驴骨胶原肽制备方法的初步建立及分子量检测

旨在建立驴骨胶原肽的制备方法,并对其分子量进行检测。以牛血清白蛋白为对照品,建立多肽含量和OD_{650 nm}值的标准曲线方程;模拟人工胃液、肠液环境,以驴骨胶原肽一级、二级酶解产物多肽含量为评价指标,在筛选胃蛋白酶和胰蛋白酶最佳酶解时间和加酶量的基础上,采用二级生物酶解技术制备驴骨胶原肽;应用SDS-PAGE法对制备的驴骨胶原肽的分子量进行检测。结果表明,建立的多肽含量和OD_{650 nm}值标准曲线方程为：y=0.163 9x-0.149 6,R²=0.998 9;应用胃蛋白酶进行一级酶解驴骨胶原肽的最适反应条件为：pH值在2.5~3.0,加酶量2.0%、酶解时间2.0 h;应用胰蛋白酶进行二级酶解驴骨胶原肽的最适反应条件为：pH值在7.5~8.0,加酶量2.0%、酶解时间2.5 h;经SDS-PAGE法检测,根据上述最适反应条件制备的驴骨胶原肽的分子量在10 kDa左右。研究结果为开发适合人体吸收分子量的驴骨胶原肽原料以及后续产品的研发提供了技术参考。

Preparation and Molecular Weight Determination of Donkey Bone Collagen Polypeptides

This study was conducted to establish a preparation technique for donkey bone collagen polypeptides,and to determine the molecular weight of the products.Using bovine serum albumin as a reference substance,the standard curve equation between polypeptide contents and ultraviolet absorption values at OD650 nm was established;under the condition of artificial gastric juice and artificial intestinal juice,using the polypeptide content of primary enzymatic hydrolysates and secondary enzymatic hydrolysates as evaluative indicator,the donkey bone collagen polypeptides were prepared with secondary bio-enzymatic hydrolysis technology after determining the optimum enzymolysis time and adding amount of pepsin and trypsin;the molecular weight of the prepared donkey bone collagen peptides was detected by using SDS-PAGE.The results showed that the established standard curve equation between polypeptide contents and ultraviolet absorption values at OD650 nm was y=0.1639x-0.1496,R2=0.9989;the optimum conditions for primary enzymatic reaction were as follows:the pH value ranged from 2.5 to 3.0,the adding amount of pepsin was 2.0%,and the enzymolysis time was 2.0 h;the optimum conditions for secondary enzymatic reaction were as follows:the pH value ranged from 7.5 to 8.0,the adding amount of trypsin was 2.0%,and the enzymolysis time was 2.5 h;by using SDS-PAGE,the molecular weight of the donkey bone collagen polypeptides prepared with the above optimal reaction conditions was determined as about 10 kDa.Our research provides a technical reference for the development of donkey bone collagen polypeptide materials with molecular weight suitable for human absorption and the subsequent products.