东北林蛙体胶原蛋白酶法制备工艺

Preparation technology of collagen from Rana dybowskii using pepsin

目的探索胃蛋白酶法制备东北林蛙体胶原蛋白的最佳工艺条件,并对提取得到的胶原蛋白的纯度及其氨基酸含量进行测定。方法采用单因素及正交试验,以提取率为指标,优化胃蛋白酶提取东北林蛙蛙体胶原蛋白的工艺条件。通过紫外光谱(UV)分析确定东北林蛙体胃蛋白酶溶性胶原蛋白(PSC)的类型,反相高效液相色谱法(RP-HPLC)测定PSC中氨基酸的含量。结果 PSC最佳提取工艺为温度37℃,E/S 20%,料液比1∶20,时间12 h;在此条件下,胶原蛋白提取率达到88.6%。经紫外光谱(UV)分析,PSC在234 nm处有最强吸收峰,反相高效液相色谱法(RP-HPLC)测定发现,PSC氨基酸含量丰富,包含人体所需的8种必需氨基酸,其中甘氨酸(Gly)、脯氨酸(Pro)、亮氨酸(Leu)和丙氨酸(Ala)等氨基酸含量较高,符合Ⅰ型胶原蛋白特征。结论优化后的制备方法提取率高、重复性好,可为PSC的工业化生产提供参考。

Objective To explore the optimum technological conditions for preparing collagen from brown frog （Rana Dybowskii） body by pepsin, and to determine the purity of collagen and contents of amino acids. Methods By determining extraction rate, single-factor trials and orthogonal experiments were used to optimize technological conditions for preparing collagen from the body of this brown frog with pepsin. The type of pepsin-solubilized collagen （PSC） was determined by ultraviolet and visible speetrophotometer （ UV ）, and the contents of amino acids in PSC were determined by using reversed phase high performance liquid chromatography （ RP-HPLC ） ; Results The optimum technological conditions of PSC were that 37 ~C for temperature,20% for E/S, 1 ： 20 for the ratio of material to solvent and 12h for time. Under these conditions ,the extraction rate of PSC was up to 88.6%, The PSC had the strongest absorption peak at 234 nm through UV analysis. Results of RP-HPLC analysis showed that PSC contained abundant of amino acids, especially 8 essential amino acids needed by human body, in which Gly,Pro, Leu and Ala had higher contents than the others, which matched with the features of collagen I ~ Conclusion The optimized preparation method had high extraction rate and good repeatability, thus could provide references for technological production of PSC.