated from pressure-treated insect meals) at different pH values (4.0, 5.5 and 7.0) and concentrations (0.5–3.0% *w*/*v*). The sample concentration (0.5–3.0% *w*/*v*) and pH values (4.0, 5.5 and 7.0) modified the protein solubility of insect meals. Regardless of pH and concentration, cricket and mealworm meals had low solubility with values ranging from 17.1 to 18.7% and from 15.8 to 20.2%, respectively. These values are consistent with those available in the literature [3,11,61,63]. More specifically, Stone [60] obtained protein solubility ranging from 29.0 to 23.4% between pH 3.0 and 7.0 for commercial cricket and mealworm meals. A study published by Kröncke et al. confirmed that oven drying of *T. molitor* larvae decreased the quality of proteins and reduced their solubility by 74% [19]. This low solubility was mainly related to the drying method (oven-drying) applied at commercial scale before the larvae grinding step. The heat treatment denatured the protein, exposing hydrophobic groups and causing protein aggregation [21,64].


**Table 3.** Solubility of cricket and mealworm meals and hydrolysates at different concentrations and pH values, generated with or without HHP pretreatment before enzymatic digestion.

M: insect meal, HT: control hydrolysate, HP: Hydrolysate generated by HHP treatment of insect meal prior to enzymatic hydrolysis. Values represent the mean of three replicates ± standard deviation. Results in the same insects-treatment with different letters (<sup>a</sup> , b , c , d ) are significantly different (*p* < 0.05).

The protein solubility of hydrolysates, which was similar for all pHs (4.0–7.0) and sample concentrations (0.5–3.0% *w*/*v*), was drastically improved compared to insect meals, with values ranging from 92.5 to 100.0% and 72.9 to 78.9%, for cricket and mealworm hydrolysates, respectively. This improvement in solubility is consistent with results obtained by Hall et al. and Wang et al. for cricket and house fly larvae after enzymatic hydrolysis by Alcalase® [3,24]. The digestion of protein into peptides increases ionizable groups, such as amino and carboxyl groups, improving hydration [65] and solubility. Furthermore, the higher solubility was also related to the fact that only the soluble fraction was freeze-dried during hydrolysate preparation [22].

Compared to the control, pressurization pretreatment of cricket meal slightly decreased the hydrolysate solubility, mainly at 1.0 and 3.0% for pH 4.0, 5.5 and 7.0 (Table 3). Protein solubility is influenced by the type of protein, protein concentration, pH and the presence of salts [66]. However, the similar compositions (Table 1) and particle sizes (Table 2) could not explain the differences in solubility between control and pressure-treated hydrolysates for both insect species. Gbogouri et al. [57], studying protein hydrolysate from salmon, showed that hydrolysates with a higher degree of hydrolysis generally had higher solubilities than those possessing lower DH. Indeed, smaller peptides from hydrolysates with high DH were considered to have more polar residues, which could enhance the quantity of hydrogen bonds with water, resulting in an increase in protein solubility in solution [37,67]. However, the similar DH values calculated for cricket hydrolysate (control and pressure treatments) (Table 1) could not explain their differences in solubility. HHP parameters, such as pressure, duration and temperature, also impact protein solubility. Under pressure, the loss of solubility is mainly related to the formation of insoluble high molecular weight protein aggregates due to exposure of hydrophobic residues and/or disulfide bond formation [68,69]. Consequently, during the enzymatic hydrolysis of pressure-treated protein, enzymes break the protein in different ways due to modifications of protein structure since some bonds became inaccessible to the enzymes and, on the contrary, others may be exposed due to conformational changes [36,70,71]. Therefore, Alcalase® hydrolysis of cricket meal pretreated by pressure could generate a different peptide profile that contains more hydrophobic peptides, which could negatively impact hydrolysate solubility. On the contrary, the solubility of mealworm hydrolysate generated from pressure-treated meals increased for all concentrations and pH values (Table 3). Kim et al., studying hemoglobin hydrolysate, mentioned that the hydrolysate solubility could be improved at low DH due to the generation of hydrophobic peptides in lower amounts [72]. Consequently, and as proposed for cricket hydrolysate, the protein unfolding and aggregation due to pressurization could generate a different peptide profile with a larger amount of hydrophilic peptides, improving hydrolysate solubility.
