胰蛋白酶酶解制备孔鳐软骨蛋白寡肽及其抗氧化活性

Peptides from the tryptic hydrolysate of cartilaginous proteins of Raja porosa and their antioxidant activities

以孔鳐软骨为材料,采用盐酸胍抽提、丙酮分级沉淀,制备孔鳐软骨蛋白;以DPPH·和HO·清除活性为导向,采用胰蛋白酶酶解、膜超滤、DEAE-52阴离子交换层析、Sephadex G-15凝胶层析和反相高效液相色谱(RP-HPLC)等技术,制备抗氧化肽,并对其活性进行系统评价。结果显示,孔鳐软骨蛋白经胰蛋白酶酶解和分离纯化得到2个抗氧化肽RCPE-A和RCPE-B,经氨基酸序列分析确定其序列分别为Gly-Glu-Glu-Gly-ProArg-Gly (GEEGPRG)和Gly-Glu-Glu-Gly-Thr-Met-Gly-Leu (GEEGTMGL),质谱(ESI-MS)测定其分子量分别为700.71和792.87 u。体外自由基清除实验结果显示,RCPE-A与RCPE-B对DPPH·(EC50 2.94和1.16 mg/mL)、HO·(EC_(50) 0.34和0.54 mg/mL)、ABTS^+·(EC_(50) 0.34和0.10mg/mL)和O_2^-·(EC_(50) 0.11和0.03 mg/mL)具有良好的清除作用,RCPE-A与RCPE-B亦显示出较强的脂质过氧化抑制作用。研究表明,孔鳐软骨蛋白酶解物及制备多肽可用于抗氧化相关的功能食品开发,也可以用作抗氧化剂延长相关产品的货架期。

In the experiment,cartilaginous proteins of Raja porosa were prepared using guanidine-HCl extraction and acetone precipitation,and two antioxidant peptides(RCPE-A and RCPE-B)were subsequently isolated from the tryptic hydrolysates of cartilaginous proteins using ultrafiltration,anion-exchange chromatography,gel filtration chromatography and reversed phase high performance liquid chromatography(RP-HPLC).The amino acid sequences and antioxidant activities including free radical scavenging activity and lipid peroxidation inhibition were also measured.The results indicated that the amino acid sequences of RCPE-A and RCPE-B were identified as Gly-Glu-Glu-Gly-Pro-Arg-Gly(GEEGPRG)and Gly-Glu-Glu-Gly-Thr-Met-Gly-Leu(GEEGTMGL)with molecular weights of 700.71 and 792.87 u,respectively.RCPE-A and RCPE-B exhibited good scavenging activity on DPPH(EC50 2.94 and 1.16 mg/mL),HO(EC50 0.34 and 0.54 mg/mL),ABTS+(EC50 0.34 and 0.10 mg/mL),and O 2(EC50 0.11 and EC50 0.03 mg/mL).In addition,RCPE-A and RCPE-B were effectively against lipid peroxidation in a linoleic acid model system.The antioxidant activities of RCPE-A and RCPE-B were due to the smaller size and the presence of antioxidant amino acids including Gly,Glu,Pro,Arg,Met and/or Leu within their peptide sequences.These results suggested that RCPE-A and RCPE-B might serve as potential antioxidants and be used as food additives and functional foods.