Purification Of Antioxidant Peptides From Frog (Hylarana Guentheri) Protein Hydrolysate And The Properties Of Peptides During Gastroinstinal Digestion

Frog is a prevalent amphibian and used as a common food in East Asia. In this research,antioxidant peptides were released from frog protein by controlled enzymatic technology andchemical models in vitro were employed to evaluate the antioxidant activities. A series ofpurification processes were done on the frog protein hydrolysate and the amino acid sequencewas identified by ESI-MS/MS. The molecular properties of two fractions with significantdifference in molecular size were studied during gastrointestinal (GI) digestion. Theconclusions in detail were listed below:Frog (Hylarana guentheri) is a kind of food material, with low content of fat and highcontent of protein (about17%), which is rich in essential amino acids, making it suitable forobtaining antioxidant peptides. The antioxidant activity related with the enzyme speciesclosely, and four proteases-Alcalase, papain, pancreatin and Flavorzyme were employed tohydrolysis the protein. Results indicated that the hydrolysate treated with papain andFlavorzyme exhibited strong antioxidant capacity, peptides with molecular weight below3kDa accounted for73%. The hydrolysate was proved to possess high nutritional value andhigh amount of antioxidant amino acids. During the simulated GI digestion, the antioxidantactivity of frog antioxidant peptides fluctuate significantly for the gastro digestion, especiallyduring0.5-1.0h the activity increased dramatically; while during2.5-4.0h, the activity of theproducts all decreased, and no significant differences were found, indicating the weak effectof pancreatin.The hydrolysate was purifed by ultrafltration, ion exchange chromatography, gelfltration chromatography and reverse phase high-performance liquid chromatography(RP-HPLC). During the research, neutral or slightly acidic peptides exerted higherantioxidant peptides and molecule with middle size contributed more to the activity. With thehelp of of ESI-MS/MS, two dipeptides were identifed as Leu/Ile-Lys (259.1607Da) andPhe-Lys (293.1446Da), respectively.The frog antioxidant peptides were fractioned into B and S by gel filtration SephadexG-25, for the purpose of studying the molecular properties during GI digestion. The resultssuggested that more small peptides were released and antioxidant activity improved moreduring intestinal digestion compared with gastric digestion; the antioxidant capacity of fraction B incresead widely, while for fraction S, it fluctuated during GI digestion.