黄鳍金枪鱼酶解物免疫活性及其氨基酸分析

本文旨在研究黄鳍金枪鱼(Thunnus albacares)碎肉中4种不同蛋白酶酶解物的3k Da超滤组分对RAW264.7细胞的免疫活性。以RAW264.7细胞为实验模型, 采用噻唑蓝比色法检测细胞增殖活性, 中性红吞噬实验检测细胞吞噬能力, Griess试剂检测细胞培养上清液中NO含量, 并分析酶解物氨基酸组成和分子量分布。实验结果表明, 胰蛋白酶酶解物在12.5~200μg·mL^-1范围内无明显细胞毒性, 能增强RAW264.7细胞吞噬能力, 浓度为400μg·mL^-1时达到最大值132.50%(P<0.01), 浓度在50~200μg·mL^-1范围内能促进巨噬细胞NO释放(P<0.01), 且NO释放量与酶解液浓度存在一定的量效关系; 胰蛋白酶酶解物中必需氨基酸(essential amino acid, EAA)含量最高, 为51.09%, 其碱性氨基酸组氨酸、赖氨酸和精氨酸的含量分别为11.59%、11.00%和6.90%, 均高于其他蛋白酶酶解物, 并且分子量小于1k Da的小肽含量高达97.48%。研究结果表明, 4种蛋白酶酶解物中, 黄鳍金枪鱼碎肉胰蛋白酶酶解物的3k Da组分具有较明显的免疫活性, 这为其作为免疫活性肽的开发利用提供了理论依据。

Immunoactivity and Amino Acid Analysis of Enzymatic Hydrolysates of Thunnus albacares

In this study, we evaluated the immune activity of ultrafiltration components from yellowfin tuna (Thunnus albacares) meat hydrolyzed by four different proteases on RAW264.7 cells. The RAW264.7 cell line was used as the cell activity screening model in vitro. Methyl thiazolyl tetrazolium (MTT), neutral red and Griess reagent methods were used to detect cell activity, phagocytosis capability and nitric oxide levels in cell culture medium. The amino-acids composition and molecular weight distribution of the hydrolysate were also analyzed. The results show that trypsin hydrolysate had no obvious cytotoxicity in the range of 12.5-200 μg·mL^-1 for RAW264.7 cells. It could enhance the phagocytic capacity, and reached the maximum value of 132.50% (P<0.01) at the concentration of 400 μg·mL^-1. In the concentration range of 50-200 μg·mL ^-1, the trypsin hydrolysate could promote nitric oxide release of macrophages (P<0.01), and there was a dose-effect relationship between NO release and the concentration of enzymolysis solution. The content of essential amino acid (EAA, 51.09%) of trypsin hydrolysate was higher than those of the other enzymatic hydrolysates, and the contents of histidine (His, 11.59%), lysine (Lys, 11.00%) and arginine (Arg, 6.90%) were also higher than those of the other enzymatic hydrolysates. The content of small peptides with molecular weight less than 1k Da was as high as 97.48% in trypsin hydrolysate. In the four protease hydrolysates, the 3k Da peptide fraction of trypsin hydrolysate of tuna meat had obvious immune activity, which provides a theoretical basis for its development and utilization as a functional ingredient.