Accumet ae150

The pH of WM and SM samples with fucoxanthin and their controls without fucoxanthin was determined throughout the four week storage period using an Accumet benchtop pH meter (Fisherbrand™ Accumet™ AE150, Fisher Scientific, Pittsburgh, PA, USA) at 22 °C. The reference buffers (4.00 and 7.00; Orion buffer solutions; Thermo Fisher Scientific, Pittsburgh, PA, USA) that were used for the calibration of pH meter and the analyzed milk samples were at room temperature (22 °C). The titratable acidity in the milk samples was determined according to the AOAC method No 947.05 [24 ]. Briefly, to each 20 mL sample of goat milk, a 40 mL of boiled and cooled distilled water was added. Phenolphthalein (1% w/v in 95% ethanol) was used as an indicator. The mixture was titrated with standardized 0.1 N NaOH until the first color change, which signals the endpoint (at pH 8.1–8.3), persisted for 30 s. One more drop of 0.1 N NaOH was added and the final volume of NaOH used for titration was recorded. The results were expressed in percentage of lactic acid. The pH and titratable acidity in the samples were determined in triplicate.

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.

The pH was determined by direct potentiometry while using a benchtop digital pH meter (Fisherbrand accumet™ AE150, Leicestershire, UK).