The Impact Of High Hydrostatic Pressure On The Digestive Properties Of Whey Protein Isolate And The Screening Of Its Antioxidant Peptides

Whey Protein Isolate（WPI）is a whey protein product with a protein content of 90%or more obtained by separation technology from whey,a by-product of cheese production.Whey protein contains a variety of functional protein components（e.g.β-lactoglobulin,α-lactalbumin,bovine serum albumin and immunoglobulins）,and whey protein-derived peptides have an important role in maintaining body functions due to their specific amino acid sequence.At present,most studies on protein bioactivity,screening of bioactive peptides and their preparation are often based on their in vitro physicochemical measurements,and their functionality and changes during gastrointestinal digestion in vivo have been less studied.As the global aging process accelerates,it is necessary to study the structure,digestive properties and biological activities of proteins in the special digestive patterns of the elderly.High Hydrostatic Pressure（HHP）has been shown to be an effective way to improve the bioactivity of food-derived proteins.Based on this,the protein structure,digestive properties,antioxidant activity and antioxidant peptides generated from the digestion of WPI modified by HHP were systematically investigated after in vitro simulated digestion of elderly people using HHP treatment at different pressures;meanwhile,the antioxidant peptides of bovine whey protein origin were screened by molecular docking technique to compare and complement the antioxidant peptides generated from the digestion of WPI simulated by elderly people.At the same time,the antioxidant peptides from bovine whey protein were screened by molecular docking technique to compare and supplement the antioxidant peptides produced by WPI during the digestion of simulated elderly.This study can provide a theoretical basis for HHP to improve the antioxidant activity of WPI during digestion in the elderly on the one hand,and provide a research method for the identification and screening of whey protein-derived antioxidant peptides on the other hand.The main findings were as follows:（1）The effects of high hydrostatic pressure treatment with different pressures on the protein structure and digestive properties of WPI during in vitro simulated digestion in the elderly were investigated.The results showed that in the undigested stage,the intrinsic fluorescence intensity and free sulfhydryl content of WPI samples increased significantly（P<0.05）with the increase of HHP treatment pressure,which extruded the original dense and small pore-like structure of the samples into a sheet-like protein structure;in the gastric digestion stage,with the increase of HHP treatment pressure,the samples showed a gradual decrease in the content ofα-helical andβ-folded,irregularly curled In the intestinal digestion stage,the effect of HHP treatment on the fluorescence intensity was weakened,and the free sulfhydryl content decreased compared with that of the sample before digestion,and the sample further decomposed into particles and fibrils,increasing the number and depth of protein pores.All electrophoretic bands disappeared almost completely,and their in vitro digestibility and hydrolysis were significantly increased compared to the gastric digestion stage（P<0.05）.（2）The effects of high hydrostatic pressure treatment at different pressures on the antioxidant activity of WPI and the antioxidant peptides produced by digestion during in vitro simulated digestion in the elderly were investigated.The results showed that in vitro antioxidant activities(ABTS⁺radical scavenging capacity,DPPH free scavenging capacity and Fe³⁺reducing power)and cellular antioxidant activities（ROS,MDA,SOD,GSH and CAT）showed significantly enhanced trends（P<0.05）with increasing treatment pressure,and in the 600 MPa-WPI intestinal digest,the identification of Fifteen specific antioxidant peptides with peptide chain profiles and bioactivity predictions showed matching bioactive peptide profiles.Among them,the LGSDMEDLR peptide sequence has the highest"-CDOCKER energy"and forms salt bridges,attractive charges,conventional hydrogen bonds,carbon-hydrogen bonds and van der Waals forces with Arg380,Arg415,Arg483,Tyr334 and Asn382,all located at the Keap1-Nrf2 binding sites for the interactions.（3）The study used the major bovine whey protein sequences from the Uniprot database as a source to screen for potential antioxidant peptides.The Peptide Ranker program was applied to predict the bioactivity score of bovine whey protein sequences,and 137 peptides with a score greater than 0.4 were screened,while the Toxin Pred,Innovagen,Expasy-compute and Pepdraw programs were used to further predict and analyze the physicochemical properties of the peptides,and the peptides were screened by pharmacokinetic ADMET and TOPKAT analyses were used to screen 14 non-toxic,non-sensitizing,non-mutagenic,non-carcinogenic and well water-soluble peptides,which were then molecularly docked with Keap1 receptor,and finally the CDEF sequence was found to have the most antioxidant activity potential by intermolecular force evaluation of complex conformation,while its antioxidant score for molecular docking was lower than that obtained by HHP pretreatment of WPI LGSDMEDLR.In conclusion,high hydrostatic pressure treatment can effectively improve the structural properties of WPI and its digestion in vitro in simulated elderly people,significantly improve its digestibility,in vitro and cellular antioxidant activity,and identify specific antioxidant peptides with antioxidant potential from its digestion products,and virtually screen bovine whey protein-derived antioxidant peptides by molecular docking technique.It indicates that high hydrostatic pressure is a potential non-thermal processing method in the dairy industry,while molecular docking technique is an effective strategy for screening antioxidant peptides.