| At this stage,the number of people who are immunocompromised or even immunosuppressed due to poor lifestyle,stress,disease and medications is increasing.Most immunomodulatory drugs are often used clinically have certain toxic side effects,while food-derived immunopeptides have become a hot spot for current research because of their natural origin,high safety and easy absorption.Spirulina platensis is a unicellular cyanobacteria with rich nutrition and wide range of functions,which is an ideal food and nutritional supplement.The C-Phycocyanin(C-PC)is the main protein in Spirulina platensis with balanced amino acid composition and various biological activities such as immunomodulation,which can be used to prepare immune peptides.In this experiment,the immunopeptides were prepared by enzymatic digestion of phycocyanin,the structure of immunopeptides was identified by UPLC-ESI-MS/MS,and their immunomodulatory activities were evaluated by in vivo and in vitro tests.The main research contents and results are summarized as follows:1.Preparation of phycocyanin immunopeptide PP by enzymatic hydrolysis: C-PC was isolated and purified from Spirulina platensis by repeated freeze-thaw and anion exchange chromatography sequentially.The yield of C-PC was 56.13 ± 3.39 mg/g,and the purity was 4.48 ± 0.31.Phycocyanin peptides were prepared by enzymatic hydrolysis of alkaline protease,papain,trypsin and pepsin,respectively.The best protease for enzymatic hydrolysis and best active fraction of phycocyanin immunopeptides was screened by RAW264.7 cell viability.The results showed that papain was the best protease for the preparation of phycocyanin immunopeptide,with the highest hydrolysis of phycocyanin of 26.24 ± 1.86%,and its enzymatic digest significantly increased the cell viability of RAW264.7(1.16 ± 0.03,p < 0.05).At a concentration of 100 μg/m L,the ultrafiltration fraction < 3 k Da(PP)had the highest cell viability up to 1.31 ± 0.02,which was significantly different(p < 0.05)compared to the other ultrafiltration fractions(hydrolysate,3-10 k Da and > 10 k Da).Therefore,the optimal active fraction PP was selected for subsequent in vivo immunoreactivity studies.2.Effect of PP on immune function and intestinal flora diversity in CTX-induced immunosuppressed mice: The body weight,immune organ index and phagocytosis of macrophages in immunosuppressed mice were significantly improved after 21 d of gavage treatment with different doses(100,200 and 400 mg/kg/b.w.)of PP.Meanwhile,PP also significantly increased the serum IL-2,IL-6,TNF-α,Ig A and Ig G levels in mice(p < 0.05).The results of 16 S r RNA sequencing of intestinal flora showed that CTX treatment led to disorder of intestinal flora in mice.α-diversity and β-diversity analysis showed that PP could increase the diversity and abundance of intestinal microflora in immunosuppressed mice and improve the structure of intestinal microflora to normal.In addition,the results of intestinal microflora phylum level and genus level analysis showed that PP increased the Bacteroidetes/Firmicutes ratio,the abundance of short-chain fatty acid producing bacteria(Alloprevotella and Anaerostipes)and beneficial bacteria(Lactobacillus).In summary,the results showed that PP enhanced immune function and improved intestinal microflora dysbiosis in mice,and could be used as a beneficial immunomodulatory component in functional foods.3.Sequence identification,in silico screening,chemical synthesis of peptides and their immunomodulatory activity of phycocyanin immunopeptides: UPLC-ESI-MS/MS was used to identify the structural composition of PP,and the immunomodulatory activity and its stability on RAW264.7 cells were investigated by computer virtual screening of immunomodulatory peptides,and the mechanism of action of peptides was studied by molecular docking technique: a total of 127 peptide sequences were obtained through identification,combining the prediction results and molecular characteristics of immunomodulatory peptides,peptides FGRFR,SPSWY,YNKFPY,GYYLRM,MFDAF and MAACLR were selected as potential immunomodulatory peptides for chemical synthesis,and to study the effects of these The effects of these peptides on the immunomodulatory activity of RAW264.7 cells were investigated.The results showed that all six peptides could increase the viability,phagocytosis and secretion of NO,IL-6and TNF-α of RAW264.7 cells to different degrees,among which the immunomodulatory activities of peptides FGRFR,SPSWY and YNKFPY were better and significantly different from those of the control group(p < 0.05).The results of stability studies of the peptides showed that all six peptides maintained high immune activity under in vitro simulated gastrointestinal digestion,heat treatment and p H treatment.The results of molecular docking analysis showed that all six peptides had a strong binding capacity with Toll-like receptors TLR2 and TLR4/MD2,which could activate cellular immune responses.Among them,the peptides had stronger binding ability to TLR4/MD2 than TLR2.Peptides FGRFR,SPSWY and YNKFPY had stronger binding ability to TLR2 and TLR4/MD2 than the other three peptides with binding energies of-8.3,-7.4,-7.3kcal/mol and-8.9,-8.2,-7.3 kcal/mol,respectively,which was consistent with the cell experimental results were consistent.In summary,PP has strong immunomodulatory activity in vitro and in vivo,which can improve the cell viability and proliferation of RAW264.7 cells,enhance the immune function of immunosuppressed mice and improve the disorder of intestinal microflora.The peptides FGRFR,SPSWY,YNKFPY,GYYLRM,MFDAF and MAACLR in PP can activate immune cells and improve immune function by interacting with Toll-like receptors TLR2 and TLR4/MD2 to activate immune cells and improve immune function,among which peptides FGRFR,SPSWY and YNKFPY can be used as novel immunomodulators for clinical treatment because of their good immunomodulatory efficacy.This study lays the foundation for the high value-added utilization of phycocyanin and the promotion of the development and application of immunopeptides. |
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