Identification of the major ACE-inhibitory peptides produced by enzymatic hydrolysis of a protein concentrate from cuttlefish wastewater

Abstract

The aim of this work was the purification and identification of the major angiotensin converting enzyme (ACE) inhibitory peptides produced by enzymatic hydrolysis of a protein concentrate recovered from a cuttlefish industrial manufacturing effluent. This process consisted on the ultrafiltration of cuttlefish softening wastewater, with a 10 kDa cut-off membrane, followed by the hydrolysis with alcalase of the retained fraction. Alcalase produced ACE inhibitors reaching the highest activity (IC₅₀ = 76.8 ± 15.2 μg mL⁻¹) after 8 h of proteolysis. Sequential ultrafiltration of the 8 h hydrolysate with molecular weight cut-off (MWCO) membranes of 10 and 1 kDa resulted in the increased activity of each permeate, with a final IC₅₀ value of 58.4 ± 4.6 μg mL⁻¹. Permeate containing peptides lower than 1 kDa was separated by reversed-phase high performance liquid chromatography (RP-HPLC). Four fractions (A-D) with potent ACE inhibitory activity were isolated and their main peptides identified using high performance liquid chromatography coupled to an electrospray ion trap Fourier transform ion cyclotron resonance-mass spectrometer (HPLC-ESI-IT-FTICR) followed by comparison with databases and de novo sequencing. The amino acid sequences of the identified peptides contained at least one hydrophobic and/or a proline together with positively charged residues in at least one of the three C-terminal positions. The IC₅₀ values of the fractions ranged from 1.92 to 8.83 μg mL⁻¹, however this study fails to identify which of these peptides are ultimately responsible for the potent antihypertensive activity of these fractions.

Figure 1

Protein recovery from cuttlefish wastewater by UF-DF at 10 kDa molecular weight cut-off (MWCO). Left: concentration of retained protein in linear relation with the volumetric concentration factor (fc) showing experimental data (points) and theoretical profiles (discontinuous line). Right: Progress of protein (○) retention with the increase of diavolume from DF process (D). For clarity, confidence intervals (in all cases less than 5% of the experimental mean value; α = 0.05; n = 2) were omitted. Equation (1) was used to fit the experimental data.

Figure 2

Left: Experimental data (symbols) for ACE-inhibitory activity of cuttlefish softening wastewater protein concentrate before (●) and after hydrolysis with alcalase for 0.5 (○), 2 (△), and 8 h (□). Right: Experimental data (symbols) for ACE-inhibitory activity of 10 kDa (▽) and 1 kDa (▼) retentates and 1 kDa permeate (▲) from the 8 h hydrolysate. The dose-response curves (lines) were obtained by modeling experimental data to the Equation (4).

Figure 3

Process of ACE inhibitory peptide production from a protein enriched fraction recovered from cuttlefish wastewater by UF-DF at 10 kDa (Step 1) followed by hydrolysis with alcalase (0.4% v/v) for 8 h at 55 °C (Step 2). Then, the hydrolysate was fractionated using a sequentially UF through membranes of 10 (Step 3) and 1 kDa (Step 4). UFP: Permeates of UF; UFR: retentates of UF.

Figure 4

Peptide profile of the 8 h hydrolysate that was permeated through the 1 kDa ultrafiltration membrane after RP-HPLC separation. Lines indicate the peaks corresponding to the four active fractions (A–D).