| As a good resource of nature protein,egg white protein（EWP）played a good functional role with high nutritional value.The peptides obtained from EWP,have been proved to showed kinds of bioactivity,including anti-bacteria,antioxidant,antihypertension,hypoglycemic,etc.It’s still a hot point of research as well as a challenge,to improve the efficiency of hydrolysis and obtain high-bioactivity peptides from EWP.The objective of this dissertation was to prepare and identify novel peptides with antioxidant and anti-inflammation activities from EWP,through the method of hydrolysis in vitro or digestion in vivo,to prevent diseases and keep health.Firstly,electron beam irradiation（EBI）technology was applied to improve the hydrolysis of egg white protein（EWP）.The results showed that the hydrolysis degree of EWP treated by EBI increased significantly with the increase of EBI dose（P<0.05）.At the dose of 3.42 k Gy,the scavenging ability for DPPH free radical and hydroxyl radical of EWP hydrolysates,increased by 2.5 % and 100 % respectively.The results of safety evaluation,including acute toxicity test,micronucleus test of bone marrow cells and sperm malformation test,indicated that the hydrolysates from EBI treated EWP（EWPH）were non-toxic and non-teratogenicity.Moreover,the results of scanning electron microscopy（SEM）and mid-infrared spectroscopy（MIR）analysis indicated that EBI treatment could lead to puncture pores on the microsurface of EWP granules,EBI treatment at doses of 3.42 k Gy and 5.40 k Gy could promote the denaturation of EWP and reduce the denaturation temperature of EWP to 25 °C,which could be the reason of EBI improving the hydrolysis of EWP.Then,EWPH was isolated and purified by ultrafiltration and sephadex gel.The results showed that the filtration with molecular weight less than 1 k Da（EWPH-III）possessed significant scavenging ability of DPPH,ABTS and oxygen free radical.And the filtration of P4 and P6 were obtain with strong radical scavenging ability.Then amino acid analyzer（AAA）,MIR and liquid chromatography and mass spectrometry（LC-MS/MS）analysis were performed to identify the peptides from EWP.Three novel antioxidant peptides with molecular weight at 627.7 Da,632.9 Da and 684.1 Da were identified and their amino acid sequence were DHTKE,FFGFN and MPDAHL,respectively.MPDAHL showed better DPPH and oxygen free radical scavenging ability.Moreover,the effect of MPDAHL on anti-oxidative activity and anti-inflammatory activity were investigated in cell models,including hydrogen peroxide（H2O2）induced oxidative stress model of HT-29 cell and Tumor necrosis factor（TNF）-α induced low grade inflammation model of 3T3-L1 adipocyte cells.The results showed that MPDAHL could improve the cellular antioxidant activity（CAA）with the increase of concentration.At the concentration of 50 μM,MPADHL showed significant inhibition effect on the the secretion of IL-8 and the formation of MDA in H2O2 induced HT-29 cells（P<0.05）.Similarly,at the concentration of 50 μM,MPADAHL had significant inhibition on pro-inflammatory cytokines in 3T3-L1 adipocyte cells,such as IL-6 and MCP-1,while promoted the anti-inflammatory cytokine of adiponectin.It indicated that MPDAHL had anti-inflammatory activity on TNF-α induced adipocyte cells.In a word,the protection of MPDAHL on oxidative stressed intestinal cell was weak and the proper concentration for anti-inflammation was high,which indicated MPDAHL was not valuable for further research in vivo and application in food industry.Next,the method of directly digest in vivo was performed and ovotransferrin（OVT）was chosen to be studied as part of EWP.The absorbable peptides digested from OVT were identified in animal model,followed by the confirmation of anti-inflammatory activity of OVT in vivo.The anti-inflammatory activity of OVT were performed by the establishment of lipopolysaccharide（LPS）-induced chronic inflammation model in mice.ELISA,gene expression and enzyme-linked western blot（WB）were applied to determine the biological factors in low-grade chronic inflammation.The results showed that the pro-inflammatory cytokines in the blood of OVT treatment mice with high dose of 150 mg/kg BW/day,including TNF-α,MCP-1 and IL-6,were significantly decreased by 59.2 %,45 % and 48 %（P<0.05）,compared with the positive group only treated by 300 μg/kg BW/ day of LPS.In the intestinal tissues,OVT could inhibit D-mannitol crossing through the gut significantly.It also up-regulated the gene expression of tight junction proteins,such as ZO-1,Claudin-2,Claudin-4 and JAMA,while decreased the gene expression of pro-inflammatory cytokines IL-6 and Claudin-7.Meanwhile,OVT at high dose could significantly improve the secretion of adiponectin and raised the expression of insulin receptor substrate（IRS-1）and PPAR-γ,which were used to inhibit insulin resistance.After all,OVT showed good anti-inflammatory activity and inhibition of insulin resistance in vivo,which had great potential in the prevention of type 2 diabetes.Finally,in order to identify the anti-inflammatory peptide digested from OVT,MALDI-TOF-MS and LC-TOF-MS were performed to detect the portal vein blood sample from OVT administrated rat after 1 h,2 h and 4 h.Compared with the control group administrated by saline,16 peaks were found existing only in the OVT administrated samples.With further analysis of LC-TOF-MS,three ion peaks at 462.24 Da,476.26 Da and 491.23 Da only appeared in OVT administrated samples.Through the sequence editor and the amino acid sequence of OVT,13 absorbable oligopeptide candidates from OVT were concluded. |
