抗氧化黑豆肽的分离纯化与结构鉴定

Isolation and Identification of Antioxidant Peptides Derived from Black Soybean Protein

以氧自由基清除能力(ORAC)为评价指标,通过超滤与大孔树脂吸附、乙醇分级洗脱对黑豆蛋白酶解产物进行初步分离,采用Sephadex G-25凝胶过滤色谱、反相高效液相色谱(RP-HPLC)与排阻色谱(SEC)逐级纯化,最终得到RP-HPLC P2组分SEC P2肽段和RP-HPLC P4组分SEC P3肽段2个纯化多肽,质量浓度为1 mg/mL的2个纯化多肽的ORAC值(Trolox当量)分别为307.5和314.6μmol/L,其氧自由基清除能力分别是超滤分离抗氧化最强黑豆肽组分(Mr<1 000)的2.68和2.74倍,经Edman降解、N-端测序进行结构鉴定,纯化后的2个抗氧化肽段的氨基酸组成序列结构分别为Tyr-Asn-Ile和Trp-Asn-Pro,N-端分别为具有酚羟基结构的酪氨酸Tyr和疏水性很强的色氨酸Trp。

The oxygen radical absorbance capacity（ORAC） has been applied as an index to isolate the antioxi- dant peptides from black soybean protein hydrolysates by ultrafiltration （UF） and Macroporous Adsorption Resin （MAR）; the fraction performed the highest antioxidant activity was further purified with consecutive methods on Sephadex G - 25 column, reversed phase high - performance liquid chromatography （ RH - HPLC ） and Size Exclu- sion Chromatography （SEC） ; two highly purified antioxidant peptides named SEC P2 and SEC P3 produced from RP - HPLC P2 and RP - HPLC P4 have been obtained with ORAC values of 307.5 and 314.6 μmol/L, respectively. The antioxidant capacities were 2.68 and 2.74 times than that of the fraction purified by UF （ Mr 〈 1 000）, respec- tively. Moreover, the amino acid sequences of the peptides with strong antioxidant activity were confirmed as Trp - Asn- Pro and Tyr- Asn- Ile by automated Edman degradation with protein sequencer. Additionally, Tyr with aro- matic residue and Trp with high hydrophobicity could be located at N -terminus.