| As the number of people with chronic diseases such as hypertension,hyperglycaemia,hyperlipemia,and hyperuric acid increases dramatically in China.With the national health strategy such as "Health China 2030" and "Big Health Industry",the demand for personalized functional factors is also increasing.Yunnan Province,as a province with large resources of special plants and animals and microorganisms,has the unique advantage of mining personalized functional factors.Yunnan Province has the second largest buffalo milk industry in China,with the Binglangjiang buffalo as the representative,which has high milk production and rich milk protein,and is the best choice to explore functional factors,especially active peptides.However,its single dairy,lack of high-end specialty dairy and insufficient mining of functional factors,the development of the Binglangjiang buffalo milk industry is hampered,and it is difficult to transform resource advantages into economic benefits.The edible whey protein hydrolysates had biological functions such as immunity enhancement,hypoglycemia and hypolipidemia,and the occurrence of these chronic diseases was often accompanied by inflammatory reactions.Whether Binglangjiang buffalo milk whey-derived active peptides can also play the above functions by inhibiting inflammatory reactions and the specific efficacy components that can play anti-inflammatory activities have not been reported.Therefore,based on multi-omics technology,this study targeted and efficiently extracted anti-inflammatory peptides from the Binglangjiang buffalo whey protein hydrolysate and revealed its anti-inflammatory mechanism in vitro,and conducted a stabilization on anti-inflammatory peptides for low gastrointestinal environmental resistance and bioavailability,and finally conducted a preliminary study on the effect of anti-inflammatory peptides in mice with ulcerative colitis induced by DSS.This study lays a foundation for achieving targeted delivery of foodborne functional factors in vivo,and thus achieving precise nutrition intervention and regulation in human body.The main research results are as follows:1.Combined multi-omics efficiently screened two novel anti-inflammatory peptides(DQPFFHYN and YSPFSSFPR)from Binglangjiang buffalo whey protein hydrolysate with high activity and safety,but weak resistance to gastric environment.(1)The functional activity of buffalo whey protein was analyzed based on Label free proteomics.label-free quantitative proteomics was used to analyze buffalo whey protein from nutrition,immunity,and anti-inflammatory aspects.It was found that the amount of immune or anti-inflammatory related proteins in the whey protein of Binglangjing buffalo was higher than that of Dehong buffalo.The anti-inflammatory effect of buffalo whey protein from Binglangjing was also better than that from Dehong buffalo.Therefore,Binglangjing buffalo whey protein was selected as the raw material for further screening of anti-inflammatory peptides.(2)Two novel anti-inflammatory peptides were screened from the hydrolysate of Binglangjing buffalo whey proteins based on peptideomics.After hydrolyzed by Procerain B protease,peptides were obtained by ultrafiltration.A total of 1483 peptides were identified from the hydrolysate of Binglangjing buffalo whey proteins by LC-MS/MS,and 12 target peptides were selected for chemical synthesis using bioinformatics analysis.Furthermore,two anti-inflammatory peptides(DQPFFHYN and YSPFSSFPR)were screened using the RAW264.7 cell inflammation model,both of which were first reported and named DN8 and YR9.(3)Structure and properties of anti-inflammatory peptides DN8 and YR9.DN8 contains 8 amino acids with a molecular weight 1067.458 Da Da,which are located at 52～59 amino acids of α-2-glycoprotein-1 protein(A0A452DK44).The hydrophobicity is 11.5 kcal/mol.YR9 contains9 amino acids with a molecular weight 1087.52 Da,which are located at 200～208 amino acids of Clusterin protein(P17697).Its hydrophobicity is 7.24 kcal/mol.Peptides DN8 and YR9 had high safety in vitro,but weak resistance to gastric environment,which were easily degraded to single amino acids or short peptides by gastrointestinal enzymes,and none of these peptides showed anti-inflammatory activity.2.Anti-inflammatory peptides DN8 and YR9 alleviate LPS-induced inflammation in RAW264.7 cells by regulating TLR2/NF-κB signaling pathway.