Effect Of High Hydrostatic Pressure Treatment On The Antioxidant Activity Of Lactoferrin Before And After In Vitro Digestion

Lactoferrin is a protein widely found in mammalian external secretions and has strong antioxidant activity.When the structure of lactoferrin is altered,the degree of exposure of its antioxidant moieties also changes,which can lead to changes in antioxidant activity.It has been shown that high hydrostatic pressure treatment can change the secondary and tertiary structures of lactoferrin,and the relationship between structural changes of lactoferrin and the expression of antioxidant activity can be explored by high hydrostatic pressure treatment.Meanwhile,the antioxidant activity of lactoferrin in vivo depends on the lactoferrin peptide produced after digestion,and the expression of antioxidant activity of lactoferrin after digestion also needs to be investigated in depth.It has been shown that appropriate high hydrostatic pressure treatment can promote the digestion of animal proteins,so the digestive characteristics of lactoferrin treated with high hydrostatic pressure also need to be investigated in depth.The aim of this experiment was to investigate the changes of lactoferrin structure and antioxidant activity after in vitro digestion by high hydrostatic pressure treatment and in vitro digestion,and to provide some theoretical basis for the high hydrostatic pressure processing of lactoferrin.In this study,after high hydrostatic pressure treatment of lactoferrin solution,lactoferrin was digested by stomach and intestine using international in vitro digestion models.Changes in molecular weight distribution and digestion of lactoferrin were assessed by two gel electrophoresis and hydrolysis;changes in lactoferrin structure were assessed by circular dichroism,fluorescence spectroscopy,zeta potential,particle size distribution,surface hydrophobicity,free sulfhydryl content,and field emission scanning electron microscopy;changes in peptide profile composition and amino acid content of small molecule fractions after lactoferrin digestion were assessed by ultrafiltration,liquid chromatography,mass spectrometry,and liquid mass spectrometry The biological activity of specific lactoferrin peptides was assessed by chemically synthesized peptides;in vitro antioxidant activities of different fractions of lactoferrin and digested lactoferrin treated with high hydrostatic pressure were evaluated by FRAP and ABTS methods;finally,the SOD activity,CAT activity,MDA,GSH content and RNA of NCM460 cells were determined by co-culture of lactoferrin and its different fractions with hydrogen peroxide-induced oxidative stress NCM460 cells.Finally,the cellular antioxidant activities of lactoferrin and its different fractions were verified by co-culture with hydrogen peroxide-induced oxidative stress NCM460 cells,and the changes of SOD activity,CAT activity,MDA content,GSH content and ROS content were measured.The results showed that high hydrostatic pressure treatment did not degrade or polymerize lactoferrin and did not alter the molecular weight distribution of lactoferrin.High hydrostatic pressure treatment promoted the hydrolysis of lactoferrin in the stomach and intestine by altering the tertiary and microstructure of lactoferrin and increasing the contact area with protease.High hydrostatic pressure treatment did not change the peptide profile composition of lactoferrin intestinal digest,but the peptide profile identification results strongly demonstrated that gastric digestion made an important contribution to the complete digestion of lactoferrin in the intestine,although lactoferrin was less hydrolyzed in the stomach.The results of in vitro antioxidant activity and cellular antioxidant activity assays showed that both in vitro digestions significantly increased the antioxidant activity of lactoferrin and that high hydrostatic pressure treatment significantly increased the antioxidant activity of lactoferrin after intestinal digestion.Finally,the antioxidant activity assay demonstrated that the fractions with strong antioxidant activity in the intestinal digest of lactoferrin were small molecule fractions,and the antioxidant activity of two synthetic lactoferrin peptides was identified.This study demonstrates that high hydrostatic pressure can improve the digestive properties and antioxidant expression of lactoferrin,which is a potentially beneficial processing method for lactoferrin.As a new lactoferrin processing method,high hydrostatic pressure has a promising application in production practice and can promote the development of sustainable,efficient and safe health food products.