南极磷虾蛋白抗氧化酶解物的制备及其活性研究

Preparation and Antioxidant Activity Evaluation of Protein Hydrolysate of Euphausia superba

以DPPH自由基(DPPH·)和羟基自由基(HO·)清除率为指标,筛选制备南极磷虾蛋白抗氧化酶解物(EPAH)的蛋白酶;利用氨基酸评分(AAS)和化学评分(CS)分析EPAH的氨基酸组成;利用脂质过氧化抑制试验和张氏肝细胞(Chang liver)氧化损伤模型评价EPAH的活性。蛋白酶筛选结果表明:胰蛋白酶和胃蛋白酶制备EPAH的HO·清除率分别为35.76%±1.58%和33.03%±1.71%,DPPH·清除率分别为44.09%±1.04%和46.7%±2.39%,显著高于中性蛋白酶、风味蛋白酶、碱性蛋白酶制备的EPAH的活性;双酶体系(胰蛋白酶+胃蛋白酶)制备的EPAH的DPPH·和HO·清除率分别为55.49%±3.05%和48.94%±1.11%,显著高于胰蛋白酶和胃蛋白酶制备的EPAH(P>0.05)。因此,确定利用双酶体系(胰蛋白酶+胃蛋白酶)制备EPAH。利用双酶体系(胰蛋白酶+胃蛋白酶)制备的EPAH总氨基酸和必需氨基酸含量分别为62.72%±0.42%和26.17%±0.16%;EPAH氨基酸评分(AAS)中苯丙氨酸+酪氨酸(Phe+Tyr)评分最高,第一限制性氨基酸为缬氨酸(Val);在化学评分(CS)中,赖氨酸(Lys)的化学评分最高,甲硫氨酸+半胱氨酸(Met+Cys)评分最低;EPAH必需氨基酸指数(EAAI)为63.02。脂质过氧化抑制实验结果表明:EPAH可以较好的抑制系统中亚油酸的过度氧化,但活性低于谷胱甘肽。细胞氧化损伤保护实验证明:在15 mg·mL^(-1)浓度下,EPAH可以显著降低张氏肝细胞氧化损伤模型中活性氧自由基的水平,降低过量活性氧自由基对细胞的破坏,将张氏肝细胞的存活率提高到57.98%±2.57%,显著高于模型组(47.61%±3.92%)(P<0.01)。因此,双酶体系(胰蛋白酶+胃蛋白酶)制备的EPAH具有较高的营养价值和抗氧化活性,可以用于抗氧化功能产品的研发。

In this paper,five kinds of proteases were screened guided by DPPH radical (DPPH·) and hydroxide radical (HO·) scavenging assays.The amino acid composition of EPAH was analyzed using amino acid score (AAS) and chemical score (CS).The antioxidant activity of EPAH was evaluated using lipid peroxidation inhibition assay and oxidative damage model of Chang liver cells.The results indicated that the HO·scavenging rates of EPAH separately using trypsin and pepsin were 35.76%±1.58%and 33.03%±1.71%,respectively,and their DPPH·scavenging rates were 44.09%±1.04%and 46.7%±2.39%,respectively,which were significantly higher than those of EPAH prepared using neutral protease,flavor protease and alkaline protease.The DPPH·and HO·scavenging rates of EPAH prepared by the double enzyme system (pepsin+trypsin) were 55.49%±3.05%and 48.94%±1.11%,respectively,which were significantly higher than those of EPAH prepared separately using trypsin and pepsin (P<0.05).Therefore,double enzyme system (pepsin+trypsin) were chosen for preparation of EPAH.The contents of total amino acid,essential amino acid and semi-essential amino acid of EPAH prepared by double enzyme system (pepsin+trypsin) were 62.72%±0.42%,26.17%±0.16%and 5.64%±0.03%,respectively.AAS results indicated that Phenylalanine+tyrosine score was the highest in EPAH and Valine was the first limiting amino acid.CS result showed that Lysine had the highest CS,while methionine+cysteine had the lowest CS among all essential amino acids.The essential amino acid index of EPAH was 63.02.The results of lipid peroxidation inhibition assay showed that EPAH had high inhibiting capability on the excessive oxidation of linoleic acid,but its activity was lower than that of glutathione at the concentration of 15 mg·mL^(-1).Moreover,EPAH could significantly increase the cell viability from 47.61%±3.92%to 57.98%±2.57%through reducing the level of reactive oxygen species (ROS) in oxidative damaged model of Chang liver cells (P<0.01).Therefore,EPAH prepared using double enzyme system (trypsin+pepsin) has high nutritional value and significant antioxidant function.Therefore,it can serve as potential antioxidants applied in nutraceutical and pharmaceutical products.