*3.5. Gelation Properties*

Gel formation was not observed with insect meals and hydrolysates (control and generated from pressure-treated insect meals) at 3% (*w*/*v*) or 10% (*w*/*v*), which is probably caused by the denaturation of proteins during the drying method used for commercial production of insect meals. Hydrolysates are also generally known to have poor gelling properties [58,74]. Protein concentration is a key factor influencing gelation properties and the gelation threshold depends on structural characteristics and gelling conditions such as pH and ionic strength [76–78]. Therefore, the concentrations used (3 and 10% *w*/*v*) in our study were probably insufficient to reach the gelation threshold.

The gelation abilities of higher concentrations of insect meals were evaluated using dynamic rheology. The results were similar for both insect meals regardless of the experimental conditions (10, 20 and 30% *w*/*v* at pH 7.0, with or without 1M NaCl). The rheological behavior of cricket meal is given as an example (Figure 1A). For cricket and mealworm meals, the elasticity modulus (G') was higher than the loss modulus (G"), which indicates that gelation occurred, regardless of pH and ionic strength. However, the higher G' values that should indicate gelation were observed before heat treatment, suggesting that a protein gel may not be responsible for this rheological behavior. The insect meals had very low solubility (15.84–20.17%, Table 3) and the insoluble particles settled quickly after agitation. In addition, certain ionic strengths could not be analyzed because the maximum axial force of the device was reached before the analysis could be performed, also suggesting that a packed precipitate formed at the bottom of the geometry. To confirm that no gelation occurred, the experimental conditions described at the beginning of this section were reproduced in glass tubes. Two distinct phases (soluble and insoluble phases) were observed (Figure 2A). The texture of the insoluble phase was similar to wet sand while the upper phase was liquid. After heat treatment, the two phases had a similar texture, but the proportion of insoluble phase seemed to increase compared to the soluble fraction (Figure 2A). From these observations, the changes in rheological behavior observed (in Figure 1A) could be explained by temperature-induced modification of the physico-chemical

characteristics of the precipitated phase. Consequently, G' and G" moduli decreased during the heating phase and increased during the cooling phase, which is representative of protein interactions where the hydrophobic interactions increase with increasing temperature and hydrogen bonds are favored with cooling [79,80]. Zhao et al. [61] also observed this phenomenon for a mealworm protein concentrate. Moreover, the G' and G" moduli were slightly lower until the cooling step and similar after this step. These authors demonstrated that the effect of the viscoelastic moduli of the salt concentration was pH-dependent [81]. In the literature, only few studies mention gelation properties of insect proteins [41,61]. More specifically, Yi et al. [41] obtained gelation of an *A. domesticus* soluble fraction (3% *w*/*v*) at pH 7.0 and gelation of different insect meal soluble fractions (*T. molitor, A. diaperinus, Z. morio* and *B. dubia*), but at high concentration (30% *w*/*v*) for pH 7.0 and 5.0. Otherwise, the authors generally obtained only an aggregation induced by the heat treatment [41]. Zhao et al. [81] obtained a weak gel after adding 2% NaCl to a mealworm protein concentrate since NaCl can improve the gelation properties of proteins by reducing the repulsive forces between proteins. migratory locust meals using rapeseed oil (1.10 goil/g). These differences may be due to the use of different oils, but many other factors such as the amino acid composition and degree of denaturation of the proteins may also have had an effect [85]. Similar to WBC, the insect OBC is generally lower than for other proteins, such as soy protein isolate (1.54 goil/g), red kidney beans (1.23–1.52 goil/g) and Indian kidney beans (2.40 goil/g) [85]. The OBC of control hydrolysates increased from 0.87 to 2.23 goil/g and from 0.77 to 1.21 goil/g for cricket and mealworms, respectively. This tendency, also observed by Purschke et al. [20] for migratory locust protein following enzymatic hydrolysis by Neutrase and Flavourzyme (alone or in combination), is due to the exposure of hydrophobic groups that were previously hidden in the edible insect protein structure [20,87]. While HHP did not impact the OBC of cricket hydrolysate, it doubled OBC for mealworm hydrolysate compared to the control (1.21 to 2.42 goil/g). The decrease in the degree of hydrolysis between control and HHP mealworm hydrolysates possibly explains this tendency, as Chalamaiah et al. [22] showed that a higher degree of hydrolysis reduces oil binding capacity. The HHP treatment probably modified the protein structures and peptide profile due to different enzyme breaks. interface [23,57,89]. Chatsuwan et al. [90] obtained EAI values ranging from 29.23 to 36.69 m2/g for *P. succinta* and *C. rosea*, respectively. Regardless of the insect species, the lower EAI values in our study can be attributed to the drying method used to produce commercial insect meals, which modifies protein solubility [19] and emulsifying properties [91]. The EAI (*p* > 0.05) of the cricket hydrolysate control was not significantly different from the meal. This tendency was also observed by Hall et al. for some enzymatic hydrolysis conditions of cricket using Alcalase® (E/S of 0.5%; hydrolysis time of 90 min and E/S of 1.5%; hydrolysis time of 30 min) compared to unhydrolyzed proteins [3]. Conversely, the EAI value of the mealworm control hydrolysate was reduced compared to the meal. Hall et al. also obtained a decreased EAI after enzymatic hydrolysis of tropical banded cricket with similar enzymatic digestion conditions (E/S of 3%; hydrolysis time of 90 min) [3]. This reduction of EAI was possibly caused by the enzymatic hydrolysis parameters which would have reduced the interfacial activity of proteins due to excessive protein degradation [57,92]. However, several studies have shown that the enzymatic hydrolysis of several proteins using Alcalase® improved their EAI values [23,38,88]. This has been explained by the enzyme's specificity in cutting aromatic residues to

