Abstract
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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Acknowledgements
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.
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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
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DOI: https://doi.org/10.1007/s00216-018-0925-x