超声辅助-碱-酶法提取鲟鱼硫酸软骨素工艺及其结构分析

Ultrasonic Assisted-Alkaline-Enzymatic Extraction and Structural Analysis of Chondroitin Sulfate from Sturgeon

为探究超声辅助-碱-酶法提取鲟鱼硫酸软骨素(Chondroitin sulfate,CS)的最佳工艺条件,以鲟鱼软骨为原料,通过单因素试验和响应面设计进行工艺优化;测定鲟鱼CS体外自由基清除能力,并通过傅里叶变换红外光谱(Fourier transform infrared spectroscopy, FTIR)、高效凝胶渗透色谱、高效液相色谱对其结构进行分析。优化得到鲟鱼CS的最佳提取工艺条件为:以碱性蛋白酶为提取用酶、料液比1:30(g/m L)、提取时间122 min、p H值为9.9、温度为45℃。在此条件下,鲟鱼CS的得率为17.06%,纯度90.16%,响应面模型拟合良好;所得鲟鱼CS对DPPH自由基有较高的清除率,IC_(50)为15.04 mg/mL;鲟鱼CS FTIR的特征吸收峰符合CS主要特征,分子质量为37ku,单糖组成以氨基葡萄糖、葡萄糖醛酸、氨基半乳糖、葡萄糖、半乳糖等为主,且同标准品单糖组成基本一致。结果表明:所获得的鲟鱼CS提取方法具有较高的得率和纯度,且能够很好地保持鲟鱼CS完整的天然结构。

In order to explore the optimal process conditions for ultrasonic assisted alkaline enzymatic extraction of chondroitin sulfate(Cs)from sturgeon.Using sturgeon cartilage as the raw material,the process was optimized through single factor experiments and the response surface design;the free radical scavenging capacity of sturgeon cs in vitro was determined,and its structure was analyzed by fourier transform infrared spectroscopy(FTiR),high-performance gel permeation chromatography(HPGPC),and high-performance liquid chromatography(HPLC).The optimal extraction process conditions for sturgeon cs were obtained as follows:alkaline protease was used as extraction enzyme,liquid ratio 1:30(g/mL),extraction time 122 minutes,pH 9.9 and 45℃.Under these conditions,the yield of sturgeon cs was 17.06%,the purity was 90.16%,and the response surface model was well fitted;the obtained sturgeon CS has a high scavenging rate for DPPH free radicals,and the IC_(50) was 15.04 mg/mL;the FTIR characteristic absorption peak of sturgeon CS conforms to the main characteristics of CS,and the molecular weight of the main component is 37 ku,and the monosaccharide composition is composed of glucosamine,glucuronic acid,galactosamine,glucose,galactose,etc.,and is basically consistent with the monosaccharide composition of the standard product.The result shows that the method for extracting sturgeon Cs has high yield and purity and can effectively maintain the intact natural structure of sturgeon cs.