Microalgae are unicellular marine organisms that have promoted complex biochemical pathways to survive in greatly competitive marine environments. They could contain significant amounts of high-quality proteins which, because of their structural diversity, contain a range of yet undiscovered novel bioactive peptides. In this work, a peptidomic platform was developed for the separation and identification of bioactive peptides in protein hydrolysates. In this work, a peptidomic platform was developed for the extraction, separation, and identification of bioactive peptides in protein hydrolysates. Indeed, extraction of proteins from recalcitrant tissues is still a challenge due to their strong cell walls and high levels of non-protein interfering compounds. Therefore, seven different protein extraction protocols, based on mechanical and chemical methods, were tested in order to produce high-quality protein extracts. Proteins obtained by means of the best protocol, consisting of milling the recalcitrant tissue with glass beads, were subjected to enzymatic digestion with Alcalase® and subsequently the hydrolysate was purified by two-dimensional semi-preparative reversed phase liquid chromatography. Fractions were assayed for antioxidant and antihypertensive activities and only the most active ones were finally analyzed by RP nanoHPLC-MS/MS. Around 500 peptide sequences were identified in these fractions. The identified peptides were subjected to an in silico analysis by PeptideRanker algorithm in order to assign a score of bioactivity probability. Twenty-five sequenced peptides were found with potential antioxidant and angiotensin-converting-enzyme-inhibitory activities. Four of these peptides, WPRGYFL, GPDRPKFLGPF, WYGPDRPKFL, SDWDRF, were selected for synthesis and in vitro tested for specific bioactivity, exhibiting good values of antioxidant and ACE-inhibitory activity.
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This work has been carried out within the framework of the Research Project “Microalgae as a source of bioactive compounds: chromatographic fractionation of peptides and lipids and their mass spectrometric characterization,” supported by Sapienza, no. RM11715C82118E74.
Moreover, the authors wish to thank Prof. Francesca Pagnanelli for providing the microalgae samples.
Conflict of interest
The authors declare that they have no conflict of interest
Published in the topical collection Discovery of Bioactive Compounds with guest editors Aldo Laganà, Anna Laura Capriotti and Chiara Cavaliere.
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Montone, C.M., Capriotti, A.L., Cavaliere, C. et al. Peptidomic strategy for purification and identification of potential ACE-inhibitory and antioxidant peptides in Tetradesmus obliquus microalgae. Anal Bioanal Chem 410, 3573–3586 (2018). https://doi.org/10.1007/s00216-018-0925-x
- Protein extraction methods
- Antioxidant peptides
- ACE-inhibitory peptides
- Off-line two-dimensional chromatography
- High resolution mass spectrometry