Abstract: 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 OD_{650 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 OD_{650 nm} was y=0.163 9x-0.149 6, R²=0.998 9; 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.

Key words: donkey bone collagen polypeptides, bio-enzymatic hydrolysis, SDS-PAGE, molecular weight