Study On The Preparation And Antioxidant Activity Of Pinctada Martensii Oligopeptide

Pinctada martensii is the pearl shell of main coastal water of shellfish aquaculture in Guangxi, Hainan and Guangdong province of China. There is a large number of Pinctada martensii meat which is rich in active substances after pearl harvest. This study took Pinctada martensii as raw materials, and prepared Pinctada martensii oligopeptides which have antioxidant activities through enzymatie hydrolysis, ultrafiltration, gel chromatography, reversed-phase high-performance liquid chromatography (RP-HPLC) and so on. This study is aim to provide theoretical evidence for making the best use of Pinctada martensii.First, under suitable conditions, Alcalase 2.4 L, subtilisin, bromelain, papain and flavourzyme were used to enzymolysis the Pinctada martensii meat respectively. Taking the total antioxidant activity, DPPH radical scavenging rate, clearance rate of superoxide anion and clearance rate of hydroxyl radical as the antioxidation index, the best single enzyme was chosen. On the basis of these, in order to optimize the enzymatic hydrolysis process of the best single enzyme, response surface methodology was carried out to study the influenence of pH value, temperature, hydrolysis time, enzyme/subsrtate ratio on the degree of hydrolysis and antioxidation activity. Finally, the mathematic model about the degree of hydrolysis and antioxidation activity was built. In the end, the technology of ultrasound-assisted enzymatic hydrolysis was used to optimize the enzymatie hydrolysis process of Pinctada martensii meat in order to increase the degree of hydrolysis. The results suggested that the hydrolysates of Alcalase had better antioxidation activity and it was chosen as single enzyme through comprehensive comparison and analysis. The results showed that the optimal hydolysis parameters were as follows: substrate concentration 3:2 (ratio of material to water, W/V), pH value 7.0, enzyme/subsrtate ratio 3.01%, temperature 61℃, ultrasonic power 360 W and 35 kHz, ultrasonic time 1h, and then continued to enzymolysis 0.5 h on water bath. The degree of hydrolysis was 33.8% under the optimum conditions. The DPPH radical scavenging rate was 67.3%, the average length of the peptides was 3.2, IC₅₀ was 15.6 mg/mL. Ultrasound-assisted enzymatic hydrolysis increased the degree of hydrolysis at the most and shorten the hydrolysis time.The oligopeptides of Pinctada martensii meat with antioxidation activity were separated and purified by ultrafiltration, gel chromatography and reversed-phase high-performance liquid chromatography (RP-HPLC). They were characteristed by matrix-assisted laser desorption ionization time of flight mass spectrometry and their molecular weights and sequences of amino acids were analyzed and determined.The results suggested that the optimal parameters of ultrafiltration were operating pressure 3.5 bar and temperature 5℃.The molecular weight distribution range of PMO was 89¹437.5 Da after Sephadex G-25 separation and than it was purified by RP-HPLC further. Each composition after purification by RP-HPLC was subjected to MALDI-TOF-MS. The results suggested that the molecular weights of composition a, b, c, d and e after purification by RP-HPLC were 1022.543 Da, 1334.660 Da, 1494.761 Da, 1085.597 Da and 1288.819 Da or 1034.828 Da, respectively. And the sequences of composition b , c and e was Arg-Gly-Glu-Arg-Arg-Asp-Gly-Tyr-Asn-Gly-Arg, Val-Ala-Thr-Gly-Leu-Arg-Lys-Glu-Gly-Val-Val-Gly-Gly-Pro-Arg and Asn-Ala-Asp-Pro-Ala-Val-Arg-Pro-Ala-Pro-Pro-Lys-Gly or Ala-Ala-Ala-Ser-Pro-Ser-Gly-His-Ala-Ala-Ser-Pro-Pro-Ala-Asn.Choosing reduced GSH and vitamin C as controls of PMO antioxidation activity, this study compared the abilities of scavenging DPPH, O^2- and .OH of the products after membrane separation. The results suggested that the abilities of scavenging DPPH, O^2- and .OH of PMO were the best among the products after membrane separation. The IC₅₀ value of scavenging DPPH, O^2- and .OH was 11.1 mg/mL, 18.9 mg/mL and 26.1 mg/mL, respectively. The total antioxidation activity of 5.23 mg/mL PMO is better than 0.1% vitamin C or 1 mg/mL GSH.