Efficient Screening of Synergistic Antioxidant and <scp>AChE</scp> Inhibitory Peptides From Sea Cucumber (<i>Stichopus japonicus</i>) Using a Novel Approach Combining De Novo Sequencing and Parallel Peptide Synthesis

ABSTRACT In this study, we report for the first time that de novo sequencing can be combined with parallel peptide synthesis to facilitate high‐throughput activity screening of biological peptides from natural food. Sea cucumber ( Stichopus japonicus ) intestine was utilized as an example, undergoing gastrointestinal digestion followed by de novo sequencing to predict potential peptide sequences. These peptides were efficiently synthesized for the first time using a readily accessible filter pipette tip as parallel reaction vessels, eliminating the use of any expensive equipment. This method enabled the synthesis of numerous peptides at a milligram scale with microfluidic amino acid flowing through the solid resin. After high‐throughput activity screening, peptide LSPGV was shown to exhibit the most potent antioxidant activity (DPPH radical scavenging activity [DRSA]: 88.96%) and strongest acetylcholinesterase (AChE) inhibition (IC 50 of 7.32 μM) simultaneously. The reduction in paralysis, amyloid‐β (Aβ) aggregation, and AChE activity in vivo was further confirmed in an animal model, Caenorhabditis elegans . 3D structure simulation and molecular docking study were also conducted for LSPGV to illustrate its potential conformation and favorable binding interactions with AChE. As a candidate for Alzheimer's disease (AD) treatment, LSPGV significantly enhances the value of sea cucumber intestine by‐products.