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

The discovery of angiotensin-converting enzyme (ACE)-inhibitory peptides from food protein hydrolysates is commonly guided by empirical fractionation or sequence-based prediction, but few screening rules have been evaluated at the hydrolysate level and independently benchmarked across peptide lengths. Here, we developed a length-aware C-terminal screening rule (Rule 5: P1’ ∈ {W, Y, F, P} and P2’ ∈ {L, I, V, K, R, H}) through an experimentally anchored framework. The rule was derived using 24 stratified protein–protease hydrolysates and showed the strongest associations with ACE inhibition (r = 0.711, p < 0.001) and log10(1/IC50) (r = 0.741, p < 0.001) among five evaluated rules. Independent evaluation in 16 composition-weighted commercial hydrolysates retained predictive utility (r = 0.608 for ACE inhibition and r = 0.581 for log10(1/IC50)). Five peptides—VF, GIF, LP, IP, and VP—were selected because they represented the intersection of in silico cleavage prediction, Rule 5 compliance, and corresponding candidate-associated low-mass MALDI features in the experimentally prepared hydrolysates. All five inhibited ACE (IC50 = 19.50–93.19 µM); VF and GIF showed mixed-type inhibition, whereas LP, IP, and VP showed competitive inhibition. External benchmarking against 1429 quantitative ACE-inhibitory peptides established a defined applicability domain: Rule 5 significantly enriched potent peptides among di- and tripeptides (2–3 residues; median IC50, 28.0 vs. 79.0 µM; padj < 0.001, Benjamini–Hochberg-corrected; enrichment factor = 2.5 at IC50 ≤ 1 µM), but enrichment attenuated rapidly as longer sequences were included. Molecular dynamics simulations (200 ns) showed persistent peptide–ACE contact for all five candidates under the simulated conditions. Rule 5 is therefore proposed as a transparent first-pass filter for prioritizing short ACE-inhibitory candidates and protein–protease combinations, rather than as a universal predictor across the full peptide-length spectrum.