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meal (1.58–1.72 gwater/g) was higher than mealworm meal (1.24–1.31 gwater/g), probably because mealworms have a lower hydrophilic amino acid content than crickets [55]. Our values for commercial cricket and mealworm meals (1.76 gwater/g and 1.62 gwater/g, respectively) were consistent with those published by Stone [60]. The WBC of insects is generally lower than several vegetable proteins such as soy protein isolate (4.47 gwater/g), red kidney beans (2.25–2.65 gwater/g) and Indian kidney beans (2.60 gwater/g) [85]. Many factors influence the WBC, such as the amino acid profile, conformation, hydrophobicity, pH, ionic strength, temperature and protein concentration [86]. The water binding capacity at pH 4.0 was significantly (*p* < 0.05) lower than at pH 5.5 or 7.0. As previously mentioned, the pI of commercial cricket and mealworm meals is close to 3.85 and 3.95, respectively. The pH affects the charge on proteins and, consequently, close to the pI, the WBC is minimal since

Figure 1B also shows the OBC of insect meals and hydrolysates (control and generated from pressure-treated insect meals). The OBC was similar for both insect meals with values ranging from

Figure 2A shows the EAI values of cricket and mealworm meals and their hydrolysates (control and generated from pressure-treated insect meals). Higher EAI values mean that the dispersed fat droplets are smaller in size and that proteins (or peptides) have more ability to absorb at the oil– water interface [88]. Globally, cricket ingredients (11.86–13.32 m2/g) had higher EAI values compared

mealworm meals (1.42 goil/g and 1.58 goil/g, respectively) and by Purschke et al. [20] for commercial

proteins and the smaller size of cricket proteins, which would allow faster diffusion at the oil–water

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protein–protein interactions are favored over protein–water interactions [86].

**Figure 1.** Water binding capacity (**A**) and oil binding capacity (**B**) of insect meals (M) and hydrolysates (HT: control hydrolysate, HP: hydrolysate generated by HHP treatment of meal prior to enzymatic hydrolysis). Different letters indicate significant difference (*p* < 0.05). **Figure 1.** Water binding capacity (**A**) and oil binding capacity (**B**) of insect meals (M) and hydrolysates (HT: control hydrolysate, HP: hydrolysate generated by HHP treatment of meal prior to enzymatic hydrolysis). Different letters indicate significant difference (*p* < 0.05). increasing pH (4.0–7.0) due, once again, to solubility modification. Pacheco-Aguilar et al. [88] obtained similar EAI values for fish (*Mercuccius productus*) protein hydrolysate at pHs 4.0 and 7.0, but higher values at pH 10.0 and a different degree of hydrolysis (DH 10–20%).

**Figure 2.** Emulsion activity index (EAI) of insect meals (M) and hydrolysates (HT: control hydrolysate, HP: hydrolysate generated by HHP treatment of meal prior to enzymatic hydrolysis) according to the insect source (cricket and mealworms) (**A**) and according to the pH (4.0–7.0) (**B**). Different letters indicate significant difference (*p* < 0.05).
