Research On The Antioxidant Activity Of Egg White Fermented By Lactic Acid Bacteria

Egg white is a high-quality food protein with antioxidant activity.In the past,the research on the antioxidant activity of egg whites mainly focused on protein and its enzymatic hydrolysates,but there was little information on the antioxidants of its naturally occurring peptides in egg white.In addition,the environment of egg white limited the development of its fermentation and processing field,which leads to few researches on the antioxidant and hydrolyzation of egg white fermented by lactic acid bacteria.This study firstly identified the naturally occurring antioxidant peptides by peptidomic;then established the technology of whole egg whites fermented by lactic acid bacteria,and explored the changes in the structure and activity of the proteins and peptides in the fermented egg whites;then studied the changes in the antioxidant activity of the fermented egg white after in vitro simulated gastrointestinal digestion;finally,an aging mouse model was used to verify the antioxidant activity of the fermented egg white in vivo and its mechanism.The main findings are as follows:(1)Based on the current albumin and the gaps in its research,this thesis identified low molecular peptides(<3 kDa)occurring naturally in egg white using a peptidomics approach and to determine their potential antioxidant activity.A total of 45 peptides were identified in unfertilized brown shells and white egg whites but surprisingly all are originated from 25 kinds of egg white minor proteins.Neuropeptide Y was identified for the first time in egg whites.Six peptides(VPPGAPEEPPR、EASNAAAAPDEAVPQGR、VPPSELKQLSAAGS、YPSKPDSPGEDAPAEDMAR、ALGEDIVDLDSFSEQH、DGVEDIFQDSTQ)were found to have antioxidant activities through the determination of ROS formation and the antioxidative enzyme levels of SOD and CAT based on cellular antioxidant assay.This result provides a new basis for further understanding of eggs as a natural antioxidant food.(2)Based on the fact that the egg white substrate is not suitable for microbial growth and fermentation,this study established egg white pretreatment technology and whole egg white fermentation technology.By measuring the number of colonies,pH,DH,SDS-PAGE electrophoresis,and molecular weight of peptide,the results showed that the pretreatment of egg white heated at 80℃ for 15 min can promote the fermentation of lactic acid bacteria and the hydrolysis of egg white protein.After the fermentation,the pH of the egg white decreased,and the ovotransferrin and ovalbumin were observed to be effectively degraded,and low molecular weight peptides were more abundant.These confirmed the feasibility of lactic acid bacteria fermented egg white technology.(3)In order to screen the lactic acid bacteria with strong hydrolytic ability to egg white,the hydrolytic effect of different lactic acid bacteria on egg white protein was compared.It was found that L.helveticus DSCAB4M1(B4),L.prantarum FNXYCHL89L1(L1)and L.praracasei CQBS21(BS)had the best hydrolysis performance.The DH of egg white increased from 5.95%to 6.89%-7.42%after fermentation by these three kinds of lactic acid bacteria.The peptide content increased by 15.12%-58.19%;the number of small peptides increased from 22 to 44,50 and 61,respectively.The peptide sequences derived from egg white high abundance proteins(such as ovalbumin,ovomucoid,ovotransferrin,etc.)and low abundance proteins(such as clusterin,ovocleidin-116,etc.)were newly identified.It is suggested that lactic acid bacteria fermentation can be used as a processing method to hydrolyze egg white protein and produce polypeptides.(4)In order to investigate the changes of protein and peptides in egg white(EW)and egg white fermentation(FEW)after in vitro digestion,the digestion of EW and FEW were prepared according to INFOGEST static in vitro simulation of gastrointestinal(GI)food digestion method.The results showed that the DH of the digestion samples was significantly increased;the proteins(>50 kDa)such as ovomucin and ovotransferrin were almost completely hydrolyzed,while peptide(<1500 Da)content increased.Compared with egg white with GI digestion(EWI),the DH of fermented egg white with GI digestion digestion(FEWI)were significantly increased(P<0.05),and the levels of FEWI-B4,FEWI-L1 and FEWI-BS were increased by 13.68%,6.88%and 7.48%,respectively.The results showed that lactic acid bacteria fermentation was beneficial to the in vitro digestion of egg white.(5)In order to explore the changes of antioxidant activity of egg white after fermentation and in vitro digestion,the in vitro superoxide evaluation model of L6 cells was used in this study.the results showed that the antioxidant activity of egg whites was gradually enhanced after fermentation and digestion,which was consistent with the change in peptide content.Compared with EW,the ROS scavenging capacity of FEWI-L1 and FEWI-BS was increased by 14.54%and 15.62%,respectively.After simulated GI digestion in vitro,the ROS scavenging capacity of FEWI-L1 and FEWIBS was further increased by 26.65%and 29.45%,respectively,and the antioxidant activity of FEWI-L1 and FEWI-BS was significantly better than that of EWI.The results indicated that the fermentation of lactic acid bacteria could enhance the antioxidant activity of egg white and its GI digestion in vitro.(6)In order to further verify the antioxidant activity of FEW after digestion in vivo,the oxidative stress model of aging mice was used in this study.It was found that FEW could effectively alleviate the oxidative stress of aging mice,and restored the relevant antioxidant enzyme defense system in the liver and blood.Among them,high-dose FEW-BS could significantly increase the activity of SOD,CAT,GSH-Px and T-AOC,and reduce the content of MDA in the liver and serum of aging mice.The expression of key proteins in the signaling pathway in mouse liver was further investigated,and the results showed that EW and FEW could be significantly up-regulate Nrf2/HO-1 protein and activated the phosphorylation level of ERK,p38 and Akt protein.The activation effect of FEW-BS was significantly better than that of EW and the other two ferments.These results suggested that egg white fermentation can improve oxidative stress in vivo.In summary,this study explored the natural antioxidant peptides present in egg whites;and created a technology for lactic acid bacteria to ferment the whole egg whites;and revealed the proteolysis and antioxidant properties of egg whites during fermentation by lactic acid bacteria and digestion in the in vivo and in vitro gastrointestinal tracts;and preliminarily explored the antioxidant mechanism in vivo.These results provide a new theoretical basis for the subsequent development of eggsource antioxidant foods,and promotes the sustainable development of the egg industry.