以鲢鱼骨为原料,经蛋白酶酶解、Sephadex G-25凝胶色谱分离纯化制备具有高抗氧化活性的胶原多肽。首先以胶原多肽得率和水解度为评价指标,从中性蛋白酶、碱性蛋白酶、风味蛋白酶、胰蛋白酶和复合蛋白酶中筛选最优酶,并采用单因素实验、响应面优化实验确定鲢鱼骨胶原多肽的最佳制备工艺;然后采用Sephadex G-25凝胶色谱进行分离纯化,得到不同分子质量多肽组分并分析其氨基酸组成;以ABTS+·清除能力和还原能力为指标,考察鲢鱼骨胶原多肽各组分的抗氧化活性。结果表明,酶解鲢鱼骨的最优酶为复合蛋白酶,制备胶原多肽的最佳工艺:酶添加量为8 220 U/g、酶解温度为51℃、酶解时间为4.5 h,此时胶原多肽得率为49.00%;经Sephadex G-25分离得到5个组分,其分子质量为14~9 788 Da,氨基酸组成符合胶原蛋白的特征;抗氧化实验表明组分Ⅳ具有最强的ABTS+·清除能力和还原能力,其IC50/OD1.0分别0.02 mg/mL和19.16 mg/mL。该研究为鱼骨资源的开发利用提供了技术参考。
Collagen polypeptide with high antioxidant activity was prepared from silver carp bone by protease hydrolyzed and Sephadex G-25 gel chromatography separation and purification. First, the optimal enzyme was screened from neutral protease, alkaline protease, flavor protease, trypsin and composite protease by taking the collagen polypeptide yield and degree of hydrolysis as the evaluation index, and the optimal preparation process of collagen polypeptide of silver carp bone was determined by single factor and response surface optimization experiment. Then Sephadex G-25 gel chromatography was used for separation and purification to obtain polypeptide components with different molecular weights and analyze their amino acid compositions. The antioxidant activity of each component of collagen polypeptide from silver carp bone was investigated by using ABTS+· scavenging capacity and reducing power as indicators. The results showed that the optimum enzyme for enzymatic hydrolysis of silver carp bone was composite protease, and the optimum process for preparation of collagen polypeptide was as follows: the amount of enzyme added was 8 220 U/g, the hydrolysis temperature was 51 ℃, and hydrolysis time was 4.5 h, and the collagen polypeptide yield was 49.00 %. Five components were separated by Sephadex G-25 with molecular weight range from 14 to 9 788 Da, and the amino acid composition accord with the characteristics of collagen. The antioxidant experiment showed that component IV had the strongest ABTS+· scavenging capacity and reducing power, with IC50/OD1.0 of 0.02 mg/mL and 19.16 mg/mL, respectively. This paper can enrich the sources of antioxidant peptides and provide technical reference for the development and utilization of fish bone resources.
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