The Preparation,purification And Identification Of Antioxidant Peptides From Changbai Mountain Pine Nuts

Changbai Mountain Pinus koraiensis Sieb.et Zucc Nuts are rich in unsaturated fatty acids, protein, vitamins, minerals, with higher nutritional value. But the development and utilization have always been limited to oil, this is a great waste of protein resources which the pine nuts rich in. Therefore, it has important significance to process pine nuts, separate and extract active ingredients, prepare bioactive peptides which is easy to be absorbed by human body and safe without side effects. In this paper, enzymolysis technology, antioxidant activity, separation and purification, structure and composition of the pine nuts antioxidant peptides were studied. Thus a reference has been provided for the comprehensive utilization of Changbai Mountain Pinus koraiensis Sieb.et Zucc Nuts and the development of polypeptide functional products.This paper taked Changbai Mountain defatted pine nuts powder as raw material, using the method of the alkaline dissolving and acid precipitating method to prepare protein isolated, and using the enzymatic hydrolysis of Alcalase to prepare antioxidant peptides. Based on the results of single factor experiment, selecting reducing power as the index to analyze response surface. Optimal technological conditions is optimized as the following: hydrolysis temperature of 56.5℃, Alcalase concentration of 7822U/g, substrate concentration of 0.021g/mL, pH of 9.0and hydrolysis time of 240 min. On this condition, reducing power of enzymatic hydrolysis liquid is 0.753.Prepare pine nuts antioxidant peptides according to the optimal technological conditions,then evaluate it’s antioxidant activity. There were six in vitro antioxidant indexes, reducing power, chelating ability of Fe2+and capacity of scavenging DPPH, ABTS, hydroxyl radicals, superoxide anion radicals of antioxidant peptides were determined respectively. The results show that, with the increasing of peptide solution concentration, antioxidant activity of antioxidant peptides presented an increasing trend, showing some dose-effect relationship. When the concentration was 4mg/mL, the radical scavenging activity of ABTS and the chelating rate of Fe2+ can reach 100%;When the concentration was 16mg/mL, the radical scavenging activity of superoxide anion can reach 31.99%, the radical scavenging activity of DPPH can reach 80.95%;When the concentration was 24mg/mL, the radical scavenging activity of hydroxyl radicals can reach 100%; When the concentration was 80mg/mL, the reducing power of antioxidant peptides could only up to 1.105.The antioxidant peptides were separated and purify by ultrafiltration, Sephadex G-25, Sephadex G-15 and reversed-phase high performance liquid chromatography successively, and the fractions were determined by the aspects of superoxide anion, ABTS, hydroxyl radical and reducing power, then selected the one most active fraction for next step separation. After four-step purification,ultimately determined that fractions D5 showed the strongest antioxidant activity.Fractions D5 were identified by Ultraflex III MALDI TOF/TOF, its molecular weight was determined to be 987.511 Da.