Preparation And Immunomodulatory Effect Of Immunomodulatory Peptides From The Heads Of Litopenaeus Vannamei

The immune system,which includes immune defense,surveillance,and immunomodulation,is capable of safeguarding human health and it also participates in the etiology of various diseases.Currently,there are many drugs available to modulate human immune function,such as levamisole,cyclophosphamide,and macrolides,but most synthetic immunomodulatory drugs have toxic side effects.Therefore,synthetic immunomodulatory drugs are not conducive to prevention or long-term use.In this area,immunoactive agents derived from natural foods possess potential value and significance for development due to their effectiveness and safety for human health.In recent years,the research of bioactive compounds in marine organisms has become a hot topic.More recently attention has been focused on providing bioactive peptides with different biological functions,such as dipeptidyl peptidase IV(DPP-IV)inhibitory peptide,angiotensin-converting enzyme(ACE)inhibitory peptide and immunomodulatory peptide.Litopenaeus vannamei is one of the major species in the global shrimp farming industry.During processing,40-50% of the by-products(heads,shells and tails)are rarely used.Among them,shrimp heads are rich in high quality proteins and essential amino acids and may act as a potential source for the extraction of bioactive peptides.However,studies on the production of immunomodulatory peptides from Litopenaeus vannamei have not been reported,especially using their discarded heads.In this study,immunoactive enzymatic digests were prepared from the heads of Litopenaeus vannamei using different types of enzymes,and immunomodulatory peptides were further obtained by purification and identification.The immunomodulatory effects of the peptides were estimated by cellular assays in vitro and animal experiments in vivo,and the stability and the action mechanism of immunomodulatory peptides were investigated.This research will support the rationale for the exploitation of functional foods with immunomodulatory peptides from the head of Litopenaeus vannamei,thus enabling the utilization of low value shrimp by-products.1.The suitable proteases and the main factors affecting the enzymolysis of immunomodulatory peptides prepared from the heads of Litopenaeus vannamei were discussed,and the enzymolysis process was optimized by response surface methodology,by using macrophage RAW264.7 relative proliferation rate and hydrolysis degree as evaluation indexes.2.Four different molecular fractions were obtained from the enzymolysis of the Litopenaeus vannamei heads by ultrafiltration membranes(10 k Da,3 k Da,1 k Da)and their effects on the proliferation rate of macrophages were measured.The results showed that the M4 fraction(below 1 k Da)showed the highest cell proliferation rate(162.15±5.27%)and could significantly promote NO production by macrophages and increase their phagocytosis rate and secretion of TNF-α,IL-2,IL-6 and IL-1β within50-150 μg/m L.The M4 fraction was passed through Sephadex G-15 to gain five fractions(M4-1,M4-2,M4-3,M4-5 and M4-5),where M4-1 exhibited the highest cell proliferation rate(162.26±10.62%)and showed a dosage of relevant immune indicators(phagocytosis,NO,TNF-α,IL-1β,IL-2,IL-6 secretion)exerting immunomodulatory effects.Therefore,peptide sequences were identified using LC-MS/MS for the optimal M4-1 fraction,resulting in 195 peptide sequences.Six immunomodulatory peptides were elected in combination with computer simulation scoring: PSPFPYFT,SAGFPEGF,GPQGPPGH,QGF,PGMR,and WQR.3.The six immunomodulatory peptides synthesized in solid phase were validated for immune effect evaluation and assessed for stability(gastrointestinal digestion,heat treatment,p H treatment),and it was concluded that PSPFPYFT,GPQGPPGH,PGMR and WQR demonstrated excellent immune activity and stability.Molecular docking was used to simulate the scoring of peptide ligands with protein receptors TLR2 and TLR4/MD-2,and the two systems with lower affinity energy values(WQR-TLR4/MD-2 and GPQGPPGH-TLR2)were selected for further analysis of their action mechanism using molecular dynamics simulation.The findings indicated that the peptides formed hydrogen bonding interactions with the binding sites on the TLR receptor,creating a strong binding interaction and allowing the peptides to exhibit better immunoreactivity.The MM/GBSA analysis also yielded binding free energies for both complexes,suggesting their stable binding.4.By evaluating the protein content,amino acid fraction and stability,it was revealed that the shrimp head peptides with below 1 k Da fraction was more stable than the enzymatic digest of Litopenaeus vannamei,while removing impurities by ultrafiltration,and was more suitable as a raw material for in vivo experiments.Therefore,the in vivo immune indexes were assayed in immunocompromised zebrafish soaked with below 1 k Da fraction,and the outcomes proved that Litopenaeus vannamei head peptides were effective in enhancing macrophage phagocytosis and T cell fluorescence intensity,upregulating IL-1β,TNF-α and IFN-γ gene expression,and enhancing the in vivo immune level of the organism.