Peptidomic profiling of in vitro digesta from diverse microalgae species

Microalgae are a sustainable protein source, yet few species are used for human nutrition. This study evaluated eight species for protein quality, digestibility, and peptide profiles after simulated gastrointestinal digestion using INFOGEST. Amino acid content, and intestinal-phase peptide mixtures were analysed by mass spectrometry. The methanol precipitation method was used to separate digested (supernatant) and undigested (pellet) peptides following in vitro INFOGEST digestion. The pellet was further separated by molecular weight into 3-10 kDa and >10 kDa fractions to isolate the lower molecular weight peptides from the higher ones. This separation method enabled the identification of potential digested peptides within the 3-10 kDa fraction that may still be present in the pellet. Cell-based and in silico analysis showed the 3-10 kDa fraction contained permeable peptides, highlighting 80% methanol's limitation in recovering all digestible peptides in the INFOGEST protocol. However, discrepancies between in silico predictions and in vitro permeability revealed that machine learning approaches alone are insufficient, underscoring the critical importance of experimental validation. The >10 kDa fraction predominantly contained structural proteins for photosynthesis, indicating digestion resistance. Pandorina sp. and Glaucocystis nostochinearum exhibited essential amino acid-rich profiles, especially their high Lysine content which is a limiting amino acid in most plant-derived proteins. These findings reveal digested microalgae's peptide composition and potential as functional food ingredients.