Improving protein extraction and peptide production from using combined mechanical/physical and enzymatic pre-treatments.

is a microalga rich in proteins with potential applications in food and feed industries. However, the presence of a cellulose-containing cell wall, which is a major barrier to protein extraction, together with fibroproteinaceous complexes, limits the bioaccessibility of nutritional and bioactive proteins and peptides from biomass. Therefore, this study aimed to evaluate the effect of different mechanical/physical pre-treatments (bead milling, extrusion, freeze-drying, heating, microwave and sonication) combined or not with enzymatic treatments (commercial trypsin and pancreatin) on protein extraction and peptide formation from a suspension. The amount of total protein and peptides released to the supernatant was quantified by Bradford and -phthaldialdehyde assays, respectively. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis was used to analyse the extracted protein fractions. The results showed that extrusion caused a 3-fold increase in total peptides (p < 0.001) compared to no-pretreatment, and trypsin increased peptides formed in bead-milled (p = 0.020) and freeze-dried (p = 0.021) microalga relative to those pre-treatments alone. Some pre-treatments, such as bead milling and microwave, were effective in releasing specific protein fractions, particularly those from 32 to 40 kDa (up to 1.2-fold), compared to control. Pancreatin combined with bead milling decreased 32 to 40 kDa- and 26 kDa-protein fractions (p < 0.010) compared with the sole use of mechanical treatment, whereas the same enzyme mixture associated with microwave produced a similar result for 26 kDa-protein fraction (p = 0.023). Pancreatin also effectively reduced the total protein fraction released after pre-treatment with sonication (p = 0.013). These findings suggest that combining different pre-treatments and enzymatic treatments could improve protein extraction from biomass, providing a useful approach for the development of sustainable protein sources. The present results highlight the need for further studies to assess the efficacy of extrusion in improving the bioaccessibility of proteins in monogastric animals' diets.