Olive Pomace Extract Contains Low Molecular Weight Peptides and Possesses ACE Inhibitory Activity

Abstract

The aim of the present study was to determine the ACE inhibitory activity of aqueous extracts of olive pomace and to understand whether they represent a good source of bioactive LMW peptides for nutritional and pharmacological applications. We produced a water extract from olive pomace (var. Picual) and obtained its low molecular weight (LMW) fraction (<3 kDa). The calculated yield of extraction was 100.2 ± 7.9 mg of LMW peptides per 100 g of olive pomace. The olive pomace LMW fraction possessed strong ACE inhibitory activity (IC₅₀ = 3.57 ± 0.22 µg prot/mL). The LMW fraction (<3 kDa) was analysed by nanoscale liquid chromatography-Orbitrap coupled with tandem mass spectrometry and de novo sequencing. Thirty new peptides, containing between 7-17 amino acids and molecular masses ranging 778-1354 Da, were identified by the Peaks database algorithm using the available Olea europaea (cv. Farga) genome database. Ten new peptides were also identified by Peaks de novo sequencing. The protein sources of twelve peptides detected in the database by Peaks DB were identified by BLAST search. The ACE inhibitory activity of the identified peptides was predicted by BIOPEP software. We conclude that olive pomace possesses ACE inhibitory activity and contains low molecular weight peptides with (predicted) biological activity. Olive pomace may represent a good source of peptides for nutritional and pharmaceutical applications. In our study, it has been shown that olive pomace possesses ACE inhibitory activity and contains low molecular weight peptides with (predicted) biological activity. Olive pomace may represent a good source of peptides for nutritional and pharmaceutical applications. More research is needed in order to identify the in vivo effects of olive pomace bioactive peptides.

Keywords: angiotensin converting enzyme (ACE); bioactive peptides; food peptides; olive pomace; peptidomics.

Figure 1

Amino acid profile measured by HPLC of olive pomace extract and its low molecular weight (LMW, <3 kDa) fraction: (A) mix of seventeen amino acid standards (at 0.1, 0.2, and 0.3 mM concentrations); (B) amino acid profile of the LMW fraction; (C) amino acid composition of olive pomace extract and the LMW fraction. AAD, L-alpha-amino adipic acid; NLE, DL-norleucine. The other large chromatographic peaks appearing in (B) originate from the phenyl isothiocyanate derivatising agent used in the analysis. Data are expressed as means ± standard deviation (SD).

Figure 2

Native polyacrylamide gel electrophoresis of low molecular weight (LMW) peptide fraction extracted from olive pomace. Panel (A), protein staining with trihalo compounds. Lane 1, protein standard of bovine serum albumin (5 µg); lanes 2–4, olive pomace LMW fraction containing 2.5, 5, and 7.5 µg of proteins, respectively. Panel (B), superoxide dismutase (SOD) activity detected in LMW peptide fraction by a photochemical method using nitrobluetetrazolium. Lanes 1 and 2, SOD activity detected in an extract of Olea europaea showing (from top to bottom) isoenzymes of Mn-, Fe- and Cu,Zn-SOD (control); lanes 3, 4, and 5, olive pomace LMW fraction containing 2.5, 5, and 7.5 µg of proteins, respectively, showing bands of superoxide dismutase antioxidant activity (see arrows).

Figure 3

Fractionation of LMW peptides extracted from olive pomace by gel filtration chromatography and FPLC. F, fraction.

Figure 4

(A) Angiotensin-converting enzyme inhibitory activity (ACEi) of water-soluble LMW peptides extracted from olive pomace and (B) calibration curve obtained from the data of panel A and used to determine IC₅₀. Data are expressed as the mean of three experiments.