2.2.2. Protein Extraction

Soluble proteins from the TMI and TMD fractions were recovered as described by Yi et al. (2013), with modifications [24]. First, TMI and TMD fractions were solubilized in a 9.5 mM ascorbic acid solution in a 1:8 (*w*/*v*) ratio and magnetically stirred overnight at 10 ◦C. The suspensions were then centrifuged at 15,000× *g* for 30 min at 4 ◦C and the supernatant containing the soluble proteins was filtered three times through a Whatman® Grade 4 filter paper in a Büchner funnel. Both filtrates were freeze-dried, producing two different fractions: a hexane-defatted soluble protein (HDSP) and a non-hexane-defatted soluble protein (NHSP) extract from *T. molitor*.

The detailed methodology used to generate the different fractions is illustrated in Figure 1.

Molecules 2021, 26, x FOR PEER REVIEW 3 of 14

different fractions: a hexane-defatted soluble protein (HDSP) and a non-hexane-defatted soluble protein (NHSP) extract from T. molitor. The detailed methodology used to generate the different fractions is illustrated in

Figure 1. Experimental design for the production of defatted and non-defatted mealworm meals and protein extracts. **Figure 1.** Experimental design for the production of defatted and non-defatted mealworm meals and protein extracts.

#### 2.3.1. Proximate Composition *2.3. Analysis*

#### The proximate composition was determined for the four T. molitor fractions (TMI, 2.3.1. Proximate Composition

2.3. Analysis

Figure 1.

TMD, HDSP, and NDSP). The protein content was measured using the Dumas Method [19,25] (Elementar rapid Micro N cube, Langenselbold, Germany), with a nitrogen-to-protein conversion factor of 4.76 for TMI and TMD, and 5.60 for HDSP and NDSP, accounting for the high chitin content of TMI and TMD fractions and therefore allowing for a more accurate protein content determination, as suggested by Janssen et al. (2017) [26]. The lipid content was determined using the Mojonnier method (AOAC 925.32). Moisture and ash content were determined by official methods AOAC 950.46 (A) and AOAC 920.153, respectively [27]. The method described by Spinelli et al. (1974) was used to determine the chitin content of the four fractions [28]. The proximate composition was determined for the four *T. molitor* fractions (TMI, TMD, HDSP, and NDSP). The protein content was measured using the Dumas Method [19,25] (Elementar rapid Micro N cube, Langenselbold, Germany), with a nitrogen-to-protein conversion factor of 4.76 for TMI and TMD, and 5.60 for HDSP and NDSP, accounting for the high chitin content of TMI and TMD fractions and therefore allowing for a more accurate protein content determination, as suggested by Janssen et al. (2017) [26]. The lipid content was determined using the Mojonnier method (AOAC 925.32). Moisture and ash content were determined by official methods AOAC 950.46 (A) and AOAC 920.153, respectively [27]. The method described by Spinelli et al. (1974) was used to determine the chitin content of the four fractions [28].
