A Length-Aware C-Terminal Rule for Prioritizing Short ACE-Inhibitory Peptides from Food Protein Hydrolysates

Area of Science:

• Biochemistry
• Food Science
• Pharmacology

Background:

• Discovering angiotensin-converting enzyme (ACE)-inhibitory peptides from food protein hydrolysates typically relies on empirical fractionation or sequence prediction.
• Existing methods lack robust screening rules evaluated at the hydrolysate level and benchmarked across diverse peptide lengths.
• There is a need for efficient and reliable methods to identify ACE-inhibitory peptides for potential health applications.

Purpose of the Study:

• To develop and validate a length-aware C-terminal screening rule (Rule 5) for identifying ACE-inhibitory peptides.
• To evaluate the predictive utility of Rule 5 at both the hydrolysate and individual peptide levels.
• To establish the applicability domain and inhibitory mechanisms of peptides identified using Rule 5.

Main Methods:

• Development of Rule 5 (P1' ∈ {W, Y, F, P} and P2' ∈ {L, I, V, K, R, H}) using 24 stratified protein-protease hydrolysates.
• Independent evaluation of Rule 5 in 16 commercial hydrolysates and benchmarking against 1429 known ACE-inhibitory peptides.
• In silico cleavage prediction, MALDI-TOF analysis, in vitro ACE inhibition assays, and molecular dynamics simulations were employed.

Main Results:

• Rule 5 demonstrated strong associations with ACE inhibition (r = 0.711) and potency (log10(1/IC50), r = 0.741) in derived hydrolysates.
• The rule retained predictive utility in independent evaluations (r = 0.608 for ACE inhibition, r = 0.581 for log10(1/IC50)).
• Rule 5 significantly enriched potent di- and tripeptides (median IC50, 28.0 µM) and identified five specific ACE-inhibitory peptides (VF, GIF, LP, IP, VP).

Conclusions:

• Rule 5 serves as an effective, transparent first-pass filter for prioritizing short ACE-inhibitory peptides and relevant protein-protease combinations.
• The rule's predictive power is most significant for di- and tripeptides, with enrichment diminishing for longer sequences.
• This approach facilitates the discovery of novel ACE inhibitors from food sources for potential use in functional foods and pharmaceuticals.