明太鱼胶原蛋白提取工艺优化及其体外抗氧化活性

Theragra chalcogramma Collagen Extraction Process Optimization and Its in vitro Antioxidant Activity

以明太鱼皮为原料,胶原蛋白得率为指标,通过单因素试验和响应面优化试验确定胃蛋白酶提取明太鱼皮胶原蛋白的最佳工艺,采用聚丙烯酰胺凝胶电泳、傅里叶红外光谱、紫外光谱技术及柱前衍生反相高效液相色谱法鉴定明太鱼皮胶原蛋白基本性质,并测定其体外对羟自由基、超氧阴离子自由基和DPPH自由基清除能力及其总还原能力,初步研究其体外抗氧化作用。结果表明,明太鱼皮胶原蛋白的最优提取工艺条件为酶用量1 500 U/g、反应温度41℃、反应时间3.9 h,该时胶原蛋白得率为48.90%。经该工艺提取制备出的明太鱼皮胶原蛋白符合Ⅰ型胶原蛋白标准品,同时在红外光谱中还含有酰胺的5个主要胶原蛋白特征吸收峰A、B、Ⅰ、Ⅱ和Ⅲ;在235 nm附近具有明显符合Ⅰ型胶原蛋白的紫外吸收峰,且该明太鱼胶原蛋白中含有除色氨酸在外的17种氨基酸,其中甘氨酸、脯氨酸、丙氨酸、谷氨酸和组氨酸含量丰富。体外抗氧化试验表明,提取的明太鱼皮胶原蛋白具有较强的抗氧化活性,其对DPPH自由基、羟自由基和超氧阴离子自由基的半数抑制浓度(IC50)分别为2.69、3.89、4.42 mg/mL。

With Theragra chalcogramma skin as raw material and the extraction rate of collagen as the index,the optimal extraction process of collagen from Theragra chalcogramma skin by pepsin was determined by a single factor test and response surface optimization test.The basic properties of collagen from Theragra chalcogramma skin were identified by polyacrylamide gel electrophoresis,Fourier transform infrared spectroscopy,ultraviolet spectroscopy,and pre-column derivation-reversed phase high-performance liquid chromatography.In addition,the in vitro scavenging capacity and total reducing capacity of the collagen to hydroxyl free radicals,superoxide free radicals,and DPPH radicals were determined,so as to preliminarily studyits in vitro antioxidant activity.The results showed that the optimal extraction process of collagen from Theragra chalcogramma skin was as follows:enzyme of 1 500 U/g,reaction temperature of 41 ℃,reaction time of 3.9 h,and the collagen extraction rate under these conditions was 48.90%.The collagen extracted from Theragra chalcogramma skin by the process met the standard of type Ⅰ collagen products.At the same time,itsinfrared spectra contained five main collagen characteristic absorption peaks of amide,namely A and B,Ⅰ,Ⅱ and Ⅲ.The ultraviolet absorption peak of type I collagen was obviously observed near 235 nm,and the Theragra chalcogramma collagen contained 17 kinds of amino acids in addition to tryptophan,with glycine,proline,alanine,glutamic acid,and histidine-rich in content.Finally,the in vitro antioxidant test showed that the extracted collagen from Theragra chalcogramma skin had strong antioxidant activity,and the half inhibitory concentration(IC50)of DPPH free radical,hydroxyl free radical,and superoxide free radical was 2.69,3.89 mg/mL and 4.42 mg/mL,respectively.