猪脾脏多肽的酶法制备及其生物活性研究

Preparation of Pig Spleen Peptides by Enzymatic Hydrolysis and Its Biological Activity

目的探索酶解法制备猪脾脏多肽的最佳条件,检测所得产物的生物活性。方法用胰酶酶解法、正交试验、甲醛滴定法考察最佳酶解条件,用乙醇沉淀法收集多肽,并分别用Fe2+诱发卵黄脂蛋白多不饱和脂肪酸过氧化体系和纤维蛋白平板法检测多肽的抗氧化活性和溶栓活性,CM-Sepharose离子交换层析纯化最佳酶解条件下的脾多肽,利用SDS-PAGE电泳确定具备促凝活性的多肽分子片段相对分子质量。结果最佳酶解条件为60℃,pH 7.0,加酶量6.0%,时间5.5 h;检测到脾多肽具备抗氧化活性和促凝活性,经纯化后的脾多肽促凝时间比正常对照提前20 s。结论可初步判断猪脾脏多肽在抗氧化和凝血方面有一定作用,且首次证实猪脾多肽具有凝血功能。

Objective To explore the best conditions of preparation of pig spleen peptides by enzymatic hydrolysis, and to detect the biological activity of enzyme reaction products. Methods Trypsin digestion method, orthogonal experiment, and formaldehyde titration method were used to study the optimal hydrolysis conditions. The peptides were collected by ethanol precipitation, then Fe2＋ was used to induce PUFA peroxidation system in egg yolk lipoprotein. The fibrin plate method was employed to assay the antioxidant activity and thrombolytic activity of peptides. The spleen polypeptides obtained under the optimal hydrolysis conditions were purified by CM-Sepharose ion-exchange chromatography and the molecular weight of the peptide fragments with procoagulant activity was determined by SDS-PAGE electrophoresis. Results The optimal enzymatic hydrolysis conditions of pig spleen peptides were 60 ℃, pH 7.0, 6.0 % enzyme for 5.5 h. The spleen peptides had antioxidant activity and procoagulant activity. The clotting time of purified peptides was 20 seconds shorter than that of natural. Conclusion It can be preliminarily concluded that the pig spleen peptides have antioxidant activity and procoagulant activity. The coagulation function of pig spleen peptides can be confirmed for the first time.