Sequence-resolved discovery and cellular validation of immunomodulatory peptides from cricket protein hydrolysate

Cricket protein hydrolysate (CPH) is a promising alternative-protein-derived ingredient, but the active peptide sequences and mechanisms underlying its potential bioactivity remain difficult to define. In this study, CPH was evaluated in LPS-stimulated BV2 microglial cells using an integrated strategy combining cellular assays, transcriptomics, LC–MS/MS-based de novo peptidomics, structure-based screening, and peptide validation. CPH attenuated LPS-induced inflammatory activation, as shown by reduced pro-inflammatory cytokine production, nitric oxide release, and ROS accumulation. Transcriptomic analysis showed that CPH broadly modulated inflammation-related pathways, including TNF, Toll-like receptor, NOD-like receptor, IL-17, and JAK–STAT signaling. JAK–STAT signaling was subsequently selected as a biologically relevant cytokine-responsive pathway for further investigation rather than as the dominant transcriptomic response. De novo peptidomics revealed that CPH contained predominantly short peptides, which were subsequently subjected to peptide characterization, bioinformatic filtering, structure-based prioritization, molecular dynamics simulations, and cellular validation. Several CPH-derived peptides showed predicted compatibility with the STAT3 SH2 domain. Selected peptides reduced LPS-induced IL-6 and TNF-α secretion, while their effects on oxidative stress, IBA1 expression, and STAT3 phosphorylation varied among peptide sequences. CPH also reduced LPS-induced STAT3 phosphorylation, providing a link between the hydrolysate-level and peptide-level findings. Together, these results suggest that selected CPH-derived peptides may modulate STAT3-associated inflammatory signaling. Collectively, this study establishes a sequence-resolved workflow for identifying immunomodulatory peptides from cricket protein hydrolysate and supports CPH as a source of candidate functional peptides with cellular anti-inflammatory activity.