A novel angiotensin I-converting enzyme inhibitory peptide derived from the trypsin hydrolysates of salmon bone proteins

Abstract

When fish are processed, fish bone becomes a key component of the waste, but to date very few researchers have sought to use fish bone to prepare protein hydrolysates as a means of adding value to the final product. This study, therefore, examines the potential of salmon bone, through an analysis of the benefits of its constituent components, namely fat, moisture, protein, and ash. In particular, the study seeks to optimize the process of enzymatic hydrolysis of salmon bone with trypsin in order to produce angiotensin-I converting enzyme (ACE) inhibitory peptides making use of response surface methodology in combination with central composite design (CCD). Optimum hydrolysis conditions concerning DH (degree of hydrolysis) and ACE-inhibitory activity were initially determined using the response surface model. Having thus determined which of the salmon bone protein hydrolysates (SBPH) offered the greatest level of ACE-inhibitory activity, these SBPH were duly selected to undergo ultrafiltration for further fractionation. It was found that the greatest ACE-inhibitory activity was achieved by the SBPH fraction which had a molecular weight lower than 0.65 kDa. This fraction underwent further purification using RP-HPLC, revealing that the F7 fraction offered the best ACE-inhibitory activity. For ACE inhibition, the ideal peptide in the context of the F7 fraction comprised eight amino acids: Phe-Cys-Leu-Tyr-Glu-Leu-Ala-Arg (FCLYELAR), while analysis of the Lineweaver-Burk plot revealed that the FCLYELAR peptide can serve as an uncompetitive ACE inhibitor. An examination of the molecular docking process showed that the FCLYELAR peptide was primarily able to provide ACE-inhibitory qualities as a consequence of the hydrogen bond interactions taking place between ACE and the peptide. Furthermore, upon isolation form the SBPH, the ACE-inhibitory peptide demonstrated ACE-inhibitory capabilities in vitro, underlining its potential for applications in the food and pharmaceutical sectors.

Fig 1. Influence of temperature (a), hydrolysis time (b), and E/S ratio (c) upon DH (●) and ACE-inhibitory activity (●) of the hydrolysate.

Each data value is presented in the form of mean ± S.D. and the tests were conducted in triplicate.

Fig 2. Response surface and contour plots presenting the influence of interactions among independent variables upon the degree of hydrolysis (%DH).

(a) influence of temperature and time, (b) influence of temperature and E/S ratio, and (c) influence of time and E/S ratio.

Fig 3. Response surface and contour plots presenting the influence of interactions among independent variables upon ACE-inhibitory activity (IC₅₀; μg/mL).

(a) influence of temperature and time, (b) influence of temperature, and E/S ratio, and (c) influence of time and E/S ratio.

Fig 4. The RP-HPLC chromatogram of the molecular weight cut-off < 0.65 kDa fraction of the trypsin hydrolysate obtained from salmon bones.

Fig 5. MS/MS spectrum analysis and amino acid sequence details for the ACE-inhibitory FCLYELAR peptide from the F₇ fraction obtained via RP-HPLC.

Fig 6

(a) The Lineweaver-Burk plot illustrating the influence of FCLYELAR uncompetitive inhibition upon ACE. The ACE-inhibitory activities were assessed when various concentrations of FCLYELAR peptide were present or absent (●, control; $●$, 0.05 mM; $●$, 0.1 mM; $●$, 0.2 mM). 1/V and 1/S are the respective reciprocals of the velocity and substrate. Each of the points is presented in the form of mean value ± Std. Deviation with tests performed in triplicate, and (b) the secondary plots showed the determination of the inhibitor constant (K_i) in the case of the uncompetitive inhibition by FCLYELAR.

Fig 7. Simulations of molecular docking for FCLYELAR binding to ACE (PDB: 1O8A).

(a) Broad overview and narrow localized perspective for the 3D structure prediction of the peptide-ACE complex; (b) Anticipated interactions involving the synthesized FCLYELAR peptides and ACE residues (Images produced using Discovery Studio Visualizer v19.1.0 software), (c) 2D representation of the anticipated interactions involving the synthesized FCLYELAR peptides and ACE molecules (Images produced using Discovery Studio Visualizer v19.1.0 software).