Study On Intracellular And Extracellular Antioxidant Activities Of Antioxidant Proteins And Peptides In Chicken Soup

In different cultures of the world,chicken soup is a healthy and conditioning food.Its anti-inflammatory and anti-oxidation effects are not only widely recognized by consumers,but also confirmed by many studies.Chicken protein-derived peptides can be obtained by enzymatic hydrolysis,and any studies have confirmed that these chicken-derived peptides have antioxidant activity.They are considered to be the main antioxidants in chicken soup,but lack data to prove the existence of these antioxidant proteins or peptides in the real food system of chicken soup.The protein and peptide components in chicken soup are extremely rich in both species and concentration,but something is rarely involved on component separation and component activity.This study first investigated the protein dissolution and protein types of chicken soup during cooking,and investigated the antioxidant activity changes of chicken soup and its different molecular weight components during cooking,and tried to establish the law between protein dissolution and antioxidant activity changes.In order to further understand the antioxidant protein and peptide components contained in chicken soup,we used ORAC as a screening method for antioxidant activity,and isolated and purified the protein and peptide components with antioxidant activity.Finally,we also characterized the intracellular and extracellular antioxidant activities of purified proteins and peptides.The main findings are as follows:(1)During the heating of chicken soup,protein dissolution has a certain regularity.At the beginning of cooking,some water-soluble protein can be detected in the chicken soup,and the protein components is thermally unstable and has a short time in the soup.After cooking for a period of time,collagens and myosin can be observed by SDS-PAGE,this part of the protein is not retained in the soup for a long time.The final dissolved protein shows higher thermal stability.The dissolution law of protein during the cooking of chicken soup is manifested by the order of dissolution time and the difference in heat stability of dissolved protein.(2)The antioxidant activity of chicken soup during the cooking process was monitored by ORAC method.It was found that the antioxidant activity increased with the prolongation of heating time,and the change of antioxidant activity reached a stable period after 180 min.The different molecular weight components of the chicken soup obtained by ultrafiltration,and the antioxidant activity measured by ORAC showed that the antioxidant activity of chicken soup was mainly derived from the small molecular weight component.The antioxidant activity and protein concentration of different molecular weight components contained in the chicken soup cooking process are consistent with time,suggesting that the antioxidant capacity of chicken soup may be mainly derived from proteins or polypeptides.There are some differences in the results of the antioxidant activity of chicken soup in vitro and in vivo,which indicates that the antioxidant effect of chicken soup is achieved by various mechanisms,the single extracellular antioxidant may be determined but does not fully reflect the antioxidant capacity of chicken soup in the body.(3)ORAC was used as a screening method for antioxidant activity.Two-step chromatography was used to separate an antioxidant active protein from chicken soup that remained soluble after prolonged heating.it was called P30 protein since its molecular weight is 30 kDa.The P30protein contains a sugar chain and its unit oxidation resistance is 1169.97?MTE/g.The protein sequence of P30 was obtained by N-terminal sequencing is SAPLISAPLIVAPPL,and P30 showed first-order structural similarity with protein tyrosine phosphatase by searching the database.The antioxidant polypeptides P1 and P2 were isolated from chicken soup by Sephadex G-25 gel chromatography and Luna 5?m NH2 normal phase chromatography.Their unit antioxidants were 1.45×10~8?MTE/g,8.32×10~7?MTE/g by ORAC,the protein sequences of P1 and P2 were obtained by N-terminal sequencing as LVPPP and LAMTT,respectively.(4)The antioxidant activities of P30,P1 and P2 were tested by Caco-2 and macrophage models,all of which showed different levels of intracellular antioxidant capacity.In normal macrophages,P30,P1 and P2did not show an effect on the normal physiological function of macrophages,and had no effect on membrane potential,mitochondrial reactive oxygen species and macrophage phagocytosis.After AAPH stimulation caused macrophage oxidative stress,all three can greatly promote cell depolarization,improve the oxidative damage state of cell membrane,and regulate the cell membrane potential.In addition,the three can also regulate the inhibition of mitochondrial oxygen respiration caused by AAPH,and to some extent alleviate the oxidative damage of macrophage phagocytosis.P1 has a more significant protective effect on macrophage membranes and mitochondria than P30 and P2.