Immunomodulatory Effects of Galleria mellonella and Alphitobius diaperinus Protein Digestates Generated Using a Modified INFOGEST Protocol – In Vitro and In Silico Evidence

Abstract Insect-derived peptides have attracted increasing attention due to their potential health-promoting properties. This study investigated peptides released from Galleria mellonella (W) and Alphitobius diaperinus (B) proteins following a modified static INFOGEST protocol. Digestion-derived peptides were identified by LC-MS/MS and evaluated for anti-inflammatory activity using in vitro assays, bioinformatic prediction, and molecular docking, focusing on IKK-β as a key regulator of the NF-κB pathway. At 500 μg protein/mL, W-I2h and B-I2h reduced NF-κB reporter activity by 28.7% and 53.5%, respectively, and decreased nitric oxide production by 31%. Digestates also reduced IL-6, TNF-α, IFN-γ, and IL-1β secretion at selected concentrations and decreased IL-6 and IL-1β mRNA expression. Conversely, 25 μg protein/mL increased IL-1β secretion by 109.4% and 102.1%, respectively. Increased HO-1 and γGCS transcripts suggested modulation of Nrf2-responsive gene expression, although pathway activation was not confirmed. These findings demonstrate concentration-dependent immunomodulatory effects and identify candidate peptides for future validation.