(1)Anti-inflammatory peptides DN8 and YR9 can significantly inhibit the secretion of pro-inflammatory cytokines NO,TNF-α and IL-6 by inflammatory cells.ELISA showed that at 200 μg/m L,anti-inflammatory peptide DN8 inhibited TNF-αand IL-6 at 50.10% and 60.44%,respectively.The inhibition rates of YR9 on TNF-α and IL-6 were62.91% and 44.34%,respectively.The results indicate that anti-inflammatory mechanisms of peptides DN8 and YR9 may be related to inhibiting the secretion of inflammatory cytokines.(2)Based on TMT proteomics,anti-inflammatory peptides DN8 and YR9 were predicted to relieve cellular inflammation by regulating the TLR2/NF-κB pathway.A total of 194 different proteins were found between the DN8 vs.LPS group,14 of which were associated with inflammation.167 different proteins were found in the YR9 vs.LPS group,14 of which were associated with inflammation.GO annotation and KEGG pathway enrichment analysis showed that these inflammation-related proteins were mainly involved in Toll-like receptor signaling pathway and NF-κB signaling pathway.Further analysis of key proteins in these two pathways showed that the expressions of TLR2,My D88,IKBb1,IKBa,p50,NOS2 and COX2 proteins were significantly decreased after the anti-inflammatory peptides treatment,which suggest that anti-inflammatory peptides DN8 and YR9 may reduce the inflammatory damage of LPS-stimulated RAW264.7 cells by mediating TLR2/NF-κBp50 signaling pathway.(3)Western blot analysis showed that anti-inflammatory peptides DN8 and YR9 alleviated cell inflammation by regulating TLR2/NF-κB signaling pathway.Anti-inflammatory peptides DN8 and YR9 could significantly reduce the expression levels of surface receptor protein TLR2,downstream protein My D88,p IKB-α,p NF-κBp50 and pro-inflammatory factor proteins i NOS,TNF-α and IL-6 in LPS-stimulated RAW264.7 cell.These results suggest that anti-inflammatory peptides DN8 and YR9 reduce the inflammatory damage in LPS-stimulated RAW264.7 cells mainly by regulating TLR2/NF-κBp50signaling pathway.3.Nano inclusion complexes CM-β-CD-DN8 and CM-β-CD-YR9 loaded with anti-inflammatory peptides DN8 and YR9 were constructed successfully.They are mainly crosslinked by electrostatic adsorption and hydrogen bond,and have good activity,stability and targeting.(1)Nanoparticles CM-β-CD-DN8 and CM-β-CD-YR9 showed potent encapsulation rate and anti-inflammatory activity.Cytotoxicity evaluation showed that CM-β-CD had no toxicity to RAW264.7cells at 0.625～10 mg/m L.The best encapsulation effect was detected by UV-Vis spectroscopy when CM-β-CD:anti-inflammatory peptide was 10:5.The encapsulation rates of CM-βCD-DN8 and CM-βCD-YR9 were found to be 82.513±4.617 and 90.974±0.421,respectively,by FITC-labeled peptide assay.Nanoparticles also showed no toxicity to RAW264.7 cells,and significantly reduced the levels of NO,TNF-α,and IL-6 secreted in LPS-stimulated RAW264.7 cells.(2)CM-β-CD was successfully included with anti-inflammatory peptides DN8 and YR9 through electrostatic adsorption and hydrogen bond.The surface of CM-β-CD-DN8 and CM-β-CD-YR9 inclusion complexes has weak negative charges,which are-26.767±2.250 and-12.137±3.048,respectively;the average particle size is75.03 nm and 41.71 nm,respectively.The structure and bond energy of the nanoparticles were characterized by FTIR and molecular docking,which found that CM-β-CD-DN8 and CM-β-CD-YR9 were combined by hydrogen bond.The results indicated that the nanoparticles CM-β-CD-DN8 and CM-β-CD-YR9 were mainly composed of electrostatic adsorption and hydrogen bond.(3)CM-β-CD improved the stability and intestinal targeted release of anti-inflammatory peptides DN8 and YR9.CM-β-CD-DN8 and CM-β-CD-YR9 have high safety,thermal stability,and pepsin resistance in vitro.Interestingly,the release of CM-β-CD-DN8 and CM-β-CD-YR9 in simulated gastric environment is controlled,but released in intestinal environment.4.Preliminary investigation of the anti-inflammatory effect in vivo revealed that the steady-state anti-inflammatory peptide effectively alleviated the symptoms of DSS-induced ulcerative colitis in C57BL/6J mice.Post-stabilization anti-inflammatory peptides DN8 and YR9 effectively alleviated mortality,weight loss,disease index,colon length,liver,and spleen enlargement in mice with ulcerative colitis.After the intervention of anti-inflammatory peptides DN8 and YR9,the number of murine karyorrhexis cells was significantly increased and tightly arranged;the infiltration of inflammatory cells in the lamina propria and submucosa was also significantly improved;In addition,the expression of ZO-1 and Occludin,the main proteins affecting colonic permeability,was increased.The results showed that the anti-inflammatory peptides DN8 and YR9 were effective in preventing anti-ulcerative colitis in mice. |
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