Bioassay-guided fractionation approach for determination of protein precursors of proteolytic bioactive metabolites from macroalgae

In the last decades, an upsurge in the occurrence of chronic diseases caused by hypertension and oxidative stress has been observed. The objective of this research was to isolate and characterize groups of antioxidant and anti-hypertensive natural bioactive peptides originating from hydrolyzed proteins of the red seaweeds Solieria chordalis and Palmaria palmata, the green seaweed Ulva lactuca and the brown seaweed Saccharina longicruris. Enzymatic hydrolysis by trypsin and chymotrypsin was performed in order to release bioactive algal peptides. Three antioxidant assays, 2,2-diphenyl-1-picrylhydrazyl (DPPH), ferric ion reducing antioxidant power (FRAP) and oxygen radical absorbance capacity (ORAC), were used to determine the activity of seaweed peptides. Angiotensin-converting enzyme (ACE) inhibition assay was used to evaluate the anti-hypertensive potential. Additionally, liquid chromatography-tandem mass spectrometry (LC-MS/MS) analyses and database searches allowed for protein identification, indicating where these bioactive peptides originated from. Fractions of red seaweeds, especially S. chordalis, produced by chymotrypsin and trypsin hydrolysis, exhibited higher in vitro antioxidant and ACE inhibitory activities than the parent proteins. Size exclusion chromatography highlighted two groups of bioactive peptides: (i) peptides with molecular weight (MW) between 1,400 and 3,200 Da displaying antioxidant activities and (ii) smaller peptides (MW < 1,000 Da) displaying both antioxidant and ACE inhibitory activities. LC-MS/MS analyses of the antioxidant subfractions of P. palmata and S. chordalis were performed, and several peptide sequences were elucidated, using tandem mass spectrometry. Of all identified peptides of S. chordalis, 61 and 43 % came from the hydrolysis of ribulose-1, 5-biphosphate carboxylase/oxygenase (RuBiSCo) enzymes. Phycoerythrin, elongation factors, photosystems and cytochrome oxidase clusters represented between 3 and 14 % of identified peptides in S. chordalis fractions. This is the first time, to the author's knowledge, that distinction between several groups of bioactive peptides, within seaweed protein hydrolysates, has been reported. In addition, proteins of origin were also identified.