Antioxidant Activity And Transepithelial Transport Of Small Peptides Derived From Porcine Hemoglobin Hydrolysates

Pig blood is an important by-product of the meat industry,which is rich in protein and has not been fully utilized.In this study,porcine hemoglobin(PBC)was used as raw material and hydrolyzed by protease to generate hydrolysates with abundant small peptides(di-/tripeptides)and biological activity.At the beginning of the study,the suitable protease was screened by virtual enzymatic hydrolysis,and the enzymatic hydrolysis conditions were optimized by single factor test and response surface methodology(RSM).The porcine hemoglobin hydrolysates(PBCH)were prepared under the optimized conditions.The antioxidant activities of PBCH before and after simulated gastrointestinal(GI)digestion were investigated,including DPPH(1,1-diphenyl-2-picrylhydrazyl)and ABTS(2,2’-azino-bis 3Ethylbenzothiazoline-6-sulphonic acid)radical scavenging activities,effect on Caco-2 cells viability and the protection against oxidative damage cells.The UPLC-Orbitrap-MS/MS technique was used to qualitatively and quantitatively analyze the small peptides in hydrolysates.Small peptides were identified by combining dipeptides standard substances and databases information.In addition,the degradation during GI digestion and the intestinal epithelial cells permeability on small peptides of hydrolysates were investigated.The results were as follows:(1)In the process of virtual enzymatic hydrolysis,porcine hemoglobin sequences were treated by various proteases,the results showed that the number of small peptides obtained by Alcalase was large.The optimal hydrolysis conditions for Alcalase were E/S of 2.5%(w/w),pH of 10,temperature of 40℃ and incubation period of 30 hours.Under this condition,the degree of hydrolysis(DH)of the hydrolysates reaches a maximum of 49.19%,which is in reasonable agreement with the predicted value(49.74%).(2)PBCH showed higher DPPH and ABTS radical scavenging activities than PBC,although the activities were weakened by GI digestion.Besides,both PBCH and GI digested PBCH(PBCH-GI)significantly improved cell viability and suppressed the production of reactive oxygen species(ROS)under oxidative stress conditions in Caco-2 cells.(3)By using UPLC-Orbitrap-MS/MS technique,there were 150 small peptides identified in the hydrolysates.The results displayed that small peptides can better resist digestion of gastrointestinal proteases.The transepithelial transport revealed that the permeability of small peptides through intestinal epithelial cells varied from 0.48%to 10.80%.These results indicate that PBCH has strong antioxidant properties and contain absorbable di-/tripeptides,which is promising for functional food additive.