Protein Extraction from BSF and Mealworm

The protein extraction process for BSF and mealworm to obtain the protein concentrates was based on that of Wang et al. [5 (link)] with some modifications. The defatted TM or partially defatted BSF powder (30 g) was mixed with 0.25 M NaOH solution at a ratio 1:5 (w/v) separately and the mixture was heated to 40 °C for one hour with constant agitation at 400 rpm on a magnetic stirrer (RCT ST, IKA, Königswinter, Germany). The mixture was centrifuged (Meditronic 7000599, J.P. SELECTA, Barcelona, Spain) at 3200× g for 15 min and the supernatant was separated to continue the protein extraction. In BSF supernatant samples the lipid fraction on the top layer was carefully separated. The pH (Accumet AE150, Fisher Scientific, Singapore) of the supernatant was adjusted to reach a value between 4.0–4.2 by adding 37% hydrochloric acid followed by centrifugation at 2233× g for 15 min. After centrifugation, the pellets were collected in aluminum petri dishes and were kept at −60 °C. The entire process was repeated twice with the pellets that remained after the first centrifugation step. The pellets from the three centrifugation processes were combined and freeze-dried (LYOQUEST-85 PLUS, Telstar, Barcelona, Spain) for 24 h at 0.2 mbar with the plates heated to 20 °C. Freeze-dried protein concentrates were stored in the fridge until further use.