Identification and Prediction of Bioactive Peptides from Anchovy (Engraulidae) Based on Processing Methods: Sub-Fragment Analysis via LC-HRMS Proteomics

Abstract

This exploratory study aimed to identify and compare peptide profiles and predicted bioactivities of anchovies (Engraulidae) in 3 forms-Raw, processed, and fortified processed-using LC HRMS based proteomics combined with BIOPEP UWM and PeptideRanker analyses. Peptides generated by tryptic digestion were analyzed by LC HRMS, matched to an Engraulidae protein database, and evaluated in silico to annotate potential biological activities and estimate intrinsic bioactivity scores. In this non replicated dataset, raw anchovies showed the highest proteomic complexity (254 protein IDs and 1,304 peptide sequences), whereas processed and fortified samples exhibited fewer proteins and peptides, particularly with chain lengths ≥ 4 amino acids. Across all samples, the most frequent predicted activities were Dipeptidyl Peptidase IV (DPP IV) (27%-31%) and Angiotensin Converting Enzyme (ACE) inhibition (± 24%), supported by antioxidant, neprilysin inhibitory (3%-6%), and other enzyme inhibitory annotations. Short peptides (2-3 amino acids) dominated the predicted bioactive profiles, especially in processed and fortified products, indicating a shift toward shorter chains. Although total peptide numbers decreased after processing, the proportion of peptides with multiple annotated activities and high PeptideRanker scores (≥ 0.8) was descriptively higher in fortified samples (14.58%). These findings provide a preliminary molecular map suggesting that anchovy proteins can act as precursors of peptides with putative multifunctional bioactivities and may represent candidates for anchovy based functional foods or nutraceuticals. However, all functional inferences are based solely on sequence level, in silico predictions from single run LC HRMS data; the patterns observed are hypothesis generating and require confirmation through replicated experiments and targeted in vitro and in vivo validation.