2.4.1. Determination of the Degree of Hydrolysis

The degree of hydrolysis (DH), which is the proportion of peptide bonds released during in vitro protein digestion, was calculated according to the method that was described by Church et al. [28] for mealworm hydrolysates collected during and at the end of enzymatic hydrolysis, for control, pretreated, and simultaneous conditions. Briefly, 150 µL of mealworm hydrolysates (5% w/V) diluted by a factor of 60 was added to 3 mL of o-phthaldialdehyde (OPA) reagent. The mixture was incubated at room temperature for 2 min, transferred to polystyrene cuvettes and the absorbance at 340 nm measured in an Agilent 8453 UV-Visible spectrophotometer (Agilent Technologies, Santa Clara, CA, USA). DL-leucine was used as a standard with concentrations ranging from 0.75 mM to 3 mM. All of the samples were analyzed in triplicate. The DH was calculated using Equation (1):

$$\text{DH} \left( \% \right) = \left[ \frac{h}{htot} \right] \times 100 \tag{1}$$

where *htot* of mealworm was determined from the amino acid composition of the protein, as the sum of mmols of the individual amino acids per g. The *htot* used in this study was 8.64 meq/g. The values of (*h*) were obtained by reference to a standard curve of absorbance at 340 nm versus mg/L amino nitrogen (using L-leucine) [29].
