Sequence-Resolved Discovery of Immunomodulatory Peptides from Cricket Protein Hydrolysate as a Functional Alternative Protein Ingredient
preprint · SSRN Electronic Journal · 2026
| Date | 2026-07-13 |
| Type | preprint |
| Venue | SSRN Electronic Journal |
| Publisher | Elsevier BV (SSRN) |
| Contribution | downstream-application |
| DOI | 10.2139/ssrn.7102266 |
| Citations (OpenAlex) | 0 |
Peer-reviewed version: Sequence-resolved discovery and cellular validation of immunomodulatory peptides from cricket protein hydrolysate (2026-09-18, Future Foods)
Abstract
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 further examined because of its biological link with IL-6-mediated microglial inflammatory responses. De novo peptidomics identified abundant short-chain peptides in CPH, followed by peptide characterization, bioinformatic filtering, structure-based prioritization, molecular dynamics simulations, and cellular validation of selected candidates. Several CPH-derived peptides showed predicted compatibility with the STAT3 SH2 domain and reduced LPS-induced IL-6 and TNF-α secretion, oxidative stress, IBA1-associated microglial activation, and STAT3 phosphorylation. Mechanistic assays further indicated that suppression of LPS-induced IL-6 production contributed to the reduced STAT3 activation observed in peptide-treated cells. Collectively, these findings establish a sequence-resolved workflow for identifying immunomodulatory peptides from cricket protein hydrolysate and support CPH as a promising source of functional peptides capable of modulating IL-6-associated microglial inflammatory signaling.
Methods and tools
- Cricket hydrolysate immunomodulatory peptides: Sequence-resolved workflow for finding immunomodulatory peptides in cricket protein hydrolysate, an alternative-protein food ingredient. The hydrolysate is analysed without a proteolytic digest (PEAKS run with a non-specific enzyme setting) on a Q Exactive, and sequenced de novo with PEAKS to 25,582 assignments at ALC above 50%. Those are filtered to unmodified peptides scoring above 80 that together account for 95% of total abundance (n = 3231), then prioritised by structure-based screening against the STAT3 SH2 domain and molecular dynamics. Selected synthetic peptides reduced LPS-induced IL-6 and TNF-alpha secretion in BV2 microglia, and the hydrolysate itself reduced STAT3 phosphorylation. The paper labels the MS step ‘de novo peptidomics’; its own methods and results call the same operation de novo sequencing, so the phrase denotes de novo sequencing of an undigested peptidome rather than a broader pipeline.
Methods it uses
- PEAKS: Commercial DP-based de novo