Extraction, purification, and mechanism of immunomodulatory peptides obtained from silkworm pupa protein hydrolysate.

Silkworm pupa, a by-product of silk reeling, is rich in protein; however, it has traditionally been used as animal feed. This study isolated and purified peptides from the enzymatic hydrolysates of silkworm pupa protein, thus effectively enhancing its utilization. The immune activity of these peptides was evaluated in macrophages, and 609 peptides were identified using LC-MS/MS. These active peptides were screened based on their toxicity, allergenic, and biological activity, and their interactions with TLR2 and TLR4/MD-2 were predicted via molecular docking. Results indicated that APFAPAPL, YLPPFNSF, and FIPNEAFAGRPF could strongly bind to TLR2 and TLR4/MD-2 through hydrogen bonding and hydrophobic interactions. These peptides were synthesized using solid-phase synthesis, and their immune activity was verified by proliferation, NO, ROS and TNF-α secretion assays. All three peptides promoted the proliferation, phagocytosis, and secretion of ROS and TNF-α by macrophages. Western blot analysis showed that the peptides activated RAW 264.7 cells via the NF-κB and MAPK signaling pathways, mediated by TLR2 and TLR4/MD-2 receptors. Therefore, this study provides a new understanding of the immunomodulatory activity of silkworm pupa proteins, implying their potential use as functional food ingredients.