School of Food and Pharmacy, Zhejiang Ocean University, Qixiangtai, Zhoushan, People's Republic of China.
Peptides. 2012 Aug;36(2):240-50. doi: 10.1016/j.peptides.2012.05.013. Epub 2012 May 28.
To get high yield of ethanol-soluble proteins (EP) and the antioxidant peptides from Sphyrna lewini muscle, orthogonal experiments (L(9)(3)(4)) were applied to optimize the best extraction conditions and enzyme hydrolysis conditions. The yield of EP reached 5.903±0.053% under the optimum conditions of ethanol concentration 90%, solvent to material ratio 20:1, extraction temperature of 40°C and extraction time of 80min. The antioxidant SEPH (EP hydrolysate of S. lewini muscle) was prepared by using papain under the optimum conditions of enzymolysis time 2h, total enzyme dose 1.2%, enzymolysis temperature 50°C and pH 6, and its DPPH radical scavenging activity reached 21.76±0.42% at the concentration of 10mg/ml. Two peptides (F42-3 and F42-5) were isolated from SEPH by using ultrafiltration, anion-exchange chromatography, gel filtration chromatography and RP-HPLC. The structures of F42-3 and F42-5 were identified as Trp-Asp-Arg and Pro-Tyr-Phe-Asn-Lys with molecular weights of 475.50Da and 667.77Da, respectively. F42-3 and F42-5 exhibited good scavenging activity on hydroxyl radical (EC(50) 0.15mg/ml and 0.24mg/ml), ABTS radical (EC(50) 0.34mg/ml and 0.12mg/ml), and superoxide anion radical (EC(50) 0.09mg/ml and 0.11mg/ml), but moderate DPPH radical (EC(50) 3.63mg/ml and 4.11mg/ml). F42-3 and F42-5 were also effectively against lipid peroxidation in the model system and peroxyl free radical scavenging in β-carotene linoleic acid assay. Their high activities were due to the smaller size and the presence of antioxidative amino acids within the peptide sequences.
为了从尖吻魟肌肉中获得高产量的乙醇溶性蛋白（EP）和抗氧化肽，采用正交实验（L(9)(3)(4)）优化最佳提取条件和酶解条件。在乙醇浓度为 90%、溶剂与物料比为 20:1、提取温度为 40°C 和提取时间为 80min 的最佳条件下，EP 的产率达到 5.903±0.053%。采用木瓜蛋白酶酶解尖吻魟肌肉 EP，在酶解时间 2h、总酶用量 1.2%、酶解温度 50°C 和 pH6 的最佳条件下制备抗氧化 SEPH（尖吻魟肌肉 EP 水解产物），其在 10mg/ml 浓度下的 DPPH 自由基清除活性达到 21.76±0.42%。采用超滤、阴离子交换色谱、凝胶过滤色谱和反相高效液相色谱从 SEPH 中分离得到两个肽（F42-3 和 F42-5）。F42-3 和 F42-5 的结构分别鉴定为色氨酰-天冬氨酸-精氨酸和脯氨酸-酪氨酸-苯丙氨酸-天冬酰胺-赖氨酸，分子量分别为 475.50Da 和 667.77Da。F42-3 和 F42-5 对羟基自由基（EC(50) 0.15mg/ml 和 0.24mg/ml）、ABTS 自由基（EC(50) 0.34mg/ml 和 0.12mg/ml）和超氧阴离子自由基（EC(50) 0.09mg/ml 和 0.11mg/ml）具有良好的清除活性，但对 DPPH 自由基的清除活性适中（EC(50) 3.63mg/ml 和 4.11mg/ml）。F42-3 和 F42-5 对模型体系中的脂质过氧化和β-胡萝卜素亚油酸测定中的过氧自由基清除也有明显的抑制作用。它们的高活性归因于肽序列中较小的尺寸和抗氧化氨基酸的存在。
